Anti-parasitic and Anti-viral Immune Responses in Insects

Anti-parasitic
and
Anti-viral
Immune Responses in Insects
Olle Terenius
Department of Genetics, Microbiology and Toxicology
Stockholm University
2004
Doctoral thesis from the Department of Genetics, Microbiology and
Toxicology, Stockholm University, Stockholm, Sweden
Abstract
Insects encounter many microorganisms in nature and to survive they have developed
counter measures against the invading pathogens. In Drosophila melanogaster
research on insect immunity has mainly been focused on infections by bacteria and
fungi. We have explored the immune response against natural infections of the parasite
Octosporea muscaedomesticae and the Drosophila C virus as compared to natural
infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were
able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly
shown that natural infections led to different results than when injecting the pathogens.
In order to search for the ultimate role of the lepidopteran protein
hemolin, we used RNA interference (RNAi). We could show that injection of double
stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to
embryonic malformation and lethality and that there was a sex specific difference. We
continued the RNAi investigation of hemolin in another lepidopteran species,
Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A
similar induction of hemolin was seen after infection with baculovirus and we
therefore performed in vivo experiments on baculovirus infected pupae. We could
show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae
showed any symptoms of infection, while a high dose led to a more rapid onset of
symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in
comparison with other Hemolin sequences, it was possible to select a number of sites
that either by being strongly conserved or variable could be important targets for
future studies of hemolin function.
© Olle Terenius, 2004
ISBN 91-7265-930-0 pp. 1-68
PrintCenter Stockholms Universitet, Stockholm 2004
2
Table of contents
List of papers included in the thesis
5
Abbreviations
6
Introduction
8
Aims of this thesis
12
Parasites in Drosophila
13
Comparative analysis of reports on anti-parasitic
immune responses in Drosophila melanogaster
16
Virus in Drosophila
21
RNA interference in insects
24
RNAi – a defence against intruding RNA
25
The RNA interference mechanism
25
Baculovirus and Hemolin in Lepidoptera
27
Background
27
Physiological protection against baculovirus infection
31
Developmental resistance
31
Developmental expression of Hemolin
36
Immunological protection against baculovirus infection
39
The importance of haemocytes in the response to
baculovirus infection
40
The importance of phenoloxidase in the response to
baculovirus infection
42
3
The involvement of hemolin in immune response to
baculovirus
45
A model for anti-viral response in Lepidoptera with hemolin
as a key player
48
Phylogenetic aspects on hemolin and baculovirus
in Lepidoptera
50
Summary of the results
54
Acknowledgement
55
References
59
4
List of papers included in the thesis
This thesis is based on the following papers#, which will be referred to in
the text by roman numerals. In addition some previously unpublished data
are presented.
I.
Roxström-Lindquist K*, Terenius O*, Faye I (2004) Parasite-specific
immune response in adult Drosophila melanogaster: a genomic study.
EMBO Reports 5: 207-212.
* These authors contributed equally to the work.
II. Bettencourt R, Terenius O, Faye I (2002) Hemolin gene silencing by
ds-RNA injected into Cecropia pupae is lethal to next generation
embryos. Insect Molecular Biology 11: 267-271.
III. Hirai M, Terenius O, Li W, Faye I (2004) Baculovirus and dsRNA
induce Hemolin, but no antibacterial activity, in Antheraea pernyi.
Insect Molecular Biology 13: 399-405.
IV. Li W*, Terenius O*, Hirai M, Nilsson AS, Faye I (2004) Molecular
characterization, immunological and phylogenetic analysis of
Hemolin, in the Chinese oak silkmoth, Antheraea pernyi. Manuscript.
* These authors contributed equally to the work.
#
The published papers were reproduced with the permission from the copyright
holders.
5
Abbreviations
20E
20-hydroxy-ecdysone
AcMNPV
Autographa californica multinucleocapsid
nucleopolyhedrovirus
ALP
alkaline phosphatase
ApNPV
Antheraea pernyi nucleopolyhedrovirus
BmNPV
Bombyx mori nucleopolyhedrovirus
BV
budded virion
DCV
Drosophila C virus
dsHemolin
double stranded RNA of Hemolin
dsRNA
double stranded RNA
EcR
ecdysone receptor
egt
ecdysteroid UDP-glycosyltransferase
FHV
flock house virus
HPLC
high performance liquid chromatography
LD50
(LD; lethal dose) the amount of a material, given all
at once, which causes the death of 50% of a group
of test animals
LdMNPV
Lymantria dispar nucleopolyhedrovirus
MALDI-TOF
Matrix-assisted laser desorption/ionisation-time of
flight
MNPV
multinucleocapsid nucleopolyhedrovirus
mRNA
messenger RNA
ODV
occlusion derived virion
6
PCR
polymerase chain reaction
per os
by mouth
phk
pherokine
PO
phenoloxidase
qRT-PCR
quantitative reverse transcriptase PCR
RNAi
RNA interference
S2
Schneider’s cell line 2
siRNA
short interfering RNA
SNPV
single nucleocapsid nucleopolyhedrovirus
USP
ultraspiracle protein
7
Introduction
Insecta is the most successful group of terrestrial animals, both by
number of individuals and number of species. Insects outnumber the
rest of the terrestrial animals by several orders of magnitude, for
example are the species of vertebrates (fish, amphibians, reptiles,
birds and mammals together) only as many as the coleopteran
family Curculionidae. However, one has to take into consideration
that in the vertebrate classes most species are described, which is not
the case for insects. It is only in the best-known insect families
where the number of newly described species (per year and total
number of species in the family) is as low as in vertebrates (O.
Terenius and S. Firle, unpublished).
Some insects can appear in gargantuan numbers as in
locust swarms, consisting of many billion individuals that eat
several tons of plant material each day. These swarms have been
reported since historic times and were one of the punishments for
Pharaoh not letting Moses and his people leave Egypt “and there
remained not any green thing in the trees, or in the herbs of the field,
through all the land of Egypt” (Exodus, 10:15). While I am writing
this, locust swarms strike sub-Saharan Africa leading to devastating
consequences. Another important torment for humans caused by the
act of insects is plague, which is spread by fleas and in medieval
times made such impact on humanity that it caused the ever-growing
world population to temporally decrease. Today, the plague is
feared, but very rare. This is however, not the case for the vector8
borne (insect-transmitted) disease malaria that is spread by
mosquitoes and ends a human life every 20 second.
In contrast, insects also may have a direct beneficial
impact on human life by producing honey and silk. In all these
cases, their impact is linked to the fact that they are so numerous.
How have the insects become so successful? Could their
rapid development and massive production of progeny suffice as
explanations, or are there other things that contribute to the success?
A short-lived species could be expected to invest more in
reproductive fitness than in life prolonging traits. However, many
insects do live for long periods of time. In the Palaearctic and
Nearctic regions, hibernation is necessary and lasts over several
months. For some species, like the currently reproducing 17-year
cicada (Boyce, 2004), the life prolonging traits are undoubtedly
crucial. One of the important traits that make the insects survive for
long enough periods of time to produce their progeny is their
defence system against invading microbes.
In the last three decades, since the inducible immune
response against bacteria was first discovered in Drosophila
melanogaster by Boman and co-workers (1972), the field of insect
immunity has expanded tremendously including also anti-fungal
response in insects (for reviews, see Hultmark, 2003; Hoffman,
2003; Steiner, 2004). Moreover, this knowledge has been integrated
with the innate immune response in mammals and today there are a
number of interesting reviews comparing the innate immune
responses in mammals and insects (for recent examples, see Boman,
2003; Beutler, 2004). The anti-parasitic responses have been studied
9
mainly in vector insects like Anopheles gambiae and Aedes aegypti,
however no direct anti-parasitic factors have been demonstrated.
Only recently genes involved in parasite-specific response
mechanisms against the malaria parasite are starting to be revealed
in An. gambiae through use of the genome microarray and RNA
interference (for a review, see Blandin & Levashina, 2004).
