Evolution and Functions of Parasitoid Wasp Venom Proteins Outline Write an outline about the evolution and functions of parasitoid wasp venom proteins base

Evolution and Functions of Parasitoid Wasp Venom Proteins Outline Write an outline about the evolution and functions of parasitoid wasp venom proteins based on science papers attached. Different wasps have very different venom proteins. I have attached 5 papers, there are more I will attach later.

Make sure to include these points in the outline.

The reason for your research:

• General background

• Hypothesis

• Which genes are you studying

-Parasitoid wasps

• Life history

• Specific information about Ganaspis sp.1 and related species

-Wasp venom Insect Molecular Biology (2003) 12(5), 527–534
A teratocyte gene from a parasitic wasp that is associated
with inhibition of insect growth and development inhibits
host protein synthesis
Blackwell Science, Ltd
D. L. Dahlman, R. L. Rana, E. J. Schepers, T. Schepers,
F. A. DiLuna and B. A. Webb
lepidopteran host physiology to support endoparasite
development.
Department of Entomology, University of Kentucky,
Lexington, KY, USA
Keywords: Microplitis croceipes, Heliothis virescens,
teratocytes, teratocyte-secreted protein, parasitoid.
Abstract
Introduction
After parasitization, some wasps induce hosts prematurely to initiate metamorphic development that is
then suspended in a postwandering, prepupal state.
Following egression of the parasite larva, the host
remains in this developmentally arrested state until
death. Teratocytes, cells released at egg hatch from
extra-embryonic serosal membranes of some wasp
parasites, inhibit growth and development when
injected into host larvae independent of other parasite
factors (e.g. venom, polydnavirus). Synthesis of some
developmentally regulated, abundantly expressed
Heliothis virescens host proteins is inhibited in hosts
parasitized by Microplitis croceipes and by teratocyte
injection. A cDNA encoding a 13.9 kDa protein (TSP14)
that inhibited protein synthesis, growth and development was isolated from a protein fraction secreted by
teratocytes. TSP14 appears to be responsible, in part,
for the teratocyte-mediated inhibition of host growth
and development. Interestingly, this cDNA encoded a
cysteine-rich amino acid motif similar to that described
from Campoletis sonorensis polydnavirus, a mutualistic virus that enables wasp parasitization of lepidopteran larvae. Moreover, TSP14 inhibited protein
synthesis in a dose-dependent manner in rabbit reticulocyte lysate and wheat germ extract translation systems. We hypothesize that some wasp parasites inhibit
translation as a general means to regulate and redirect
Parasitization of lepidopteran larvae by hymenopteran
endoparasites significantly impacts host immune, growth
and developmental systems. These disruptions are not
mediated by the parasite itself, but rather by products
from accessory glands of the female wasp’s reproductive
tract. Along with the egg, endoparasites inject venom,
ovarian proteins and in some cases an unusual symbiotic
segmented DNA virus (polydnavirus or PDV) during oviposition (Webb et al., 2000). In some braconid parasites, teratocytes (extra-embryonic cells derived from the serosal
membrane of some parasite eggs) are released into the
haemolymph at egg hatching. These teratocytes remain in
circulation and secrete proteins involved in disrupting host
physiology (Dahlman & Vinson, 1993). Teratocytes have
trophic and immunosuppressive roles in some species and
regulate host growth and development in others (Dahlman
& Vinson, 1993).
Microplitis croceipes teratocytes inhibit growth, alter
development and affect related physiological parameters
of Heliothis virescens larvae. Injection of teratocytes
from one parasite egg caused the characteristic postwandering, prepupation developmental arrest and eventual
death normally observed in naturally parasitized larvae
(Zhang & Dahlman, 1989). Some biochemical effects of
parasitization were also mimicked by teratocyte injection.
Juvenile hormone esterase and ecdysone titres were
suppressed to a degree similar to those in parasitized
