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Cotesia kariyai larvae need an anchor to emerge from the host Pseudaletia separata.

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Archives of Insect Biochemistry and Physiology 66:1–8 (2007)
Cotesia kariyai Larvae Need an Anchor to Emerge From
the Host Pseudaletia separata
Y. Nakamatsu,1* T. Tanaka,1 and J. A. Harvey2
Mature larvae of the gregarious endoparasitoid Cotesia kariyai construct cocoons for pupation approximately 10 days after
parasitization and emerge from their host Pseudaletia separata under a long day photo-regime (16L8D) at 25 ± 1°C. The
parasitoid larvae make capsules in the host hemocoel just prior to their emergence. These capsules function as “anchors,”
which enable them to press against the host integument from inside the host. It was predicted that this anchor might be
composed of silk proteins secreted from the parasitoid larvae, because a previous study showed that the anchor was made up
of a glycoprotein and that the silk gland of parasitoid larvae developed from 2nd larval stage. Fibroin-like proteins in
C. kariyai larva mainly consist of two proteins with molecular masses of the 300.6 and 46.7 kDa estimated by SDS-PAGE.
The fibroin-like proteins with the same molecular mass were detected from the anchor proteins just prior to parasitoid emergence. These results indicate that the anchor was assembled with fibrion-like proteins and was formed just before parasitoid
emergence while in the host body cavity. Injection of bovine pancreatic trypsin inhibited the emergence of parasitoid larvae
from the host because the anchor was decomposed by trypsin. Trypsin activity in the parasitized host hemolymph increased
only after parasitoid emergence. Arch. Insect Biochem. Physiol. 66:1–8, 2007. © 2007 Wiley-Liss, Inc.
KEYWORDS: anchor; silk; fibroin; parasitoid emergence; parasitoid
INTRODUCTION
Mature endoparasitoid larvae within several
subfamilies of the large family Braconidae (e.g.,
Microgastrinae) emerge from a still-living host to
construct their cocoons and pupate (Askew, 1971;
Godfray, 1994). Approximately 10 days after parasitization, third instar larvae of Cotesia kariyai
emerge from their host and pupate (Tanaka et al.,
1992; Nakamatsu et al., 2001). The mature endoparsitoid larvae make holes in the host integument
just prior to their emergence (Askew, 1971). For
example, larvae of C. kariyai perforate the host cuticle with their mandibles and enlarge the holes
by physically moving their head capsules from side
to side (Nakamatsu et al., 2006). In order to
emerge from the host, some basal pressure (from
terminal body segments) needs to be applied by
the wasp larvae from within the host. Larvae of C.
kariyai construct capsules for this purpose by using their own exuviae.
A silk gland in the body cavity of second instar
C. kariyai larvae is well developed (Nakamatsu et
al., 2002). The viscosity in the parasitized host
hemolymph is higher in proportion to the parasitoid development because a glycoprotein is secreted
into the host by the parasitoid larvae as they mature (Nakamatsu et al., 2006). Mature larvae of the
silkworm Bombyx mori secrete silk proteins (Akai,
1983; Magoshi et al., 1985). Two proteins, fibroin
and sericin, are the main components of silk in B.
mori (Voegeli et al., 1993; Kato et al., 1998). Seri-
1
Applied Entomology, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan
2
NIOO-KNAW, Centre for Terrestrial Ecology, Heteren, The Netherlands
*Correspondence to: Yutaka Nakamatsu, Applied Entomology, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601,
Japan. E-mail: nakamatu@agr.nagoya-u.ac.jp
Received 13 June 2006; Accepted 7 February 2007
© 2007 Wiley-Liss, Inc.
DOI: 10.1002/arch.20189
Published online in Wiley InterScience (www.interscience.wiley.com)
2
Nakamatsu et al.
cin is a protein that covers fibroin fibers and functions as an adhesive material in the cocoon of silkworms (Takahashi et al., 2003). The high viscosity
in the hemolymph of parasitized Pseudaletia separata
larvae also appears to be based on adhesive proteins that function like sericin.
If parasitoid larvae need to make an anchor
around their body, a large amount of protein
would be required for this purpose. It is conceivable that the well-developed silk glands of parasitoid larvae are the leading candidate as an organ
that can provide enough proteins to enclose their
entire bodies. In this study, we investigate the structure and functions of the anchor.
