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Journal of Science and Technology
1 (1), 2007, 146-156
©BEYKENT UNIVERSITY
EFFECTS of PARASITISM by
ENDOPARASITOID Pimpla turionellae on
HEMOLYMPH PROTEINS of HOST
Galleria mellonella
Süleyman KALELI 1*, M. Yaşar AKSOYLAR 2,
Abdurrahman AKTUMSEK 3 , C. COKMUS 4
1
Department of Biology, Isparta Healty College, Suleyman Demirel
University, Isparta, Turkey
2
Department of Biology, Sciences and Arts Faculty, Suleyman
Demirel University, Isparta, Turkey
3
Department of Biology, Sciences and Arts Faculty, Selcuk
University, Konya, Turkey
4
Department of Biology, Sciences Faculty, Ankara University, Ankara,
Turkey
ABSTRACT
The plasma proteins were determined under the effect of parasitism by
endoparasitoid, Pimpla turionellae
(Hymenoptera: Ichneumonidae) on
hemolymph proteins of host, Galleria mellonella (Lepidoptera: Pyralidae) by
the colorimetric and SDS-PAGE (Sodium dodecyl sulfate- polyacrylamide gel
electrophoresis) methods. In the colorimetric analysis it was showed a decrease
in the total plasma protein levels in parasitized host during the first hour
compared with unparasitized controls whereas increased total plasma protein
levels were determined during the third hour. However, In the SDS-PAGE
analysis it revealed that the levels of 15.5, 17.5, 67 and 82 kDa proteins
decreased in parasitized host, compared to unparasitized controls. After 72 h of
treatment, plasma protein in the parasitized host was different to that of
unparasitized controls. In conclusion, because of parasitization protein levels
were found as a different in the colorimetric and the electrophoresis analysis.
Keywords: Hemolymph protein, SDS-PAGE, parasitization,
Galleria mellonella
Pimpla turionellae,
Özet
Bu çalışmada Endoparasitoid Pimpla turionellae
(Hymenoptera:
Ichneumonidae) tarafından parazitlenmiş konak Galleria
mellonella
146
Süleyman KALELİ, M. Yaşar AKSOYLAR, Abdurrahman AKTUMSEK and
C. COKMUS
(Lepidoptera: Pyralidae)'da parazitlemenin etkileri kolorimetrik ve SDSPAGE (Sodium dodecyl sulfate- polyacrylamide gel electrophoresis)
yöntemleri ile hemolenf proteinler araştırılmıştır. Kolorimetrik analizlerde,
parazitlenmemiş kontrolle ilk saat karşılaştırıldığında parazitlenmiş konağın
total plazma proteinlerinde bir azalma ve üçüncü saat süresince belirlenmiş
total protein düzeylerinde ise artış görülmüştür. Bununla birlikte, SDS-PAGE
analizinde, kotrollerle karşılaştırıldığında parazitlenmiş konakta 15.5, 17.5, 67
ve 82 kD protein düzeylerinin azaldığı ortaya çıkmıştır. Parazitlemeden
sonraki 72. saatte parazitlenmiş konağın plazma proteini kontrole göre farklı
olduğu görülmektedir. Sonuç olarak parazitlemeden dolayı protein düzeyleri
kolorimetrik ve elektroforetik analizlerde farklı bulunmuştur.
Anahtar kelimeler: Hemolenf protein, SDS-PAGE, parazitleme,
turionellae, Galleria mellonella
Pimpla
1. INTRODUCTION
Use of parasitoids to regulate the density of a particular pest insect is an
attractive way in biological control programmes and integrated pest
management systems, thus creating a need to expand our knowledge of hostparasitoid relationship (DeBach and Rosen, 1991; Dorn, 1993; Ockroy et al.,
1997).
Eggs of endoparasitoids are laid directly into the hosts hemocoel.
Ichneumonid and braconid parasitoids co-inject eggs, venom and
polydnaviruses into the host (Lavine and Beckage, 1995; Strand and Pech,
1995; Shelby and Webb, 1999). During parasitization, the presence of
parasitoid- derived factors evokes a striking re-programming of host
behaviour, reproductive potential, development and immunity (Beckage, 1993;
Lawrence and Lanzrein, 1993).
When endoparasitoid insects parasite their hosts, especially,
lepidoptereous species, qualitative and quantitative changes in the host plasma
protein profile often occur. In some cases, the levels and timing of appearance
of naturally-occurring host proteins may be altered. For example, parasitism of
Trichoplusia ni by Hyposoter exiguae decreased the concentration of several
host hemolymph proteins (Richards and Edwards, 1999). Similarly, parasitism
of Pieris rapae by Apanteles glomeratus ( Smilowitz and Smith, 1977)
resulted in decrease in the concentration of host storage protein, while
parasitism of Manduca sexta by A. congregatus (Beckage and Templeton,
1986) decreased the arylphorin concentration. By contrast, arylphorin levels
increased in T. ni parasitized by Chelonus sp. (Kunkel et al., 1990).