larvae (Zhang et al., 1992). Hemolymph protein titres
were reduced, with a major effect on storage protein particularly evident (Zhang et al., 1997). Synthesis of storage
protein in the fat body seemed to be inhibited at the level of
translation, as storage protein mRNA levels did not change
even though protein synthesis declined precipitously after
Received 21 October 2002; accepted after revision 1 July 2003. Correspondence: Dr Bruce A. Webb, Department of Entomology, University of Kentucky,
Agric. Sci. Bldg. North, S-225, Lexington, KY 40546–0091, USA. Tel.: +1 859
257 7415; fax: +1 859 323 1120; e-mail: bawebb@.uky.edu
Data deposition: the sequences reported in this paper have been deposited
in the GENBANK database (accession nos. AY145084).
© 2003 The Royal Entomological Society
527
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D. L. Dahlman et al.
injection of teratocytes (Dong et al., 1996). Juvenile hormone esterase synthesis also was inhibited at the level
of translation although transcription may also be affected
(Dong et al., 1996).
Teratocytes do not undergo cell division subsequent to
their release into the haemocoel of the host but do become
polyploidal. Ultrastructural studies show that teratocytes
possess cellular architecture necessary for efficient protein
secretion (Zhang et al., 1994; de Buron & Beckage, 1997).
Teratocytes collected from eggs hatched in vitro produced
responses similar to those collected from parasitized larvae
(Zhang et al., 1997). Moreover, when cultured in vitro, teratocytes secrete proteins (TSPs) that, when injected into
host larvae, inhibit growth and development in a manner
similar to injection of the cells themselves and to natural
parasitization (Schepers et al., 1998). TSPs were shown to
inhibit protein synthesis in fat body, testes and salivary
glands but had no significant effect on protein synthesis by
midgut tissue. Fat body and testes both synthesize the storage protein p76 in abundance and synthesis of this protein
is clearly inhibited in both tissues whereas reductions in
synthesis of other proteins is not evident by SDS-PAGE
analyses of labelled proteins. These studies led to development of in vitro assays for purifying TSP biological activity
based on the ability of various TSP fractions to inhibit
protein synthesis in H. virescens fat body or testes assay
(Schepers et al., 1998). Ultimately, the testes assay proved
most reliable and sensitive, because of the ability to collect
and label identical amounts of tissue for the assay. Routinely, a single testis was labelled in the presence of a
fraction of TSP proteins while the contralateral testis was
labelled as a matched control. These bioassays allowed the
inhibitory activity to be associated with the 3–30 kDa
fraction and only four low molecular weight proteins were
detected in this fraction (Schepers et al., 1998), the most
prominent of which is 14 kDa. In this communication we
report the cloning, sequencing and expression of the
14 kDa TSP and show that this protein, TSP14, inhibits protein synthesis in organ culture bioassays and also inhibits
translation in in vitro translation systems.
There is considerable interest in isolating and expressing
genes from parasitic insects and their associated viruses
that disable the host immune system, inhibit growth and
prevent parasitized insects from completing their development. These genes may allow specific targeting of physiological systems unique to insects and may also provide
novel reagents to study the affected systems. TSP14 is
such a protein. It inhibits growth and development of herbivorous insects when expressed in transgenic plants
(Maiti et al., 2003). Thus, biochemical and molecular
characterization of genes that insect endoparasites employ
to disrupt insect physiological systems can both elucidate
the physiological abnormalities and identify genes that
prove useful for insect control.
Results
Isolation and sequence analysis of TSP14
To isolate one or more active proteins from the 3–30 kDa
TSP fraction that inhibited protein synthesis in organ culture