MATERIALS AND METHODS
Insect Rearing
The endoparasitoid Cotesia kariyai (Watanabe)
emerged from Pseudaletia separata (Walker) larvae
that had been collected in cornfields in Kanoya,
Kagoshima Prefecture. They were thereafter maintained as a colony in our laboratory. Adult wasps
were kept for mating under a long-day condition
at 25 ± 1°C with a 20–30% sugar solution as food
in a glass tube (12 cm long × 5 cm in diameter)
for 2 days. Fifth instars of P. separata were individually presented to single female parasitoids that
were allowed to sting once to avoid superparasitism. In addition, a laboratory colony of P. separata
was established using larvae collected in cornfields
of the agricultural field station at Nagoya University. Pseudaletia separata larvae were reared on an
artificial diet (Insecta LF®, Nihon Nosan, Kanagawa)
under a long-day photo-regime (16 h light:8 h
dark) at 25 ± 1°C. It was designated as day 0 when
each larva ecdysed to the next stadium. The P.
separata larvae parasitized by C. kariyai were also
maintained under the conditions described above.
Extract of Fibroin-Like Proteins
Initially, a cocoon protein was prepared by the
method of Takasu et al. (2002) but with a slight
modification. A combined total of 25 mg of para-
sitoid cocoons was put into 1 ml of 60% lithium
thiocyanate (LiSCN pH 7.0) to obtain the whole
cocoon protein solution. Dissolved protein solution
was dialyzed at room temperature against 20 mM
Tris-HCl buffer, pH 8.0, containing 5M urea for 3 h
with exchanges of the buffer at 1-h intervals. It was
then kept in the dark at room temperature.
Solid fibroin-like proteins and liquid fibroinlike proteins were prepared by the method of
Sumida et al. (1993) but also with a slight modification. Second instar larvae of C. kariyai were dissected in ice-cold 1.15% KCl solution and the silk
glands were isolated. The glands were washed in
ice-cold fresh 1.15% KCl solution to remove
hemolymph, and immersed in ice-cold 30% ethanol. After being kept in 4°C, the surrounding fixed
silk gland tissues were separated by stirring the solution in a small beaker with a magnetic stirrer.
Coagulated fibroin-like proteins were then collected. They were washed several times in ice-cold
distilled water and immersed consecutively in 50%,
99.5% ethanol and ethyl ether for desiccation to
give solid fibroin-like proteins. Solid fibroin-like
proteins were kept in desiccated dark conditions
at room temperature. Liquid fibroin-like proteins
were prepared as follows. Briefly, 0.2 ml of 60%
LiSCN, pH 7.0, was added to 0.01 g of solid fibroin-like proteins. This was kept for 6 h at room
temperature to dissolve the solid fibroin-like proteins. Dissolved solid fibroin-like proteins were dialyzed at room temperature against 20 mM Tris-HCl
buffer, pH 8.0, containing 5M urea for 3h with
exchanges of the buffer at 1-h intervals. This was
then kept in the dark at room temperature.
Collecting Hemolymph from the Host
Day 0–L6 instars of P. separata were prepared
for parasitization. The hemolymph from Day 10
host larvae after parasitization was collected. The
proleg of the host was cut with scissors, and from
the wound 100 µl of hemolymph per host was collected on parafilm (American Can Co. LTD) and
put on ice. A saturated phenylthiourea solution
added 8% to the hemolymph volume.
Archives of Insect Biochemistry and Physiology
September 2007
doi: 10.1002/arch.
C. kariyai Larvae Need Anchor to Emerge From Host
3
Fibroin-Like Protein Extract From Anchor
Observation of Anchors in the Host
A cluster of mature parasitoid larvae was removed from the host just before emergence by incising the dorsal skin with scissors after CO 2
anesthesia. A 0.1-ml solution of 60% lithium thiocyanate (LiSCN, pH 7.0) was added to the mass of
parasitoid larvae. The solution containing the clusters was mixed with a stirrer, until the parasitoid
larvae were physically separated from each other.
The dissolved solution was dialyzed at room temperature against 20 mM of Tris-HCl buffer (pH 8.0)
containing 5M Urea for 3 h with exchanges of the
buffer at 1-h intervals. The dialyzed sample solution was diluted with 2 volumes of the fresh buffer
used for dialysis.
