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Premature production of late larval storage proteins in larvae of trichoplusia ni parasitized by euplectrus comstockii.

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Archives of Insect Biochemistry and Physiology 26:97-109 (1 994)
Premature Production of Late Larval Storage
Proteins in Larvae of Trichoplusia ni
Parasitized by Euplectrus comstockii
Thomas A. Coudron, Davy Jones, and Grace Jones
Biological Control of Insects Research Laboratoy, U S . Department of Agriculture, A R S ,
Columbia, Missouri (T.A.C.); Graduate Center for Toxicology (D.J.),and School of Biological
Sciences (G.J.), University of Kentucky, Lexington
Investigations were conducted to determine the titer of storage proteins in larvae
of the cabbage looper, Trichoplusia ni (Hubner), that were parasitized by the
ectoparasitoid Euplectrus comstockii Howard (Hymenoptera: Eulophidae). A gradual increase was noted in the titer of the storage proteins present in the hernolymph
of parasitized third and fourth instar larvae and in the hemolymph of isolated
thoracic and abdominal tissues of fourth instar larvae. The final amount present in
parasitized third and fourth instar larvae was similar to thatfound in nonparasitized
fifth instar larvae. The stimulation of storage proteins in envenomed larvae demonstrates the ability (competence) of early larval stages to produce a gene product
that normally occurs in the last larval stadium of the lepidopteran larval host. The
gene expression necessary for storage protein production in isolated tissues may
be altered by mechanisms separate from inherent developmental processes and
the intact endocrine system. o 1994 WiIey-Liss, Inc.*
Key words: Hymenoptera, venom, host hemolymph, plasma proteins, regulation
Peptides and proteins found in the hernolymph of insects are known to serve
numerous functions that include intercellular communication, transport and
defensive mechanisms, and energy storage 11-31. Storage proteins have been
found in many species of insects and are commonly thought to supply peptides
and/or amino acids required for late larval development and metamorphosis
[1,4-61. Other roles attributed to these proteins include cuticular associations
171 and ligand binding and the transport of xenobiotics [8].
Acknowledgments:A.I. Soldevila, J. Derks, and S. Brandt are recognized for their technical assistance.
Received January 21, 1993; accepted April 5, 1993.
Address reprint requests to Dr. T.A. Coudron, USDA ARS BCIRL, P.O. Box 7629, Columbia, M O
Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by
the U.S. Departmentof Agriculture and does not imply its approval to the exclusion of other products
or vendors that may also be suitable.
0 1994 Wiley-Liss, Inc. *This article is a US Government work
and, as such, is in the public domain in the United States of America.
Coudron et al.
In their native form, the storage proteins often have a molecular weight
greater than 400,000 and are composed of glycosylated subunits in hexamer or
octamer aggregates. The molecular weights of the subunits range from 70,000
to 90,000 [3,9-111. The fat body of feeding larvae and nymphs has been shown
to be the primary site of synthesis and secretion of the storage proteins [3,9,101.
Although the fat body is accepted as the primary site of synthesis, secondary
sites of synthesis have been implicated [12-151, which suggests there may be
several regulatory mechanisms controlling the expression of these genes [161.
Sequestration of the storage proteins by the fat body of holometabolous insects
occurs just prior to the larval to pupal molt [3,17,181.
Production of storage proteins is thought to be under a developmental
regulation. This regulation allows for an abundant accumulation of storage
proteins, to as much as 80% of the total hemolymph protein by weight in the
hemolymph of the last larval instar of homometabolous insects [9,10,19,201.
Trichoplusia ni (Hubner) is one of the lepidopteran insects studied in which
this phenomenon has been reported [21].However, our understanding of the
regulation, processing, transport, and biological functions of the storage
proteins is limited.
Parasitic Hymenoptera are known to alter the physiology, biochemistry,
and behavior of the host, resulting in the enhancement of the host as a source
of nutrients [22]. One alteration is in the constituent protein profile of the
host. These alterations include changes in the normal concentrations as well
as alterations of the time of protein occurrence in the hemolymph of the host.
Parasitism of larvae of T. ni by Hyposoter exiguae caused a decrease in the
concentration of several proteins normally found in the hemolymph of the
host [23,24]. Parasitism of Spodoptera frugiperda (J.E. Smith) by Cotesia mayginiventris caused an early production of several high molecular weight
proteins that were not characterized further [251. A recorded decrease in the
concentration of storage proteins in larvae of Pieris rapae (L.), parasitized by
Cotesia glomevata [26], was proposed to be a result of uptake of host hemolymph proteins by the parasitoid [27].The reduced production of arylphorin
by larvae of Manduca sexta (L.), parasitized by C. congvegata, was proposed
to be a result of the inhibitory effects of parasitism on host fat body, food
consumption, and growth [281. In contrast, an increase in the concentration of arylphorin was reported in larvae of T. ni parasitized by Chelonus
SPP. 129-311.