and translation in in vitro systems, approximately 1500 LE
(LE = the amount of TSP secreted in vitro over 3 days)
were collected from teratocytes cultured in vitro. At least four
major proteins were visualized by SDS-PAGE in this fraction
(Schepers et al., 1998). The most abundant of these proteins,
14 kDa, was separated from the other TSP proteins by
reversed phase HPLC. It was digested with the Lys-C protease
and re-chromatographed on a C18 column. Two peptide fragments were selected for amino acid sequencing. One fragment
provided a sequence of only five amino acids (VTWYN) and
the other fragment was sequenced through twenty-eight
positions (HPFDFSDDGNQSCAPASGICHRVGLEIT).
To amplify a cDNA encoding this protein (TSP14), two
degenerate primers (terat 1 and terat 2) were synthesized
taking advantage of inferred lysine residues N-terminal to the
derived peptide sequence (inferred by Lys-C digestion). The
3′ end and subsequently the 5′ end of the cDNA were amplified
by rapid amplification of cDNA ends (RACE) (Frohman, 1990;
Clackson et al., 1991). The degenerate oligonucleotide
primers, terat 1 and terat 2, were used in parallel reactions
to prime second-strand teratocyte cDNA synthesis and then
amplified in a conventional PCR reaction [terat 1 + oligo-d(T)
and terat 2 + oligo-d(T)]. The terat 1 + oligo-d(T) reaction
produced a single major amplimer of about 150 bp, whereas
the terat 2 + oligo-d(T) reaction produced a major product of
approximately 540 bp. The 540 bp amplimer was cloned and
sequenced (Fig. 1A). The signal peptide and untranslated
leader sequence were obtained from a 5′ RACE product.
The composite sequence encodes all thirty-three amino
acids known from peptide sequence data and shows that
the terat 2 peptide sequence is N-terminal to the terat 1
peptide, as suggested by the initial size of the amplification
products. The complete cDNA contains an open reading
frame of 426 nucleotides that encodes a twenty-two amino
acid signal peptide and a total of 129 amino acids with a
predicted molecular weight of 13 903 Da (Fig. 1A). After
the removal of the signal peptide, the predicted weight of
the secreted protein is 11 607 Da. There are two potential
glycosylation sites in this sequence with lectin blots using
concanavalin A indicating that the protein is glycosylated
(data not shown). Glycosylation would increase the molecular weight of the secreted protein, making it more consistent with the estimated size of 14 kDa for the most abundant
protein in the 3–30 kDa fraction. The ‘inferred’ lysine residue used in construction of the terat 2 oligonucleotide was
not present in the predicted peptide sequence because this
peptide sequence was from the N-terminus of the mature
protein. The poly A tail is preceded by two consensus
polyadenylation signals (Fig. 1A).
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 527– 534
Wasp protein inhibits host protein synthesis
529
Figure 1. (A) TSP14 cDNA and predicted
amino acid sequence (GENBANK accession no.
AY145084). Nucleotide sequence is shown in
capital letters for coding and lowercase for
noncoding. The determined (underlined) and
predicted amino acid sequence is shown. The first
thirty amino acids after the signal peptide have
weak similarity (BLASTX, P = 0.0092) to the
zebrafish hox-114 protein. The six cysteine
residues in the cys-motif are in bold type with the
intron position indicated (*). The position of an
N-glycosylation site is also indicated (bold italics).
(B) Comparison of TSP14 cysteine-motif with
similar motifs from the Campoletis sonorensis
ichnovirus. Cysteine and absolutely conserved
residues are in bold type. CsIV: Campoletis
sonorensis ichnovirus. Molecular weight markers
are indicated in kilobases.
Inspection of the predicted protein sequence of the
14 kDa cDNA revealed a cysteine motif similar to those
previously described from the Campoletis sonorensis
ichnovirus (CsIV) (Summers & Dib-Hajj, 1995) (Fig. 1B).
Alignment of the TSP-cysteine motif with the six cysteines
in CsIV (Fig. 1B) shows that all six cysteine residues were
conserved at a similar spacing to those observed in
CsIV (Blissard et al., 1987; Dib-Hajj et al., 1993; Cui et al.,