Morphological observations in the body cavity
of the parasitized hosts were performed to investigate the structure of the anchor. Parasitization was
performed on day 0–L5 host larvae. Coincident
with larval wasp emergence, parasitized hosts were
fixed with 2.5% glutaraldehyde and postfixed with
Bouin’s solution for 24 h, dehydrated and then
embedded in paraffin. Eight-micrometer sections
were stained with Mayer’s hematoxylin and 1%
eosin Y solution (Sano, 1965).
SDS-PAGE Analysis
Samples for SDS-PAGE were prepared by adding the same volume of sample buffer (0.1M sodium phosphate buffer of pH7, containing 6M
urea, 1% SDS, 2% 2-mercaptoethanol, and 0.025%
bromophenol blue). Samples were incubated at
60°C for 15 min and put on 8 to 15% gel. The
gels were stained after the run with Simply Blue
(Invitrogen).
Trypsin Treatment
The host moved away from the parasitoid cocoons soon after parasitoid emergence. If the anchor is not disrupted, the host would cease to
function. In the Euplectrus separatae–C. kariyai system, before pupating the matured parasitoid larvae secrete trypsin-like enzyme into the host body
cavity (Nakamatsu and Tanaka, 2004). It is possible that the C. kariyai larvae also secrete the same
enzyme.
Fifty microliters of trypsin solution (635 U/µl
Tris-Saline buffer; 50 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 1 mM CaCl2, 0.1 mg/ml BSA)
was added to the 0.2 ml of the liquid fibroin-like
proteins solution. The mixed solution was incubated for 1 h at 37°C. One unit activity was hydrolyzed in 1.0 µmol of substrate/min at 37°C.
Archives of Insect Biochemistry and Physiology
September 2007
doi: 10.1002/arch.
Effect of Collapsed Anchor on the Parasitoid
Larvae and Their Hosts
To clarify the relationship between the destruction of the anchor and the success/failure of parasitoid emergence, we examined the effect of bovine
pancreatic trypsin on parasitoid emergence behavior. A trypsin solution (10 µl at 635 U/µl) was injected into day-10 parasitized larvae just prior to
parasitoid emergence. The trypsin-injected larvae
were incubated at 37°C for 30 min and were maintained until parasitoid emergence at 25°C. We
counted the number of parasitoid larvae that failed
to emerge from hosts that had been injected with
trypsin. The number of parasitoid larvae that remained in the host were also counted when these
larvae were injected with Tris-saline buffer as a control after parasitoid emergence. The total number
of eggs laid was calculated by combining the number of parasitoid larvae that remained in the host
with the number of parasitoid pupae in cocoons.
A 10-µl trypsin solution (635 U/µl) was injected
into non-parasitized larva to check for effects of
toxicity. One activity unit shows the ability to hydrolyze 1.0 µmol of substrate per min at 37°C.
Measurement of Trypsin Activity on Fluorescence
Spectrophotometer
The parasitized host larva was fixed as the parasitoid emerged and was thus not moved. This immobility may be caused by a solid anchor formed
4
Nakamatsu et al.
in the host hemocoel. After parasitoid larvae
emerge from the host, the host caterpillar usually
crawls up to several centimeters away from the cocoon cluster. If the activity of trypsin-like enzymes
increases after the completion of parasitoid larval
emergence, the destruction of the anchor might facilitate host movement.
One hundred microliters of hemolymph from
day 4–L6 control and parasitized larvae were collected immediately prior to and after parasitoid
emergence in a 1.5-ml plastic tube on ice. The saturated 1-phenyl-2-thiourea (PTU) solution was added
immediately to adjust to 8% of the final volume.
Twenty microliters of 0.05 mM MCA-substrate (BocGln-Ala-Arg-MCA, Peptide Institute Inc.) and 0.4
ml of each buffer were put into a cuvette, and then
8 µl of hemolymph from healthy and parasitized
larvae was added in the cuvette. A fluorescence
spectrophotometer was used to record emission
fluorescence at 440 nm at 37°C for 10 min.
RESULTS
Figure 1.