Many endoparasitoids have been shown to interfere with the
development of their host insect- and parasitoid- associated factors such as
polydnaviruses, venom and teratocytes appear to play important roles in this
respect (Lawrence and Lanzrein, 1993). Polydnaviruses have been described in
certain members of the families Braconidae and Ichneumonidae of
147
Effects of Parasitism by Endoparasitoid Pimpla Turionellae on Hemolymph Proteins of
Hosts Galleria Mellonella
Hymenoptera (Stoltz et al., 1995), whereas, teratocytes were found only in
certain members of the families Braconidae, Platygasteridae and Sclonidae of
Hymenoptera (Dahlman and Vinson, 1993).
Parasitism-specific proteins (PSPs) occur in insect hosts after attack by
a variety of parasitic hymenopterous species, most notably the braconid and
ichneumonid parasitoids of lepidopteran hosts (Soldevila and Jones, 1991;
Harwood and Beckage, 1994; Li and Webb, 1994). A limited number of such
species have been evaluated with respect to their molecular compositions or
their origins (Harwood and Beckage, 1994; Li and Webb, 1994). So far PSPs
from only one dipteran host species have been reported (Lawrence, 1990;
Rolle and Lawrence, 1994a,b). However, their origins, molecular compositions
or their functions are unknown.
In addition, new proteins which are not of host origin may sometimes
appear with their site of synthesis and timing of appearance may vary or
depend on the particular endoparasitoid hymenopterous species and host
concerned (Beckage et al., 1987; Jones, 1989; Beckage, 1993; Beckage and
Kanost, 1993).
The effect of parasitism by parasitoid species on the plasma protein
profile of their insect hosts are less well known. The current study was
undertaken to investigate how parasitism of host, Galleria
mellonella
parasitized by endoparasitoid, Pimpla turionellae.
2. MATERIALS AND METHODS
2.1. Insects
Hosts, greater wax moths, Galleria mellonella
L. (Lepidoptera:
Pyralidae) were mass cultured at 25 o C, 60 % relative humidity and 12 hours
light: 12 hours dark on semiartificial diet (Bronskill, 1961). Pimpla turionellae
L. (Hymenoptera: Ichneumonidae) is a solitary endoparasitoid which can
develop successfully in Galleria pupae. Therefore, they were reared routinely
on pupae of G. mellonella in insect laboratory of Biology Department,
Sciences and Arts Faculty, Selcuk University, Konya, Turkey. Control and
parasitized insects were held under the same conditions.
2.2. Hemolymph
collection
G. mellonella
pupae were divided into two groups, namely a)
parasitized pupae: G. mellonella pupae were parasitized by mature females of
endoparasitoid P. turionellae, b) unparasitized pupae (control). Hemolymph
samples were taken at 1, 3, 6, 12, 24, 36, 48, 60 and 72 after parasitism.
Samples were collected by cutting at abdomen and drawing into
glass
capillary tubes. Samples were pelletted at 5000 g for 4 minutes at 4 o C and
stored at -18 ° C until use. A small amount of phenylthiourea was added into
148
Süleyman KALELİ, M. Yaşar AKSOYLAR, Abdurrahman AKTUMSEK and
C. COKMUS
all eppendorf tubes for hemolymphs collected to prevent melanisation and
oxidation (Zupko et al., 1993).
2.3.
Electrophoresis
Hemolymph samples were removed f r o m freezer, diluted in a buffer
containing 0.05 M Tris-HCl (pH 6.8) and centrifuged at 12.500 g for 10
minutes. Supernatant and SDS sample buffer were mixed in the ratio of 3:1
followed by denaturation of the proteins in boiling water for 5 minutes.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed as described by Laemmli (1970), using 4 % (w/v)
stacking and 10 % (w/v) resolving gel. Protein bands were visualized by
Coomassie Brillant Blue R250 (Sigma). The molecular weight of each protein
band could then be calculated according to the standard curve of purified
marker proteins: • -lactalbumin - cow's milk (14.2 kDa), trypsin inhibitor soybean (20.1 kDa), carbonic anhydrase - bovine erythrocytes (29 kDa),
ovalabumin - egg (45 kDa), albumin - bovine (66 kDa), phosphorylase B rabbit muscle (97.4 kDa), • -galactosidase - E. coli (116 kDa), myosin - rabbit
muscle (205 kDa) from Sigma (Cat. No. M. 2789). The current study was
carried out using Technicon RA-XT autoanalyzator. 2.5 ^l protein extract was
loaded into each well of gels and the electrophoresis was run from cathode to
anode for 1 h at 20 mA and 3 h at 35 mA at 4 °C.