The key steps in innate immune responses are 1) uniquely
microbial pattern molecules recognized by 2) soluble or membrane
bound pattern receptors like Toll (TLRS) or PGRPs that forward
signals starting a chain of 3) intracellular signalling events that
results in 4) effector molecules and mechanisms acting against the
microbes. It is interesting that the structural differences that exist in
the pattern molecules include nucleotides, peptides, sugars and
lipids or combinations of these in peptidoglycans phospholipids,
lipopolysaccharides, etc. Consequently, one could expect many
pattern receptors, however, strikingly the innate immunity seem to
rely on rather few receptors and the potential for specificity in the
response is still not clear (Beutler, 2004).
Until recently, few attempts have been made to elucidate
the immune responses to virus and parasites in non-vector species.
The result from this thesis and a few other studies show that it is
difficult to incorporate the anti-viral and anti-parasitic responses in
D. melanogaster as well as the anti-viral response in saturnid moths,
in the known signalling pathways of insect immunity. In addition,
the relatedness to these responses in humans is still an open
question. In my summary, I will present the scarce data on antiparasitic and antiviral responses in D. melanogaster, and some of
10
the current knowledge of baculovirus ecology and physiology that
may be of importance for anti-viral defence. As much of these fields
are still unravelled, there are many interpretations that should be
regarded as provisional.
11
Aims of this thesis
• To explore the immune response in Drosophila melanogaster
after natural infection of different microorganisms with special
emphasis on parasites.
• To investigate the function of Hemolin in the saturnid moths
Hyalophora cecropia and Antheraea pernyi.
12
Parasites in Drosophila
In Drosophila melanogaster (Diptera: Drosophilidae) studies on
unicellular parasites have mainly been performed in two species:
Nosema kingi (Nosematidae) and Octosporea muscaedomesticae.
The Microsporidea is a group of eukaryotic organisms that are
obligate intracellular parasites of other eukaryotes, but have been
difficult to place phylogenetically (at kingdom-level) among the
eukaryotes in the tree of life. They have previously been included in
the kingdom Apicomplexa, where organisms such as Toxoplamsa
and Plasmodium belong. Now, however, they have their own
kingdom, Microsporidea, which is considered as the closest relative
of fungi (Hirt et al., 1999; Baldauf et al., 2000). Microsporidea are
obligate parasites that infect representatives from all insect orders.
When the host eats the microsporidium, it infects the host by
sending a harpoon-like organelle, the polar tube, into the midgut
epithelial cell and injects its sporoplasm through the polar tube.
N. kingi is mainly transmitted horizontally, but also
vertically, that is either by feeding on food contaminated with
spores, or via eggs laid by heavily infected females. In Drosophila
willistoni the transmission of N. kingi to offspring were 28% when
the parents mated directly after exposure of the parasite, but 43-93%
if there was a delay of two weeks from exposure to mating
(Armstrong, 1977). The time from infection to egg laying seem to be
important, as in D. melanogaster only 4% of the offspring from 513
days-old infected females were parasitized while the horizontal
transmission by feeding larvae with parasites resulted in 100%
infection (Armstrong & Bass, 1989). The effect of N. kingi infection
on D. melanogaster is largely asymptomatic although the adult life
was reduced from 38 days to 28 days in laboratory cultures
(Armstrong et al., 1986). This reduction in adult life span should not
have any implications in the wildlife situation since the mean life
expectancy of an adult D. melanogaster is only 3 days (Rosewell &
Shorrocks, 1987). We tried to obtain N. kingi, but unfortunately the
stocks used in these former studies could not be brought back to life.
Instead, we succeeded in obtaining another microsporidean parasite,
Octosporea muscaedomesticae.
O. muscaedomesticae was originally isolated from the
housefly Musca domestica (Diptera: Muscidae). J. P. Kramer
performed several studies in the 1960’s; in one study he could show
that O. muscaedomesticae isolates from wild caught dipteran species
were able to infect D. melanogaster (Kramer, 1964). O.
muscaedomesticae was shown to be able to persist in the gut for at
least 9 days and was never found in the fatbody or reproductive
tracts among 300 dissected flies. In another study D. melanogaster
was found to be one of the most susceptible flies for O.
muscaedomesticae infection (Kramer, 1973). Any effect on survival
was not reported, however all flies were kept for 10 days before
dissections, which I assume implies that they were alive at the time
for dissection. This encouraged us to use O. muscaedomesticae for
our study on anti-parasitic response in D. melanogaster (Paper I).
14
When starting the project on anti-parasitic immune
response in D. melanogaster there was no published study on which
we could base our investigation and therefore, an approach that
would detect any type of change in gene expression seemed
appropriate. We chose to use Affymetrix GeneChips, which do not
require a microarray facility, something that did not exist at
Stockholm University in those days. While performing our
experiments on a microsporidean parasite, a study on the effect of an
infection by Crithidia, on D. melanogaster was published by
Boulanger and co-workers (Boulanger et al., 2001). Crithidia is a
genus of flagellate protozoans (Euglenozoa: Kinetoplastida:
Trypanosomatidae) and belonging to the same family as some
tropical and vector-borne microorganisms that cause infections in
humans, namely Trypanosoma cruzi, T. brucei and Leishmania.
Since there is only one previous study on anti-parasitic immune
responses in D. melanogaster, I will take the opportunity to compare
it with our genome study on anti-parasitic response. However, one
has to keep in mind that we study two different parasites and that the
focus of the paper by Boulanger and co-workers (2001) is on genes
mainly involved in anti-bacterial and anti-fungal immune responses,
rather than on anti-parasitic genes.
15
Comparative analysis of reports on antiparasitic immune responses in Drosophila
melanogaster
Boulanger and co-workers (2001) infected adult D. melanogaster
OregonR by feeding them for 24 hours with Crithidia fasciculata or
C. bombi parasite cultures in Schneider’s medium. They extracted
RNA from whole flies and analyzed the gene expression of the antimicrobial genes Cecropin A, Defensin, Diptericin, Drosocin and
Drosomycin by Northern blot. The infection did not result in any
detectable expression, possibly due to long maintenance of OregonR
in a laboratory environment, and therefore a new fly strain was
established from field-collected flies, the ILL97. When ILL97 was
fed with Crithidia, Drosocin and Drosomycin showed the strongest
induction and it was somewhat lower for Diptericin. As a positive
control, they also infected the flies orally with a mix of the Gramnegative bacteria Escherichia coli and the Gram-positive bacteria
Micrococcus luteus. In our study (Paper I), we fed the D.
melanogaster strain Canton-S on O. muscaedomesticae in 1% sugar
solution for 24 hours and we also fed the flies with a Gram-negative
bacterium, Serratia marcescens Db11 (Flyg & Xanthopoulos, 1983).
Of the genes that we can compare from the two studies, no one was
induced in our study in all four comparisons, which was the criterion
for selecting them (for explanation of how chips were compared see
Supplementary information text part A belonging to Paper I).
16
Generally the bacterial mix induced the anti-microbial peptides
more than the parasite did. Here we observe the same pattern,
although the differences are small between our parasite O.
muscaedomesticae and bacteria used (Table 1). However, the gene
most strongly induced by S. marcescens and O. muscaedomesticae
was Diptericin, which only was weakly induced by Crithidia or the
bacterial mix. Drosocin, which was strongly induced by E. coli and
M. luteus, and also Crithidia, had around two-fold up-regulation in
both types of infections in our study, but as the comparisons do not
give a clear picture it is difficult to draw any conclusions. The gene
that has the best correspondence between the two studies is Defensin
that was not significantly induced by any infection.
Interestingly, Boulanger and co-workers (2001) separated
the gut tissue from the haemolymph and analysed the peptides in the
different tissues by HPLC and MALDI-TOF mass spectrometry.
They found a single specific peptide peak after feeding parasites as
compared to feeding or injecting bacteria. In our study we found
unique up-regulation of 48 genes after feeding the flies with O.
muscaedomesticae, 12 of which we regard to be secreted (Table 2 in
Paper I). Both our data confirm the hypothesis that insects can
discriminate between different pathogens (Lemaitre et al., 1997).