1997). A four amino acid core (KPCC) was also present in
the TSP motif. This finding is either evidence of homology
between ichnoviruses and teratocyte genes or convergent
evolution, suggesting a functional and/or genetic relationship between polydnaviruses and teratocytes. All of the
polydnavirus cysteine motif genes contain an intron just
prior to the second cysteine residue in the motif. To determine if the TSP14 gene had an intron at this position, primers
were designed to amplify across the cysteine motif. The
size and sequence of the resulting amplimer confirmed
the existence of an intron at this position, suggesting that
these cysteine-rich viral and teratocyte genes may have a
common ancestral origin.
The TSP14 cDNA was used to probe Northern and
Southern blots of teratocyte and control RNAs or
M. croceipes genomic DNA and bracovirus DNA with
M. croceipes (McBV), respectively. The cDNA encoding
TSP14 was expressed only from teratocytes with a single
mRNA of about 600 bp detected (Fig. 2A). The TSP14
cDNA hybridized only to the wasp genome, apparently as
a single copy gene. Two hybridizing bands were detected
after digestion of M. croceipes genomic DNA with HindIII
and EcoRI (Fig. 2B).
recombinant baculovirus (data not shown). The purified
antiserum reacted only to TSP14 on Western blots of
haemolymph from parasitized H. virescens from 3 days
following parasitization (1 day after egg hatch) through the
time of parasite egression (Fig. 3, day 9). Interestingly,
TSP14 signal declined rapidly once parasite larvae had
exited, suggesting that wasp larvae may contribute to
teratocyte viability.
Immunological studies
Assays with crude TSP
Antibody prepared to a TSP14 peptide reacted specifically
with crude TSP, TSP fractions and TSP14 expressed by
Parasitization and injection of teratocytes decreased
insect growth, haemolymph protein titres and synthesis
Figure 2. Northern (A) and Southern (B) blots probed with TSP14 cDNA.
(A) Northern blot with teratocyte RNA (Terat, 5 µg) and RNA from
unparasitized Heliothis virescens larvae (Hv, 20 µg). (B) Southern blot of
M. croceipes genomic DNA (Mc, 10 µg) and DNA from the polydnavirus
isolated from the same species (M. croceipes bracovirus; McBV, 1 µg).
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 527– 534
530
D. L. Dahlman et al.
Figure 3. Western blot of pooled haemolymph samples from H. virescens
parasitized by M. croceipes for various lengths of time. Lane 1, 0.5 µl
haemolymph from nonparasitized larvae; lane 2, 0.25 LE of crude TSP;
lanes 3–12, 0.5 µl of haemolymph from days 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 postparasitization, respectively. Parasite larva emerged from hosts by day 9.
Figure 5. Dose–response of rabbit reticuloctye lysate assay using
luciferase mRNA with M. croceipes TSP14 protein generated from a
baculovirus expression system. Each data point is the mean of at least four
replications ± SEM.
and in rabbit reticulocyte lysate assays with mRNA from
H. virescens testes, where a dose of 3.5 LE/50 µl caused a
51.3 ± 1.4% decrease in [35S]-methionine incorporation
(n = 3).
Analysis of TSP14 produced from recombinant system
Figure 4. Effects of crude TSP on protein synthesis in H. virescens larval
testes. See Experimental procedures for details of the assay. Lines
calculated as second order regression; dpm, disintegrations per minute.
of protein by the fat body (Zhang & Dahlman, 1989;
Zhang et al., 1997). An in vitro assay was developed to
determine if proteins secreted from teratocytes inhibited
protein synthesis and to facilitate purification of the
active protein. Proteins synthesized by testes from an
individual larva were labelled with [35S]-methionine
after TSP treatment of a single testis in a pair, and the
other, untreated testis in a pair served as a matched
control. TSP treatment consistently reduced protein
synthesis relative to controls. For example, 3 LE of
crude TSP significantly decreased [35S]-methionine
incorporation (e.g. 81.3 ± 6.1% at 4 h, n = 25). Controls
showed a linear increase in the incorporation of [35S]methionine into protein over 8 h (Fig. 4, control values).