Protein Extracts from Parasitoid Cocoon
Polypeptides with various molecular masses
from parasitoid cocoons were detected on SDSPAGE analysis (Fig. 1). The silk protein-extract consisted of nine proteins. The estimated molecular
masses of the nine major proteins were 300.6 kDa
(F1), 46.7 kDa (F2), 17.5 kDa (S1), 16.2 kDa (S2),
15.3 kDa (S3), 14.8 kDa (S4), 14.2 kDa (S5), 13.0
Fig. 1. Electrophoretic profile of silk protein (S), fibroinlike (F) proteins extracted from the cocoons of the Cotesia
kariyai. Thirty micrograms total protein was run on 15%
for silk protein and 10% SDS-PAGE gels for fibroin-like
proteins, and stained with Simply Blue (Invitrogen). Numbers on the right and left indicate molecular masses (kDa)
of standards. F: fibroin-like proteins.
Fig. 2. Electrophoretic profile of the fibroin-like proteins
(F) extracted from the cocoons of the C. kariyai and of
the hemolymph of day-10 parasitized host larvae (P10).
Protein (30 µg total) was run on 10% SDS-PAGE gel and
stained with Simply Blue (Invitrogen). Numbers on the
right and left indicate estimated molecular masses (kDa).
Figure 2.
Archives of Insect Biochemistry and Physiology
September 2007
doi: 10.1002/arch.
C. kariyai Larvae Need Anchor to Emerge From Host
Fig. 3. The parasitized larva (P) is just beginning to
emerge from the host. Each parasitoid larva is enclosed
by an anchor (FH). The parasitized host was pre-fixed by
injection of 2.5% glutaraldehyde solution and post-fixed
Archives of Insect Biochemistry and Physiology
September 2007
doi: 10.1002/arch.
5
with Bouin’s solution for 24 h. An 8-µm section was
stained with Hematoxylin and Eosin dyes. I: Host integument. Scale bar = 500 µm
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Nakamatsu et al.
kDa (S6), and 9.5 kDa (S7). The fibroin-like protein-extract consisted of two proteins, 300.6 kDa
(F1) and 46.7 kDa (F2).
Proteins in the Parasitized Host Hemolymph
We could not detect the proteins with the same
molecular mass as fibroin-like proteins (300.6 kDa,
46.7 kDa) in the day-10 parasitized host hemolymph (Fig. 2).
Structure and Proteins of Anchor
Morphological observations with paraffin sections revealed that the parasitoid larvae were only
able to emerge from hosts by using the anchor.
The parasitoid larvae attained a stable anchor by
gluing the remaining exuvium on the parasitoid
larval body just before emergence (Fig. 3).
The protein with the same molecular masses as
300.6 kDa (F1) in fibroin-like protein was detected
from the extract of the anchor (Fig. 4).
Fig. 4. Electrophoretic profile of the fibroin-like proteins
(F) extracted from the cocoons of the C. kariyai, the anchor extract (Anchor), and the hemolymph of day-10 parasitized host larvae (P10). The arrows show the bands of
fibroin-like proteins (300.6kDa) in the F and the anchor.
Thirty micrograms total protein was run on 15% SDSPAGE gel and stained with Simply Blue (Invitrogen). Numbers on the right indicate molecular masses (kDa) of
standard marker.
Treatment of Trypsin
Treatment with trypsin reduced the fibroin-like
protein of 300.6 kDa on the gel (Fig. 5). Injection
of trypsin solution inhibited the emergence of almost all parasitoid larvae (Table 1). All of the nonparasitized larvae injected with the same amount
of trypsin solution survived and pupated (n = 20).
The emergence of parasitoid larvae from the saline-injected parasitized hosts as control was also
not affected.
Trypsin Activity in the Parasitized Host Hemolymph
Although we have not characterized the enzyme
detected in host hemolymph as trypsin in this
study, this enzyme in the hemolymph shows an
Fig. 5. Electrophoretic profile of the fibroin-like proteins
(F) extracted from the cocoons of the C. kariyai and the
fibroin-like proteins, which were treated by 50 µl of trypsin
solution (Bovine pancreatic trypsin 3 mg/0.2 ml Tris-saline buffer) (T). There are no fibroin-like proteins in the
trypsin-treated. Total protein (30 µg per lane) was stained
with Simply Blue (Invitrogen) after running on 15% SDSPAGE gels. Numbers on the right and left indicate molecular masses (kDa) of standard marker.
Archives of Insect Biochemistry and Physiology
September 2007
doi: 10.1002/arch.
C. kariyai Larvae Need Anchor to Emerge From Host
TABLE 1. Number of Parasitoid Larvae That Could Not Emerge From
Trypsin Solution (10 µl at 635 units/µl)-Injected Host†
Treatment
No. of host
larvae used
No. of eggs
oviposited*
(Mean ± S.D.)