The semi-quantification of the protein concentration of the samples was
determined by using the method of Esen (1978). The protein extracts were
applied as 2.5 ^l onto the surface of a set Whatmann paper (No 1) cut out as
bands and were dried.
2.4. Statistical
Analysis
Experiments were repeated at least three times. All data were
analysed using a computerized statistical package (MSTATC).
3. RESULTS
Total plasma protein values of host, Galleria mellonella pupae were
clearly altered by the parasitism of endoparasitoid Pimpla turionellae (Table
1).
There was a dramatic decrease in the total protein concentration on
the first hour of the parasitized host following the parasitism (p < 0.01).
Similarly, a dramatic decrease was also observed on the 6 th hour after
parasitism. However, decreases during the 12 th and 36 th hour were significant
(p < 0.05). O n the other hand, the total plasma protein concentrations of
parasitized host were increased during the 48 th and 72 th hour (p < 0.05),
although, no significant changes were observed during the 3 rd , 24 th and 60 th
hour between total plasma protein levels of parasitized and unparasitized hosts
(p > 0.05).
149
Effects of Parasitism by Endoparasitoid Pimpla Turionellae on Hemolymph Proteins of
Hosts Galleria Mellonella
The proteins with size of > 205, 46, 42, 36, 34, 26 and 18 kDa
decreased in parasitized host compared to controls (Fig. 1). Proteins at 36 and
34 kDa sizes decreased remarkably after the 24 hours, and 36 kDa sizes
disappeared f r o m the host after 72 hours parasitism. In contrast, protein at 36
kDa size remarkably increased on unparasitized host on 72 hours following
parasitism. Proteins at 67 and 82 kDa sizes were easily visualised on the gel as
a single band due to their higher densities. Protein at 82 kDa size decreased
until the 24 th hours and increased after the 24 th hours in parasitized host.
Protein at 67 kDa size was found to decrease in the first hour but it was seen to
increase after the first hour in parasitized host. Proteins at 17.5 and 15.5 kDa
sizes decreased until the 24 th hour and increased in parasitized host thereafter.
Table 1. Total plasma protein values at different time of parasitized by endoparasitoid
turionellae and unparasitized host G. mellonella pupae.
Total plasma proteins of "host fp'dl) 1
Unparasitized host
Time
Mean± S.D.
Parasitized "host
Mean = S.D.
First hour (h)
8.530 - 0.466 '"
4.667 = 0.186 ""
3'J
h
6.400 - 0.327
6.27 = 0.466
6^
i
6.470 - 0.488 "
6.400 = 0.336™
8.800 = 0.377 "
7.867 = 0.136"
6.400 = 0.864
6.667 = 0.186|
36^ h
8.933 = 0.377 "
7.467 = 0.377"
48a h
6.00 = 0.0.327 "
7.200 = 0,327 "
60th h
7.207 = 0.166
7.067 = 0.754
72"d h
6.267= 0.377 "
8.267 = 0.754 "
12 a h
h
!
H i e average of three Experiments.
*: p<0.05, ** : p<0.01
150
P.
Süleyman KALELİ, M. Yaşar AKSOYLAR, Abdurrahman AKTUMSEK and
C. COKMUS
Figure 1. Protein profiles of pupae hemolymph proteins of G. Mellonella
parasitized by P.
turionellae and unparasitized host G. mellonella at different periods. (Lane 2, 4, 6, 8, 10, 12, 14,
16 and 18 are parasitized hosts; lane 3, 5, 7, 9, 11, 13, 15, 17 and 19 are unparasitized
hosts;
Lane 1 and 20 are the molecular weight standards. And lanes 2-3 at first h, lanes 4-5 at 3rd h,
h
th
th
th
lanes 6-7 at 6 h, lanes 8-9 at 12 h, lanes 10-11 at 24 h, lanes 12-13 at 36 h, lanes 14-15 at
48th h, lanes 16-17 at 60h h, lanes 18-19 at 72nd h).
4. DISCUSSION
Earlier studies showed that the total amount of hemolymph protein is
reduced during parasitism, beginning almost immediately after the host is
parasitized. Simultaneously, parasitism induces synthesis of large amounts of
novel proteins that appear in the blood as early as 1-2 hours post-parasitization.
The present report confirms earlier studies describing the presence of novel
proteins in larvae and pupae, and also characterizes the effects of parasitism in
altering the titers of several endogenous host hemolymph proteins normally
produced by the fat body and other tissues (Beckage and Kanost, 1993).
Previous reports presented the physiological arrestment and alterations in
hormone and hemolymph protein titers of lepidopteran larvae of permissive
host following parasitization by Euplectus species (Coudron et al., 1990,
1994).
