Anti-parasitic and Anti-viral Insect Immunity - DiVA Portal

69 Pages·2004·0.82 MB·English

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
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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
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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.

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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
LD (LD; lethal dose) the amount of a material, given all
50
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

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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 vector-
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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
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