By contrast, the rate of synthesis in the TSP treatments
was greatly reduced relative to controls over the first
hour. Thereafter, the rate of protein synthesis declined so
that beyond 2 h relatively little additional incorporation of
radiolabelled methionine occurred (Fig. 4). Most of the
reduction in protein synthesis in fat body organ assays
was associated with the TSP 3–30 kDa fraction (Schepers
et al., 1998) with TSP14 the most abundant protein in this
fraction. This fraction had similar activity in testes bioassays
The TSP14 protein expressed from the baculovirus system
was purified for analyses of protein synthesis inhibition in
the testes bioassay. A TSP14 dose of 0.5 µg/100 µl resulted
in a 27.1 ± 2.2% (n = 11) reduction in [35S]-methionine
incorporation by testes. Based on the predicted molecular
weight (11 466 Da), the concentration of the recombinant
protein would be approximately 0.436 µM. Three LE of
crude TSP/100 µl used in a parallel set of experiments
produced a similar [31.0 ± 6.0% (n = 9)] reduction in [35S]methionine incorporation.
When the recombinant TSP14 was assayed in a rabbit
reticulocyte lysate system, an inverse dose-dependent
effect on protein synthesis was observed over a range of
0.2–0.8 µg protein/25 µl reaction (Fig. 5). At the highest
dose, the TSP14 concentration was approximately 2.79 µM.
To determine if TSP14 inhibited protein synthesis in other
in vitro translation systems, 0.5 µg of TSP14 was assayed
in the wheat germ system (Fig. 6). A significant reduction of
[35S]-methionine incorporation (23%) was observed.
In conclusion, recombinant TSP14, purified after expression in a baculovirus system, inhibits protein synthesis
in organ cultures, rabbit reticulocyte lysate assays and
wheat germ extract assays. Inhibition of translation was
similar to that observed when the 3–30 kDa TSP fraction
was assayed (data not shown). Activity of TSP14 in in vitro
translation assays may allow direct determination of the
effect of TSP14 on translation and elucidation of the biochemical pathway through which teratocytes disrupt protein synthesis and thereby target selected physiological
systems of their lepidopteran hosts.
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 527– 534
Wasp protein inhibits host protein synthesis
Figure 6. In vitro inhibition of protein synthesis by TSP14 in a wheat germ
extract translation system. See Experimental procedures for details of the
assay. Each bar represents the mean of three replications ± SEM.
Discussion
Parasitization by many endoparasitic wasps disables the
immune system of their insect hosts, inhibits host growth
and may arrest their development. Identification of genes
disrupting insect immunity, growth and development systems may allow direct targeting of insect physiology to
control pest insects or interrupt transmission of infectious
diseases. Understanding the mechanisms through which
insect physiological systems are controlled by parasitic
wasps also may suggest fundamentally novel approaches
to insect control.
Because of their striking effects on host development,
we have focused on the effect of teratocytes on insect
growth, development and related physiological parameters
(reduced titres of storage proteins, juvenile hormone esterase and ecdysteroids). Teratocyte injection alone arrested
host development in a postwandering, prepupation stage
and caused the eventual death that is characteristic of
parasitism (Zhang & Dahlman, 1989; Zhang et al., 1994,
1997; Schepers et al., 1998). Proteins secreted by teratocytes selectively inhibited synthesis of several abundantly
expressed proteins in a tissue-specific manner. Protein
synthesis in H. virescens fat body, labial glands and testes
were all inhibited by TSP, but protein synthesis in midgut
tissues was not inhibited (Schepers et al., 1998). Fat body
and midgut from a nonhost, M. sexta, also were unresponsive. However, TSP14 inhibited protein synthesis in in vitro
rabbit reticulocyte lysate and wheat germ extract translation systems, regardles…
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