Boulanger and co-workers (2001) also could show that there was a
difference between feeding and injecting bacteria. Likewise, we also
proposed that this could be the difference for obtaining many
contrasting results as compared to another study where Affymetrix
GeneChips were used (De Gregorio et al., 2001) and bacteria were
17
18
D
D
I
I
I
D
NC
I
I
I
I
D, decrease; FC, fold change; I, increase; NC, no change.
Induction as judged from the Northern blot presented in Boulanger and co-workers (2001)
NC I NC I 1.5
I
b
++
+++++
Drosomycin
NC I NC I 2.3
NC
Fold change of GeneChips is an average of Cecropin A1 and Cecropin A2.
++
+++++
Drosocin
I
1.4
1.1
1.9
2.5
NC NC NC NC 0.9
I I NC I 2.7 NC
NC I NC NC 1
NC I NC I 1.5 NC
a
+
+++
Diptericin
+
+
+++++
Defensin
Cecropin Aa
E. coli + M. luteusb Crithidia spp.b S. marcescens FC O. muscaedomesticae FC
Table 1. Comparison of induction of anti-microbial peptides after feeding of bacteria or parasites.
injected instead of infected per os (see Speculation in Paper I and
also Supplementary information text part B belonging to Paper I).
The use of Affymetrix GeneChip technology is very
powerful, but also expensive. To limit the number of samples we
used one time point (24 hours) for the analysis. It appeared that our
choice of time point was reasonable, since in the Crithidia study,
gene expression of Diptericin, Drosocin and Drosomycin at 24
hours and 48 hours post-injection was compared and there was an
induction seen at 24 hours, but not at 48 hours. Similar to our data
(Fig. 3 in Paper I), Boulanger and co-workers (2001) could not
observe lethality when feeding either of the two species of parasites,
however, when they injected C. fasciculata, originating from
mosquitoes, all flies died within 4 days. As mentioned above
Kramer did not report on any lethal response to heavy O.
muscaedomesticae infection in D. melanogaster. While we for the
other microorganisms could see that the pathogen was present by
observing the decline in survival of the flies, this was not the case
for O. muscaedomesticae. We did not verify the presence of
parasites in the flies after dissection, but as mentioned above,
Kramer (1964) observed O. muscaedomesticae in the gut of D.
melanogaster for 9 days after infection. A method for detection of
microsporideans by PCR appeared in July 2002, and would be very
useful for future studies (Hogg et al., 2002).
The final conclusion of Boulanger and co-workers (2001)
was that since there was a difference in survival of the flies
depending on which of the two parasite species that was injected,
but no species-specific difference, neither for anti-microbial peptide
19
expression nor for haemocytic phagocytosis, there had to be other
mechanism(s) that led to the difference in survival. This further
strengthens our approach of using microarrays for exploring the
immune response when introducing a novel type of pathogen.
20
Virus in Drosophila
In addition to investigating the anti-parasitic immune response in D.
melanogaster, we also infected the flies with Drosophila C virus
(DCV). DCV is a small single-stranded RNA virus, which is
horizontally transmitted. When feeding 1st instar larvae of D.
melanogaster with DCV, it persists through all stages until
emergence of adults (Filipe et al., 1998), thus unlike baculoviruses
in Lepidoptera (see page 31) DCV does not seem to be lost during
metamorphosis. Infection with DCV has different outcomes
depending on which developmental stage that is affected. If larvae
are infected, their survival diminishes, but if adults are infected,
their reproductive capacity increases (Thomas-Orillard, 1996).
Recently, Sabatier and co-workers (2003) reported on
DCV infection of Drosophila adults and the isolation of hemolymph
peptides after 72 hrs, a time point chosen based on observations of
morphological defects in the fatbody that appeared two to three days
after injection. The haemolymph was analysed by MALDI-TOF
mass spectrometry and in contrast to the plethora of induced
proteins that are seen after injections of bacteria or fungi, one single
molecule was clearly induced 72 hours post-injection. This
molecule, CG11390 (named Phk-1, pherokine 1), was not induced
by septic injury of mixed bacteria or by fungal infection. Notably,
the DCV infection did not induce any anti-microbial peptides, and
flies that were mutant for the Toll or Imd pathways did not
experience any difference in survival as compared to wild-type flies.
21
At the transcriptional level there was no up-regulation of
Phk-2, which also was seen in our data (Supplementary Table 1 in
Paper I, not included in the thesis); and constitutive expression by
UAS/GAL4 flies of Phk-2 did not lead to protection against DCV
infection. In our studies we saw that survival after feeding flies with
DCV was about 60% at day 7 (Fig. 3 in Paper I), which corresponds
to a dilution of DCV of between 10-6 and 10-8 of 1011.5 LD50/ml when
injecting 5 nl into each fly (Sabatier et al., 2003). However, it is well
known from previous studies that DCV is much more lethal when
injected than when it enters per os and therefore it is difficult to
estimate the amount of virus imbibed. Recently (January, 2004)
Cherry and Perrimon (2004) demonstrated that the pathogenicity of
DCV infection is dependent on the endocytosis pathway. Flies
mutated in genes involved in endocytosis are protected from viral
pathogenesis.
The pattern of few proteins responding to infection with
DCV was confirmed at the genomic level by our data (Paper I). As
compared to infections by fungi or parasite, that generated 298 and
127 up-regulated genes respectively, there were only ten genes upregulated by DCV (Fig. 1 in Paper I), and five of these were above
2-fold (Supplementary Table 2 in Paper I). The experience of the
Affymetrix data analyses is that up- or down-regulation above 2-fold
can always be confirmed with qRT-PCR. However, there are many
genes with lower fold changes that are biologically relevant. Three
of the five genes with more than 2-fold up-regulation are serine
proteases, CG8869, Ser 4 and Ser 99Dc, interestingly the two latter
genes show strong down-regulation by Beauveria bassiana (fungus)
22
that since long is known to down-regulate the immune defence of D.
melanogaster.
The down regulation of transcription as an antiviral
mechanism leading to global translational arrest and apoptosis is
well studied in mammals. In insect systems this phenomenon has
also been demonstrated in recent years, particularly in Lepidoptera,
where also counter defence mechanisms of baculoviruses are
successfully studied (Thiem & Chejanovsky, 2004).
23
RNA interference in insects
In 1997, the first report on RNA interference (RNAi) in animals
appeared. It described how an increasing number of copies of a gene
in Drosophila melanogaster reduced rather than increased the
expression of that gene (Pal-Badhra et al., 1997). In 1998, Fire and
co-workers announced the discovery of RNA interference [(in
Caenorhabditis elegans (Nematoda: Rhabditida: Rhabditidae)],
which as a natural phenomenon is very intriguing, but that has
become even more important as a research tool and through the
years generated a large number of highly ranked scientific
publications. For a recent review (July, 2004) see a paper published
by Novina and Sharp (2004).
RNAi has introduced a general tool to explore the world
of genetics. Earlier, only a few species of insects allowed creation of
mutants to explore what difference a missing protein would make in
the animal, but now RNAi is becoming increasingly important.
When we started our work on RNAi in the Cecropia moth (Paper II),
few reports were published on the existence and mechanism of
RNAi. Today this is routinely used in many laboratories throughout
the world, and a publication (Gatehouse et al., 2004) recently
reported on the use of RNAi in the honeybee, Apis mellifera
(Hymenoptera: Apidae). As RNAi has a fundamental role for the
results in both Paper II and Paper III, I will use some space to
explain the mechanism.
24
RNAi – a defence against intruding RNA
The present understanding is that there are at least two ways that
cells can use RNAi. It can be a way for the cell to hinder
transposons from moving around in the genome; C. elegans
defective in RNAi have a higher degree of transposon mobilization
(Sijen & Plasterk, 2003). Endogenous short interfering RNA
(siRNA) from both Trypanosoma brucei (Kinetoplastida:
Trypanosomatidae) and D. melanogaster has been shown to be
identical to transposon sequences (Djikeng et al., 2001, and Elbashir
et al., 2001, respectively).
Li and co-workers (2002) showed that RNAi also could
work as an anti-viral response in insects. When Drosophila S2 cells
were infected with the flock house virus (FHV) the production of
FHV-specific siRNA appeared rapidly. Decreasing amounts of FHV
RNA a few days’ later indicated effects from RNAi. Therefore, to be
able to infect the S2 cells, the FHV produces an anti-silencing
protein, which provided evidence for RNAi as an anti-viral defence
in D. melanogaster.