Successful endoparasitic development requires suppression of the
humoral system, but not its irreversible shutdown. The invading parasitoid has
to ensure that it is not destroyed by the immune processes, but that immunocompromised host maintains the ability to fight secondary infection. It is
critical that the immune reaction is not undermined to such an extent, that
death of both host and developing parasitoid cannot be avoided (Ockroy et al.,
2002).
151
Effects of Parasitism by Endoparasitoid Pimpla Turionellae on Hemolymph Proteins of
Hosts Galleria Mellonella
Invasion by parasitoids or by bacteria induces arrays of new
hemolymph proteins in the host insects (Beckage et al., 1989; Soldevila and
Jones, 1993; Rolle and Lawrence, 1994a). In Pieris brassicae upon parasitism
by Cotesia glomerata also considerable changes in the hemolymph protein
pattern occurs. These changes, however, were clearly distinct from
hemolymph protein patterns obtained after bacterial infection on wounding.
For example, a new 38 kDa protein was found in P. brassicae two days after
oviposition of parasitic wasp (Ockroy et al., 1997). New proteins appears in
the hemolymph at defined times post-oviposition. It is suggested that such
proteins have a function linked to alterations in behaviour, development and
immunity of the host (Ockroy, 1999).
Many studies demonstrating that parasitization by Cotesia
congregata
induces synthesis of novel hemolymph proteins that begins immediately after
the host is parasitized, specifically to show that titers of endogenous host
proteins also are dramatically affected by parasitism (Beckage et al., 1987,
1989; Harwood, 1993).
Parasitism-specific proteins have been detected in numerous
endoparasitoid-host model systems, their sources are variable, and many help
to condition the host to ease parasitism. For example "new" host proteins
might be the result of parasitoid-mediated rediction of host protein synthesis,
which may make the host plasma more nutritious for the endo- or
ectoparasitoid (Thompson, 1993; Coudron et al., 1994). Alternatively, many
parasitism-specific proteins are the products of polydnavirus genes, which
suppress and distrupt the host immune and endocrine systems, respectively
(Cook et al, 1984; Vinson, 1990a; Beckage, 1993, 1997, 1998; Strand and
Pech, 1995; Harwood et al., 1998).
In the study confirms that protein profiles of hemolymph of host insects
affected by parasitism. Results f r o m this experiments indicated that 72 hours
of treatment, plasma from parasitized pupae was different to that unparasitized
controls. At the end of first hour of parasitization, table and figure showed that
total protein levels and protein profiles in parasitized hosts were dramatically
decreased. All proteins were reduced by parasitoid at the beginning of the first
hour after parasitization. > 205, 46 and 42 kDa proteins, and 36, 34, 26 and 18
kDa
proteins at the following hours were reduced. Titers of various
hemolymph proteins in parasitized hosts were increased in the different times.
For example, 67 kDa protein after 3 hours, 15.5 and 17.5 kDa proteins after 24
hours and 82 kDa protein after 36 hours were different than controls. 36 and 34
kDa are probably major protein or larval specific protein because of these
molecular weight protein was exist in both of host and endoparasitoid
hemolymph protein profiles. Beckage et al (1989) reported that 33 kDa
proteins on host-parasitoid systems were, also, seen after 3 hours of
parasitizing the host, Manduca sexta by parasitoid C. congregata. However,
152
Süleyman KALELİ, M. Yaşar AKSOYLAR, Abdurrahman AKTUMSEK and
C. COKMUS
it was reported that the reason of the formation of 33 kDa proteins in
parasitized M. sexta hemolymph was the infection of polydnavirus which was
secreted f r o m ovarian calyx of C. congregata. Luckhart and Webb (1996)
pointed that polydnavirus did not occur in ovarian calyx, but 29-36 kDa
proteins groups in ovarian calyx fluid were found. These proteins were present
f r o m 3 to 96 hours in parasitism of parasitized host hemolymph, and are
reported to be in the form of the glycoform. In this study, new proteins were
not seen due to parasitization, suggesting that there was no virus secreted
ovarian calyx of P. turionellae. This situation, also, supports the study of
Luckhart and Webb (1996). According to the study of parasitized hosts were
shown in A. suspense pupae at the 24 th and 36 th , and this protein was described
to be a specific protein bound to parasitism. Roll and Lawrence (1994b)
reported the 24 kDa protein was in the nature of a glycoprotein. Glycoproteins
left into host by parasites can induce the formation of patogenity since they can
potentially be dangerous factor to host and glycoproteins are the sources of
nutrient for parasite feedings.
In conclusion, the hemolymph proteins were affected in parasitized host
as seen in the table and the figure. The study shows the effects of relation
between parasite and host. We think that the effects of parasite on host may be
studied as amino acid and hormone levels in the future.
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