No. of parasitoid
larvae not
emerged*
(Mean ± S.D.)
20
14
148.6 ± 58.8 a
144.7 ± 49.1 a
142.9 ± 63.0 a
0.7 ± 1.1 b
Trypsin solution-injected
Saline-injected control
†
All parasitoid larvae involved not emerged parasitoids in the trypsin solution-injected hosts were living. Non-parasitized hosts which were injected with trypsin
solution did not die (n =15).
*Means within a column followed by the same letter are not significantly different
(P < 0.01; one-way ANOVA).
activity similar to bovine trypsin. Trypsin-like enzyme activity was detected in the parasitized host
(Table 2). Especially after parasitoid emergence, the
trypsin-like enzyme activity in the host hemolymph
became about 12-fold higher than that in the
hemolymph before emergence.
DISCUSSION
Silk proteins are made up of fibroins and fibrous
adhesives (Craig and Riekel, 2002). Although the
silk proteins of moths (Lepidoptera) are used primarily for forming cocoons, predatory arthropods
such as spiders use their silk for many functions,
including the capture of prey, shelter construction
or reproduction (Vollrath and Knight, 2001). One
type of spider silk consists mainly of glycoproteins
(Vollrath, 1999), which are able to maintain high
levels of plasticity by retaining a high water content (Vollrath and Tillinghast, 1991). The anchor
of C. kariyai was composed of glycoproteins
(Nakamatsu et al., 2006) and also appeared to require some flexibility to enhance the successful
emergence of the parasitoid larvae.
TABLE 2. Trypsin Activities in the Parasitized Host Hemolymph Before
and After Parasitoid Emergence on Day 10 After Parasitization†
Host hemolymph
collected
No. of host
larvae used
Enzyme activity*
Means ± S.D.
(×10–3 units)
4
4
4
286.0 ± 18.7 b
22.2 ± 7.2 a
3.2 ± 0.2 c
After parasitoid emergence
Before parasitoid emergence
Control
†
The day 4-6th instar hosts were used as control.
*Means within a column followed by a different letter are significantly different (P
< 0.01; one-way ANOVA).
Archives of Insect Biochemistry and Physiology
September 2007
doi: 10.1002/arch.
7
The fibroin in C. kariyai’s silk was composed of
two proteins with a large molecular mass (300.6
kDa) and a small molecular mass (46.7 kDa). The
fibroin of B. mori is also composed of two proteins with large (350 kDa) and small molecular
masses (25 kDa) (Shimura, 1988). By contrast,
Yamada et al. (2003) reported that fibroin-like protein extracted from cocoon of Cotesia glomerata was
comprised of a single polypeptide with a molecular mass of over 500 kDa. This variation may reflect differences in the cocoon structure of the two
Cotesia species, even though they are closely related.
C. kariyai makes a cocoon cluster that is composed
of two different layers: an inner layer that surrounds
each individual pupa, and a thick outer layer that
covers each cocoon entirely, whereas C. glomerata
larvae only construct individual cocoons after
emergence from the host (unpublished data).
The 300.6-kDa protein, which has the same
molecular mass as fibroin-like proteins in C. kariyai
cocoons, was detected in the anchor extraction, suggesting that the fibroin-like protein was a component of the anchor. However, they were not detected
in hemolymph of parasitized host larvae from day
0 to 10 after parasitization except just prior to parasitoid emergence at day 10.
A fibroinase in the silk gland of B. mori larvae
decompose the fibroin (Sumida et al., 1993). In this
study, we used bovine trypsin, because the activities
of trypsin-like enzymes in the host larvae increased
just after parasitoid emergence and the anchor was
left in the host hemocoal. The fibroin-like protein
was not detected on SDS-PAGE gels after treatment
with trypsin. Over 96% of the parasitoid larvae failed
to emerge from the host when the trypsin was injected into the host 10 days after parasitization. The
parasitoid larvae are unable to perforate the host
cuticle probably because they cannot gain sufficient
basal support to exert enough pressure on the host
integument due to the collapse of the anchor.
ACKNOWLEDGMENTS
We thank M. Nishioka and colleagues at the
Kagoshima Agricultural Experiment Station for
their help in collecting Pseudaletia separata larvae.
8
Nakamatsu et al.
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Archives of Insect Biochemistry and Physiology
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