The RNA interference mechanism
When a cell encounters a long double stranded RNA (dsRNA) it
leads to triggering of RNAi. The long dsRNA can come from a
transposon, a virus or being manmade and subsequently injected or
transfected. The RNA interference mechanism can be divided into
five steps (Fig. 1): i) Initially the long dsRNA is cleaved into 21-25
25
nucleotides long double-stranded fragments called short interfering
RNAs (siRNAs) by an enzyme called Dicer. The length of the
siRNAs is dependent on organism; ii) An RNA-induced silencing
complex (RISC) distinguishes between sense and anti-sense RNA.
iii) The sense strand is degraded, iv) and the anti-sense strand can
either be amplified (in worms and plants, not shown) or incorporated
in RISC and target a complementary mRNA; v) When RISC has
bound the anti-sense siRNA it can repeatedly participate in mRNA
degradation, which silences the gene.
dsRNA
Cleavage by Dicer
siRNA duplex
Sense strand degradation
RISC
Target recognition
mRNA
mRNA degradation
mRNA
Figure 1. The mechanism of RNA interference.
26
Baculovirus and Hemolin in
Lepidoptera
Background
Also insects are targets for viruses. This is especially true for insects
of the order Lepidoptera (moths and butterflies) where the number
of insect species affected by viruses is found to be more than twice
as high as in all other holometabolic orders together (720 vs. 320;
Martignoni & Iwai, 1986). This is very high regarding the fact that
the number of described insect species in the other holometabolic
orders (Coleoptera, Diptera and Hymenoptera) are 5 times as many
(Fig. 2). The high number of lepidopteran species infected with
viruses is to a large extent due to a certain type of virus, the
baculovirus (Baculoviridae), which is a family of double-stranded
DNA viruses that infect arthropods, mainly insects and in particular
Lepidoptera, but also some Hymenoptera and Diptera (Cory &
Myers, 2003). Recently, it has been shown that the baculoviruses
have co-evolved with their hosts since ancient times (Herniou et al.,
2004), possibly since the different holometabolic insect orders split.
Baculoviruses have in general narrow host specificity,
especially in the lepidopteran family Lymantriidae, where the
baculoviruses are restricted to one host species. However, one of the
most well studied baculoviruses is AcMNPV (Autographa
27
Other insect orders 100/150 000
Coleoptera 60/370 000
Hymenoptera 110/150 000
Holometabola
Diptera 150/120 000
Lepidoptera 720/150 000
Figure 2. Phylogeny of holometabolic insect orders. The values
indicate number of viruses per order and total number of described
species per order, respectively.
californica multinucleocapsid nucleopolyhedrovirus) from the
alfalfa looper, A. californica (order Lepidoptera: superfamily:
Noctuoidea: family Noctuidae), mainly due to its importance in
protein expression systems. The AcMNPV has a broad infectious
host range and infects larvae from at least 15 families of
Lepidoptera. Baculoviruses with wide host ranges are usually not
equally infective to every species they infect. The wide host range
viruses are mainly isolated from noctuid moths (Lepidoptera:
Noctuoidea: Noctuidae; Cory & Myers, 2003).
Baculoviruses sometimes cause widespread epizootics
that can lead to >90% death in larval populations (Federici, 1993).
28
The baculoviruses only infect larval stages where they form
occlusion bodies, which are proteinaceous structures that contain the
virus particles. Many baculoviruses include multiple genomes in
each infective occlusion body. When the occlusion bodies enter the
midgut they are dissolved upon exposure to proteases and alkaline
pH. Most baculoviruses (in Lepidoptera) spread from the midgut to
other tissues via the tracheoles. By the end of the infection cycle, the
body of the lepidopteran larvae is liquefied and transformed into
millions of new occlusion bodies that are spread into the
environment.
As a consequence of the large viral load on lepidopteran
insects, they have evolved both immune responses and physiological
countermeasures against viral infections. Some of the results from
studies on baculovirus ecology and physiology could be interpreted
in the light of Hemolin as involved in viral defence. Hemolin is a
protein involved in immunity belonging to the immunoglobulin
superfamily that has only been found among Ditrysia moths.
Hemolin was first identified after bacterial injection of the cecropia
moth Hyalophora cecropia (Lepidoptera: Bombycoidea:
Saturniidae; Faye et al., 1975; Rasmuson and Boman, 1979). For a
review on hemolin see Faye & Kanost (1998). On the following
pages I will present circumstantial evidence for possible Hemolin
involvement in several host-baculovirus interactions. The main
outline is presented in Fig. 3 where observations from the
baculovirus system and the Hemolin system are shown as coincided
observations.
29
30
Aggregating
haemocytes
involved in anti-viral
defence
Hemolin mediates
binding between
haemocytes and
pathogens
Midgut a key location
for Hemolin expression
RNAi of HemolinÆ
Phenoloxidase↓
Phenoloxidase↑ Æ
Resistance↑
Entrance of
baculovirus via
the midgut
Adding ecdysone Æ
Hemolin↑
Adding ecdysone Æ
viral suppression
Age↑ Æ
Hemolin↑
Hemolin
Immunology
Development
Figure 3. Coinciding observations from studies on baculovirus and hemolin.
Baculovirus
Immunology
Development
Age↑ Æ
resistance↑
Physiological protection against baculovirus infection
Developmental resistance
Developmental resistance is the phenomenon that as the larva age, it
becomes increasingly resistant to fatal baculovirus infection; this has
been known since long in lepidopteran species (Kirkpatrick et al.,
1998, and references therein). One example is that there was a
40 000-fold increase in LD50 from 1st to 4th larval stage in Mamestra
brassicae (Lepidoptera: Noctuoidea: Noctuidae), and a 1300-fold
increase for Helicoverpa armigera (Lepidoptera: Noctuoidea:
Noctuidae; Rovesti et al., 2000) [Rovesti and co-workers (2000) are
using the synonymous name Heliothis armigera]. Two explanations
of developmental resistance have been presented, one is cellular and
the other is coupled to hormone regulation.
The major route of baculovirus infection is via the
midgut; therefore protection against the virus should be present
there. Indeed, the midgut changes its structure in the late larval
instar to accustom the midgut to a dietary change from solid food
(leaves) during the larval stages, to liquid (nectar) in the adult, and
this can be utilized for expelling virus infected cells. Stem cells that
are to be pupal gut cells replace the larval cells in a process called
sloughing. (Also metamorphoses between the larval stages induce
sloughing.) A number of studies have shown that the mechanism of
a midgut-based resistance involves an increased sloughing of the
31
baculovirus infected midgut cells, which would prevent the
baculoviruses from spreading throughout the body. For example,
when blocking sloughing chemically in Trichoplusia ni
(Lepidoptera: Noctuoidea: Noctuidae) and Heliothis virescens
(Lepidoptera: Noctuoidea: Noctuidae), the AcMNPV pathogenesis
increased (Washburn et al., 1998). On the contrary, the mortality of
AcMNPV-infected H. virescens larvae decreased due to high rate of
sloughing after feeding on cotton, thereby decreasing the utility of
baculovirus as biological control on this plant (Hoover et al., 2000).
What has evolved among baculoviruses that make them avoid being
expelled as a result of the sloughing? One apparent strategy has been
to include several virions in the occlusion body. Viruses can have
one (MNPV) or several (SNPV) nucleocapsids per virion. By having
multiple virions it is possible for the MNPV-virus to avoid the insect
defence. Washburn and co-workers (1999) found that a SNPV
established more primary midgut cell foci, but MNPV infected twice
as many tracheal cells. Since the sloughing cleared the midgut from
baculovirus, more SNPVs than MNPVs were needed to reach the
same level of larval mortality.
Developmental resistance has mainly been coupled to
sloughing of midgut cells and been possible to overcome by intrahemocoelical injection of baculovirus. However, in Lymantria
dispar (Lepidoptera: Noctuoidea: Lymantriidae) developmental
resistance persisted despite injection of baculovirus into the
hemocoel and therefore suggested a systemic component of
baculovirus defence (Hoover et al., 2002).
32
As mentioned previously, the virions of the baculovirus
are released in the larval midgut by the combined action of alkaline
gut pH and proteases. Therefore, also physiological changes that
create an unsuitable environment could have an impact on
baculovirus proliferation. Although the investigators did not suggest
a connection to viral defence, these kind of changes were found in L.
dispar by Lee and co-workers (1998). When L. dispar larvae were
about to enter diapause, the protease activity was substantially
lowered and remained low throughout the diapause. Conversely, the
alkaline phosphatase (ALP) activity increased at the same time and
remained high during the whole diapause. The ALP isolated from
20-days-old pharate larvae had two peaks of activity, at pH 9.0 and
pH 10.6 indicating the presence of two types of ALP. During
diapause, however, there was a strong band of only one diapauseassociated ALP, that was proposed to have a specific but unknown
function (Lee et al., 1998). The physiological function of ALP is not
well understood. In 4th instar of L. dispar larvae the pH ranges from
8.5 in larvae to 11.5 at 3-5 hours post feeding (Schultz &
Lechowicz, 1986). Thus, in the non-feeding diapause stage, the pH
may be suitable for the ALP that has the optimal activity at pH 9.0,
which would indicate a lowering of pH during diapause, which
might lead to a slower development of baculovirus. Notably, in a
parallel case larvae of the Japanese beetle Popillia japonica
(Coleoptera: Scarabaeidae) lowered the midgut pH upon entry into
diapause, which hindered solubilization of crystal endotoxin of the
Gram-positive bacterium Bacillus thuringiensis (Sharpe & Detroy,
1979).
33
The apparent correlation of increased resistance against
baculovirus with developmental age could also suggest that the
resistance would be hormonally regulated. There are two types of
hormones that regulate development in insects, ecdysone and
juvenile hormone. When ecdysone is increasing it leads to moulting
(e.g. transformation from one larval stage to next or from larva to
pupa). The juvenile hormone, on the other hand, hinders the larva
from developing into a pupa, and if the larva experiences an
overdose of juvenile hormone it will continue to grow and become
gigantic instead of entering the pupal stage.
Recently, the long lasting question of how juvenile
hormone antagonizes ecdysone has been clarified (Maki et al.,
2004). Since before it is known that 20-hydroxy-ecdysone (20E)
binds to the ecdysone receptor (EcR) in the heterodimer complex
with USP (ultraspiracle protein), which then regulates insect
development via secondary responses. It has now been shown that
juvenile hormone can counteract the effects of ecdysone by binding
to the USP and recruiting a co-repressor complex without inhibiting
the EcR/USP heterodimerization (Fig. 4).
34
20E
USP
Corepressor
complex
JH
USP
Ecdysone
response
EcR
20E
No ecdysone
response
EcR
Figure 4. A model of inhibition of ecdysone by juvenile hormone
interpreted from Maki and co-workers (2004).
Since a long larval period leads to prolonged baculovirus
multiplication, it would be of advantage for the baculovirus to
decrease the impact of the moulting hormone ecdysone. Therefore,
the baculovirus has evolved towards changing the environment of its
host and as this affects the developmental period. Baculoviruses
encode a gene involved in hormonal manipulation: the ecdysteroid
UDP-glycosyltransferase (egt) gene that produces an enzyme that
conjugates ecdysteroids with galactose or glucose (O’Reilly, 1995).
When egt is expressed the level of ecdysone is radically reduced,
which leads to a longer duration of the larval stage and substantially
increased yield of virus progeny (Slavicek et al., 1999).
Consequently, by knocking out the egt gene it has been possible to
make the baculovirus more efficient as a biological control tool as it
rapidly kills the larvae without prolonging the larval period.
35
In Bombyx mori (Lepidoptera: Bombycoidea:
Bombycidae) on the other hand, a high level of ecdysone has a
protective effect against baculovirus infection. It was possible to
suppress susceptibility to Bombyx mori nucleopolyhedrovirus
(BmNPV) by adding β-ecdysone to the culture medium of B. mori
cells (Su et al., 1989) or by injecting β-ecdysone into B. mori larvae
(Hou & Yang, 1990) and thereby presumably counteracting the egt
gene. However, apart from shortening the larval period, there is no
explanation for how a high level of ecdysone could rescue the larva
from a viral infection.
Developmental expression of Hemolin
There are a number of reports showing that Hemolin is present in the
midgut of larvae and pupae and also that it is increasing with
developmental age. In Manduca sexta (Lepidoptera: Sphingoidea:
Sphingidae) the mRNA level of Hemolin was increased during
larval development without microbial challenge (Yu & Kanost,
1999). Hemolin did not appear on Western blot from haemolymph
of 1st, 2nd or 3rd instar larvae, but started to be seen in 4th and 5th
instar larvae with a larger expression of protein in haemolymph and
fatbody at the wandering stage. Hemolin expression was even more
pronounced in the larval midgut (Yu & Kanost, 1999).
In Hyalophora cecropia (Lepidoptera: Bombycoidea:
Saturniidae) Hemolin levels increased during the 5th instar and
continued to be high during the pupal stage (Trenczek, 1998).
Hemolin was also shown to be present in the oocytes and embryos.
The latter had hemolin in the stomodeal and proctodeal epithelium
36
as well as in neural and epidermal tissue (Bettencourt et al., 1997,
2000). Hemolin was also present in H. cecropia meconium of
emerging adults (Bettencourt, unpublished). Injection of Hemolin
dsRNA into diapausing pupae gave rise to malformation and
lethality in next generation embryos (Paper II).
In L. dispar Hemolin was also appearing in large amounts
before it entered diapause, and it was also present in midgut and
hindgut of diapausing larvae (Lee & Denlinger, 1996). However, in
L. dispar the diapause occurs during the 1st larval instar and not in
the pupal stage as in H. cecropia and M. sexta. In L. dispar the
diapause period starts 15-20 days after oviposition when the embryo
has developed into a pharate larva (this means that embryogenesis is
completed and the larva has consumed extra-embryonic yolk). It
hatches from the egg after completion of diapause.
The correlation between ageing and increasing Hemolin
levels suggested a hormonal regulation of Hemolin expression. This
was shown to be the case in L. dispar larvae where Hemolin was upregulated by 20E (Lee et al., 2002). Hemolin increased when the
diapause started and continued to be expressed at a high level
throughout the diapause. When using KK-42, which inhibits
ecdysteroid biosynthesis, the diapause was prevented and Hemolin
expression was abolished. Moreover, adding 20E to culture media
resulted in Hemolin up-regulation. The fact that the abundance of
Hemolin is increasing when there is a change of alkaline conditions
in the midgut could indicate that as the L. dispar larva develops, it
can protect itself from baculovirus infections by at least two means.
37
In H. cecropia Hemolin was also shown to be upregulated by injection of ecdysone or bacteria into diapausing pupae
(K. Roxström-Lindquist, Y. Assefaw-Redda, K. Rosinska and I.
Faye, unpublished). Ecdysone and bacteria led to up-regulation of
Hemolin in both fatbody and midgut, whereas ecdysone alone led to
up-regulation only in fatbody, thus, the presence of a microorganism
was needed for midgut induction, which is different from the
findings in L. dispar. Interestingly, conserved response elements for
both interferon regulatory factors and hormones in the upstream
region of H. cecropia and M. sexta Hemolin genes were also found.
Another observation that may be related to developmental
resistance of viral infection is the finding that injection of double
stranded RNA of Hemolin (dsHemolin) into developing H. cecropia
pupae led to embryonic malformation and lethality in next
generation (Paper II). We could show that injecting male pupae with
dsHemolin significantly increased the severity of malformation in
the embryos. In D. melanogaster the antimicrobial peptide Andropin
is found constitutively expressed in male seminal fluid and is
thought to function as a protection against bacterial infections
(Samakovlis et al., 1991). Although baculovirus enters while
feeding during the larval stage, it may persist in a latent form in the
pupae and there is a possibility that this infection was manifested in
the next generation embryos. The anticipated need of protection
during the embryonic stage was recently (February, 2004) confirmed
by Gorman and co-workers (2004) who reported that extraembryonic yolk in M. sexta contains antimicrobial proteins as well
as Hemolin.
38
The developmental resistance seen in a number of species
could thus consist of three different mechanisms where the first,
sloughing of midgut cells, is present in all species and may work as
a virus protection for all lepidopterans. Second, changes of pH and
enzyme activity would lead to an environment that keeps the virus
proliferation down. The third mechanism, which may be restricted to
only some species, would be systemic and could involve hemolin as
an anti-viral protein (see below and Paper III) utilizing the high level
of ecdysone produced at the time for the moult as inducer.
Immunological protection against
baculovirus infection
Several cellular anti-viral mechanisms exist; one is the
incompatibility of viruses with hosts. Croizier and co-workers
(1994) showed that recombinant viruses of AcMNPV and BmNPV
had a wider host range than either parental strain. They obtained the
recombinant viruses by first growing them in Sf9 cells from
Spodoptera frugiperda (Lepidoptera: Noctuoidea: Noctuidae) and
then in Bm5 cells from B. mori. The recombined viruses that
originated from AcMNPV, with ability to grow in both cell types,
had a few amino acid changes as compared to the parental strain and
thereby could also infect B. mori larvae, which the parental strain
could not.
Other anti-viral mechanisms are apoptosis and the
shutdown of protein synthesis. Some species of Lepidoptera can
respond to viral infections by inducing apoptosis in infected cells
39
(Clarke & Clem, 2003). As anticipated the baculovirus has
developed countermeasures, in this case using the anti-apoptotic
gene p35 (Clem et al., 1991). Infection by AcMNPV with mutant
p35 leads to global shutdown of protein synthesis in L. dispar cells
(Du & Thiem, 1997) and apoptosis in cells from S. frugiperda (Clem
& Miller, 1993) leading to 1000-fold higher LD50 for S. frugiperda
larvae infected by AcMNPV mutated in p35. However, in T. ni there
is no difference in survival whether the larva has been infected by
the wild type or the p35 mutant forms, indicating that T. ni lacks the
anti-viral apoptotic response. Thus, one of the reasons for the wide
host range of AcMNPV may be its ability to inhibit the apoptotic
immune response (Clem & Miller, 1993). Recently, a novel host
range gene, hrf-1 involved in counter defence against baculoviruses
has been described by Thiem and Chejanovsky (2004). Disruption of
systemic viral resistance in L. dispar was demonstrated by coinfecting baculovirus (LdNPV) and polydnavirus (PDV); the latter is
known to protect the eggs of parasitoid wasps by immune
suppression (McNeil et al., 2004).
The importance of haemocytes in the response to
baculovirus infection
Haemocytes are involved in clearing microorganisms from the
haemolymph of insects by forming melanotic capsules (for a review,
see Ashida & Brey, 1997). However, haemocyte behaviour in viral
infections does not seem to be consistent among different
lepidopteran species. The haemocytes can have different roles, from
40
actively spreading the baculoviruses, via avoiding being infected, to
actively clearing the haemolymph from the viruses.
Barret and co-workers (1998) showed that AcMNPV
could infect T. ni haemocytes, and that the haemocytes caused a
secondary infection within the tracheal epithelial cells. Clarke and
Clem (2002) compared the difference of haemocyte action between
T. ni and S. frugiperda after injection of AcMNPV into the
hemocoel. They found that T. ni was extremely susceptible to viral
infection and that the infection occurred at 12-18 hours in parallel in
several susceptible tissues such as fatbody, tracheal and body wall
epithelium, and also haemocytes. In S. frugiperda the infection
started instead in the fatbody and subsequently spread to tracheal
and body wall epithelium. The haemocytes, though, resisted the
infection and only 50% were infected at the time of larval death (5
days post-infection). However, the haemocytes in S. frugiperda did
not form any melanotic capsules (see below).
The first description of an effective immune response in
insects against viral infection was a study where Helicoverpa zea
(Lepidoptera: Noctuoidea: Noctuidae) was infected with AcMNPV
(Washburn et al., 1996). They showed that haemocytes were
aggregating around midgut-associated tracheae infected by
baculovirus. Baculovirus-infected cells were then encapsulated by
haemocytes and thereafter cleared from the hemocoel. In a followup study they compared the differences between the pathogeneses of
H. zea and Heliothis virescens after AcMNPV infection and found
that encapsulation and melanization of viruses, that only occurred in
H. zea, resulted in a 1000-fold difference in susceptibility to mortal
41
infection (Trudeau et al., 2001). Also, in H. virescens the
haemocytes were infected by the baculovirus.
We know from several studies that one of the main
features of hemolin is the ability to bind to haemocytes and to each
other, thus indicating that hemolin could be a mediator between
microorganisms and haemocytes, and thereby stimulate
phagocytosis (Ladendorff & Kanost, 1991; Bettencourt et al., 1997;
Faye & Kanost, 1998). However, whether hemolin can mediate the
binding between haemocytes and baculovirus remains an open
question.
The importance of phenoloxidase in the response
to baculovirus infection
The phenoloxidase system is a defence system in arthropods that has
as its end product melanin (for reviews, see Soderhall & Cerenius,
1998; Cerenius & Soderhall, 2004). Melanization in insects is used
for healing wounds and also for encapsulation of microorganisms
(Ashida & Brey, 1997). This has also been shown for baculovirus
infection in Lepidoptera. When the cotton bollworm H. armigera
was infected with baculovirus there was a general decrease in
number of haemocytes (Kalia et al., 2001). The haemolymph of
infected larvae melanized slowly indicating phenoloxidase
depletion. Moreover, the cellular fraction had high levels of
phenoloxidase activity, but upon baculovirus infection the activity
was transformed to the plasma. In an early study, the immune
response of T. ni to AcMNPV was investigated by Andersons and
co-workers (1980). Injection or feeding of AcMNPV did not induce
42
anti-microbial peptides, instead, the viral infection resulted in
decrease of haemocyte numbers leading to a lowering of
phenoloxidase in haemolymph, to levels below what appeared when
injecting bacteria (E. cloacae β12). A possibility is that as in H.
armigera, the haemocytes were depleted due to their need in the
anti-viral defence reaction.
Transmission of pathogens is much more efficient when
the hosts are living in close proximity. Therefore, lepidopteran
species that fluctuate in population size may be flexible in their
investment in the immune system; this is called density-dependent
prophylaxis. When this phenomenon was investigated in Spodoptera
littoralis (Lepidoptera: Noctuoidea: Noctuidae), larvae reared in
high density had a higher phenoloxidase activity in haemolymph and
cuticle and also responded more strongly to an artificial parasitic
infection (Cotter et al., 2004). Previously it was shown that a high
level of phenoloxidase in haemolymph was correlated to a high
phenoloxidase level in cuticle and midgut (both being pathogen
entry locations, Cotter & Wilson, 2002). Also, examination of the
level of phenoloxidase in different individuals of S. littoralis showed
that it varied greatly and this difference of phenoloxidase levels was
inherited, which in the long run could lead to an evolutionary
adaptation. Goulson and Cory (1995) saw that rearing M. brassicae
at high or low density led to increased susceptibility to baculovirus
infection, while for larvae reared at intermediate density, both
growth and resistance were stimulated. In Spodoptera exempta
(Lepidoptera: Noctuoidea: Noctuidae) low rearing density was
shown to lead to significantly higher transmission of baculovirus
43
(Reeson et al., 2000), while high rearing density was correlated to
more resistance against baculovirus infection and to higher
phenoloxidase activity in the haemolymph (Reeson et al., 1998).
The level of viral infection in terms of LD50s for the susceptible and
resistant larvae differed by 10 times (1300 viruses vs. 14 200
viruses). This large difference led to that the resistant larvae
survived 1 day longer (8.5 days vs. 7.5 days), which is similar to our
data from Antheraea pernyi (Lepidoptera: Bombycoidea:
Saturniidae; Paper III) where we could see that baculovirus infected
pupae that had been “vaccinated” with control dsRNA showed signs
of infection 1 day later than the pupae that only received baculovirus
(M. Hirai, O. Terenius, W. Li, and I. Faye, unpublished).
In a study on the function of hemolin as an immune
protein, we injected dsHemolin and E. cloacae β12 into H. cecropia
pupae. We injected 6 pupae with dsHemolin and bacteria and 6
pupae with bacteria alone. After 24 hours the haemolymphs were
collected and applied to a nitrocellulose filter (Fig. 5) according to
the method of Brey and co-workers (1991). On the membrane it is
Normal haemolymph
dsHemolin haemolymph
Figure 5. Injection of dsHemolin leads to lowered phenoloxidase
activity. Haemolymphs from H. cecropia pupae injected with E.
cloacae β12 alone (top panel) or with E. cloacae β12 and dsHemolin
(bottom panel).
44
clearly seen that the phenoloxidase activity in dsHemolin-injected
pupae was on average much lower than in the controls.
From this we can conclude that by using dsRNA and
thereby interfering with the mRNA production of hemolin, we could
reduce the phenoloxidase activity (O. Terenius, R. Bettencourt and I.
Faye, unpublished).
The involvement of hemolin in immune response
to baculovirus
We wanted to analyze the function of hemolin from A. pernyi by
injecting dsHemolin and infecting A. pernyi pupae with E. cloacae
β12 (Paper III). When performing these experiments, the results
were difficult to interpret and we realized that the injection of
dsRNA triggered the expression of A. pernyi Hemolin. The
induction pattern of Hemolin was similar when we injected
Antheraea pernyi nucleopolyhedrovirus§ (ApNPV, Fig. 2 in Paper
III) and we could also show a dose-dependent up-regulation of
Hemolin after increasing amounts of control dsRNA (Fig. 5 in Paper
III). We also analyzed the effect of dsRNA on ApNPV infection in
vivo. Injection of 2 µg of dsRNA induced an amount of Hemolin that
delayed the appearance of symptoms observed as a white/transparent
“window” on the anterior part of the pupae (Fig. 6 in Paper III).
§
The genome of ApNPV has been sequenced and found to be 130 kbp (Huang
et al., 2002).
45
However, while injection of 10 µg of control dsRNA still delayed
the progression of the virus infection, injection of 10 µg dsHemolin
resulted in dark pupal windows indicating that the status of the viral
infection was similar to the infection in pupae infected with ApNPV
alone.
Apart from the work in Paper III, there are a number of
studies that could be interpreted as hemolin functioning as an
immune response protein involved in baculovirus infection. In L.
dispar, larvae with confirmed infections of LdMNPV recovered
from the infections indicating the presence of an active immune
system (Bakhvalov et al., 1982; Yu et al., 1992). Also, observations
of Hemolin up-regulation after baculovirus infection come from two
species in the superfamily Bombycoidea where the presence of
Hemolin has been reported. Hemolin was one of the two genes
uniquely up-regulated, according to microarray data, after injecting
B. mori with BmNPV (M. Iwanaga and T. Shimada, unpublished)
and in H. cecropia, Hemolin was up-regulated by AcMNPV (K.
Roxström-Lindquist, unpublished). Our unpublished results from H.
cecropia on the possibility to interfere with phenoloxidase
production with dsHemolin (see above), and the elevated
phenoloxidase activity related to virus resistance in S. exempta,
might suggest that hemolin could act via the phenoloxidase system
in anti-viral defence.
The baculovirus have two forms of virion structures, the
occlusion derived virion (ODV) and the budded virion (BV). The
BV spreads the infection between cells in the insect or in cell
culture, while the ODV is present in occlusion bodies and spread the
46
infection between different insects. One major difference between
the virion types is their envelope proteins; the BV has an envelope
glycoprotein that is very abundant on the surface. The surface of
AcMNPV contains the protein GP64, whereas LdMNPV has a
similar protein, LD130, that also is N-glycosylated (Rohrmann &
Karplus, 2001). Inferred from the homophilic binding properties of
Hemolin, it could be regarded as a lectin and bind to glycosylated
surfaces (Bettencourt et al., 1999).
47
A model for anti-viral response in
Lepidoptera with hemolin as a key player
Figure 6. Summary of tentative physiological and immunological
protection to baculovirus infection.
A. The increase of ecdysone before diapause leads to an increase
of hemolin, which protects the pupae from baculovirus
infection during the diapause. Also the pH becomes more
alkaline and enzymatic activity is changed in order to hinder
the development of baculovirus.
B. As the larva is growing, the risk of acquiring an infection per
os increases. Therefore, the level of hemolin increases with
developmental age and sloughing function as a back up if the
baculovirus have circumvented the defence systems. In a
gregarious species living in high density the risk of acquiring
a virus increases due to the limitation of food. In some species
a high density leads to increased levels of phenoloxidase (not
illustrated).
C. In a species where hemolin functions in anti-viral protection,
the baculovirus encounters hemolin already in the gut. The
hemolin binds to the glycosylated surface of the baculovirus
and mediates the binding to haemocytes that clear the
baculovirus from the body. Baculoviruses that escape the
defence and succeed to infect midgut epithelial cells faces the
possibility of apoptosis (not illustrated), which hinders the
proliferation of the baculovirus.
48
A
Ecdysone
Hemolin
pH
Protease activity
Pre-diapause
Diapause
B
Sloughing
Sloughing
C
Baculovirus
Hemolin
49
Haemocyte
Phylogenetic aspects on hemolin and
baculovirus in Lepidoptera
The most detailed studies on baculovirus in Lepidoptera have been
performed in Noctuoidea. The species that seems to be most
susceptible to infections is T. ni where the haemocytes actively
spread the baculovirus (Barret et al., 1998) and whose cells lack
apoptotic response to viral infection (Clem & Miller, 1993). In
contrast to the susceptible T. ni, H. zea actively encapsulates and
clears the haemolymph from baculovirus, and S. frugiperda has
haemocytes that resist infection and epithelial cells that undergoes
active apoptosis when infected. Although these data are compiled
from various reports, they were all performed with the same virus
(AcMNPV) and could thus give an indication of the species-specific
susceptibility to viral infections. Support for the data on different
resistance levels depending on species comes from a study where a
new baculovirus isolate (“new species”) was recovered from the
diamondback moth Plutella xylostella (Lepidoptera:
Yponomeutoidea: Plutellidae) and tested for pathogenicity in the
original host species as compared to different noctuids (Kariuki &
McIntosh, 1999). Out of the species present in the P. xylostella
study, that also were previously covered in this thesis, the order of
resistance (from the highest to the lowest) was S. frugiperda, H. zea,
T. ni, H. virescens and P. xylostella, which at large confirms the
order of susceptibility that others have reported.
50
A recurring theme is the inconsistency of the observations
of viral response in different lepidopteran species, and any attempts
to link the viral susceptibility with phylogenetic relationship give a
complex answer. Following the phylogeny of Noctuoidea proposed
by Mitchell and co-workers (2000), species susceptible for
baculovirus infection are clustered together with species that are
more resistant (Fig. 7). Noctuidae is paraphyletic with respect to
Arctiidae and Lymantriidae and most of the noctuoids where
baculovirus studies have been performed are in the trifine
Noctuidae.
Different responses to baculovirus infection may be
linked to the presence of hemolin. As shown in Fig. 7, the immune
protein hemolin has so far been found in one clade of
Macrolepidoptera in the superfamilies Noctuoidea and
Bombycoidea. For Noctuoidea there is most data on baculovirus
infection in the trifine noctuids, however, hemolin has until now
(August, 2004) been found in the families Arctiidae (H. cunea) and
Lymantriidae (L. dispar), which are separated from the trifine
noctuids and make direct inference of hemolin involvement in antiviral defence tentative. There are though, the two previously
mentioned reports on baculovirus infection in L. dispar where the
existence of host mechanisms for clearing viruses was suggested
after the finding that some of the larvae survived an LdMNPV
infection (Bakhvalov et al., 1982; Yu et al., 1992). T. ni belongs to
the subfamily Plusiinae and could have a basal placement in the
trifine noctuids (Mitchell et al., 2000). When immune haemolymph
from T. ni was probed with antibody against hemolin from H.
51
cecropia, no cross-reactivity was observed indicating that hemolin is
not induced and secreted in the hemocoel of T. ni (Andersons et al.,
1990). It is premature to speculate about the putative presence of
Hemolin in trifine noctuids as the phylogeny of the
Macrolepidoptera points to an unsolved evolutionary question of
where the hemolin started to function as an immune protein. Either it
was gained with divergence of the clade comprising Noctuoidea,
Bombycoidea and Lasiocampoidea and subsequently lost in the
trifine noctuids or lost and regained twice, both in the clade of
Arctiidae and Lymantriidae and in the clade of Bombycoidea and
Lasiocampoidea. Another alternative is of course that hemolin exists
in all species of Noctuoidea, but at a low level, maybe only at cell
surfaces, and that its function as a soluble anti-viral protein differs at
the species level. Future studies can hopefully settle this question.
Figure 7. Hemolin and baculovirus in Macrolepidoptera. All
lepidopteran species mentioned in the thesis are placed in the tree.
The phylogeny is based on the following sources: Macrolepidoptera,
Tree of life, http://tolweb.org/tree/phylogeny.html; Bombycoidea,
Regier and co-workers (1998); Noctuoidea, Mitchell and co-workers
(2000). Species where the presence of hemolin has been shown are
underlined.
* Samia cynthia (Lepidoptera: Bombycoidea: Saturniidae) hemolin
was identified already in 1975 by Faye and co-workers, and recently
the cDNA was partially sequenced (W. Li and I. Faye, unpublished).
52
53
Lasiocampoidea
Bombycoidea
Noctuoidea
Trichoplusia ni
Mamestra brassicae
Helicoverpa armigera, H. zea
Heliothis virescens
Spodoptera exempta, S. frugiperda,
S. littoralis
Saturniidae
Saturniinae
Attacini
Antheraea pernyi
Samia cynthia*
Saturniini Hyalophora cecropia
Sphingidae Manduca sexta
Bombycidae Bombyx mori
Noctuidae ”Quadrifine”
Arctiidae Hyphantria cunea
Lymantriidae Lymantria dispar
Plusiinae
Noctuidae ”Trifine”
Drepanomoidea
Geometroidea
Hesperoidea
Papilionoidea
Summary of the results
• Discovery of novel genes specifically involved in antiparasitic and anti-viral immune response in Drosophila
melanogaster
• Demonstration of RNAi as a tool for gene silencing in
Lepidoptera
• Discovery of hemolin as a protein crucial for embryonic
development
• Discovery of Hemolin up-regulation by dsRNA and
baculovirus
• Evidence for hemolin involvement in anti-viral defence in
Lepidoptera
• Analysis of the inter-species variation in Hemolin that may
serve as a basis for future functional studies
• Sequencing and immune expression of Hemolin in a new
species, Antheraea pernyi (Lepidoptera: Saturniidae)
54
Acknowledgement
First, I want to thank my supervisor Ingrid Faye for all your support
and inspiration during the years and for accepting me as a PhD
student in your group.
I have been able to collaborate with a number of people
and got help from numerous more. To all of these I am very thankful
for all scientific joy we have shared along the years. The most
fruitful collaborations have so far been with short men or tall
women. In order of collaboration: First, Raul Bettencourt with
whom I started the adventure on RNAi and who always had a bright
idea of how experimental procedures could be improved, second,
Katarina Roxström-Lindquist, who also being a devoted and open
minded scientist, it was so great to finally find a project were we
could work together, third, Jenny Lindh with whom I have shared so
much fun including the excitement of working with mosquitoes and
malaria, fourth, Wenli Li who introduced the Chinese oak silkmoth
and mastered the PCR, and last Makoto Hirai who always worked
hard and never stopped believing in my ideas. I hope that the fact
that this thesis is written does not stop us from pursuing the
collaborations onwards.
I also want to thank the other members in Ingrid’s group,
Yohannes Assefaw-Redda and Katarzyna Rosinska for being good
friends and for teaching me many valuable things for the future.
The former department of Genetics has been an open and
friendly working place, much due to Elisabeth Haggård-Ljungquist’s
55
leadership. Many people have passed by, but in particular I want to
thank: Richard, Sara and Jesper for taking care of the senior
student’s responsibilities, Alex and Clara for being such wonderful
girls (imaging the department of Genetics without them…), Irja and
the late Latif for many memorable moments, Agneta, Christina and
Hans for all help and support, Björn for invaluable help with
computers, Anders for tremendous help with phylogenetics, Joakim
for using his words very preciously, and Anette, Carlos, Lina, Mina,
Mirtha and Petri for immense contribution to the friendly
atmosphere.
A big thank you to all staff members of GCT for all
joyful interactions and especially to: Fredrik for organizing the
innebandy that facilitates the fusion of the departments, Igor for
interesting discussions and collaborations, and Jesper and Gunilla
for now and then showing up in our room for a couple of minutes’
distraction.
The best teachers are often your students and therefore, I
want to thank all the project students whom I have had the privilege
to supervise (in order of appearance): Elin Sial, Jenny Lindh,
Kehmia Titanji, Helen Bergquist, Haleh Ghasriani, Karolina
Eriksson-Gonzales and Linda Böhme for everything you have taught
me.
I also want to thank all the Innate Immunity people for
broadening my view of insect immunology and helping out with
many things, in particular: Anna, Carina, Eva, Fia, Gunnel, Hanna,
Håkan, Jenny, Peter, Uli and Ulla-Maja.
56
For fruitful discussions on the possibility to integrate different
fields of immunology, I am thankful to Carmen Fernández and Uli
Theopold, and I wish you good luck in the important work.
Although this thesis has not covered the aspects of malaria, I
have had the opportunity to participate in the development of the Swedish
Malaria Network and later to co-ordinate it. I want to thank all the seniors
for lots of encouragement during the years, especially Klavs Berzins,
Anders Björkman, Andreas Heddini, Thomas Jaenson, Hedvig and Peter
Perlmann, and Mats Wahlgren.
From the very start I have had a faithful companion in Jenny
Lindh who in 2001 came back from a long journey in Asia and received
the question: “Do you want to participate in organizing a national
workshop on malaria” and responded: “Yes, definitely I will! Who many
are we in the organizing committee?”. When I said: “Great! With you, we
are two”, you did not hesitate, but did a marvellous job together with me.
Since then there have been four more SMN conferences and you have
always been a great support!
I also want to thank all the members of the Junior SMN for
enthusiastically participating in our activities and in particular the board
members for the loads of work that have been performed. Also, our
supporters Anna Färnert and Hans Rosling have made the work of JrSMN
much more smooth.
As an extension of the SMN, we have together with the Swedish Network
on Tuberculosis Research (SNOTR) been developing a foundation for
research on AIDS/HIV, tuberculosis and malaria (the World Infectious
disease Foundation, VIF), and in
57
this work I have had the opportunity to work with Anders Björkman
(malaria) Gunilla Källenius and Stefan Svenson (tuberculosis), and
also with Birgitta Dahl. Thank you all for your enthusiasm and good
work!
I want to thank Sascha Firle, with family, for all fun
together and for all years of counting the number of insect species
on earth, I hope we can finish our important task one day…
A great support in life has been all my relatives in the
families Brändén, Terenius, Unge and Åkerblom, you all know how
much you mean to me. However, due to space limitations I will here
highlight only those who have participated closely in the scientific
journey. I thank my brother Petter with Karin for building the
website of the Swedish Malaria Network (www.smn.nu), my
brother-in-law Johan Unge for lots of input on prediction of protein
structures in Paper IV, my mother Malin Åkerblom for all love and
support, for being my first co-author, and for many discussions on
science in general and science in the third world in particular. And,
finally my three fathers who, in addition of being very good friends,
also have helped me with many scientific aspects through the years:
my father Lars Terenius, professor in Clinical Neuroscience, my late
step-father Carl-Ivar Brändén and my father-in-law Torsten Unge,
both protein crystallographers and professors in Molecular Biology.
Most important for everyday enthusiasm and joy has been
my own family, Rachel, Joel and Hannele. Thank you for
everything!
58
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