The eulophid, Euplectrus comstockii Howard is an ectoparasitoid of larvae
of several lepidopteran insects 1321. As an ectoparasitoid, it relies entirely on
a venom to induce alterations in certain physiological and biochemical
events of its larval hosts. The venom, which is injected into the host during
a stinging process that precedes oviposition [32], alters the development of
the host by arresting the larval-larval ecdysal process [331. The arrestment is
the result of action of the venom on the epidermal tissue and is distinct in its
action from the action of other venoms of ectoparasitoids that cause paralysis
of the host. In the present paper, we report the effect of the venom from E.
comstockii on the expression of storage proteins in the hemolymph of envenomed larvae of T . ni.
Storage Proteins in Envenomed larvae
The E. comstockii colony was derived from a stock collected in Missouri during
1985 to 1989. Stock material was reared from field collected larvae of T. ni,
Helicoverpa zed (Boddie), Plathypena scabra (Fabricius),and AZypia octomaculata
(Fabricius). A continuous colony of the parasitoid was maintained in the
laboratory since 1985 according to the procedure described by Coudron and
Puttler [32].Both parasitized and nonparasitized larvae of T. ni were maintained
on a semisynthetic modified wheat germ diet [34], and reared at 23-26"C,
40-60% RH*, and 12:12 (L:D) photoperiod 1331.Under these conditions, each of
the third and fourth larval instars of T. ni lasted for 48 h, and larvae in the fifth
larval instar began wandering after 54 h, spinning a cocoon within 72 h, and
pupated within 90 h.
Unsexed larvae of T. ni that were in synchronous growth were used for
control and treatment studies [33]. Larvae that were ligated, parasitized, or
ligated and parasitized were handled in the same manner as control (nonligated
and nonparasitized) larvae. Nonligated parasitized larvae were parasitized
within 4 h of ecdysis into the designated stadium. For ligated larvae, a short
strand of sewing thread was used to create a ligature block between the thorax
and the abdomen at 20 h post-ecdysis into the fourth stadium 1331. This process
provided for the isolation of thoracic tissue (anterior to the ligature) or abdominal tissue (posterior to the ligature) from the remainder of the body prior to the
endogenous release of 20-hydroxyecdysterone.Ligated larvae that were to be
parasitized were exposed to the parasite until either the thoracic tissue or the
abdominal tissue was parasitized. Parasitism of the isolated tissue occurred
within 2 h of ligation.
To remove the parasite, T. ni were anaesthetized with carbon dioxide within
24 h of oviposition, and the parasite eggs punctured thereby killing the parasite
approximately 24 h before egg eclosion. No attempt was made to remove the
eggs since they were firmly attached to the host cuticle, and the removal process
would greatly disturb the host larvae.
Protein Preparation and Determination
Host hemolymph was obtained from a severed proleg according to the
procedure described by Kelly and Coudron 1351. Ten microliters of hemolymph
were collected at each time point without dilution, and stored at -20°C. The total
number of insects bled for each time point was approximately 6 for third instar
larvae, 4 for fourth instar larvae, 2 for fifth instar larvae, 8 for ligated larvae bled
from the thorax, and 6 for ligated larvae bled from the abdomen. Three replicate
samples were taken for gel electrophoresis analysis and two replicate samples
were taken for immunoblotting analysis.
*Abbreviations used: IgG = immunoglobulin with a molecular weight of 150,000; JH = juvenile
hormone; L:D = 1ight:dark; RH = relative humidity; SDS-PAGE = sodium dodecyl sulfatr-polyacrylamide gel electrophoresis.
Coudron et al.
Hemolymph proteins were separated by SDS-PAGE as described by Laemmli [361. Hemolymph samples were first centrifuged at 12,0009for 8 min (4°C)
to remove hemocytes and any debris. Equal volumes containing various concentrations of the sample supernatant were applied to 20 cm x 20 cm, 10%
SDS-PAGE gels and electrophoresed at 150 V for 5 h. Protein bands within the
gel matrix were visualized with either a Coomassie blue stain or by a silver
staining method as described by Morrissey 1371.
Production of three separate polyclonal antibodies against one acidic and two
basic storage proteins found in the hemolymph of T. ni has been previously
described [38,39]. Each of these three antisera is specific for one of the three
storage proteins. The immunoblotting techniques used, including electroblotting onto nitrocellulose and the use of 12% labeled goat anti-rabbit IgG as a
secondary antibody and autoradiography to detect the presence of storage
protein in the hemolymph samples, has been previously described by Soldevila
and Jones [40]. Immunoblots were probed with a mixture of the three antisera
at a 1:1,000 antiserum dilution to give a strong signal.
Hemolymph Proteins in Non Ligated Larvae
A comparison of the electrophoretic pattern of hemolymph proteins in control and nonligated parasitized larvae is shown in Figure 1. The growth time
between ecdysis to the next larval stadium was approximately 48 h for both
third and fourth instar nonparasitized larvae, and approximately 90 h in the
fifth instar prior to pupation for the nonparasitized larvae. Examination of
hemolymph protein samples from control fifth instar larvae revealed the presence of relatively abundant proteins, which appeared as an intense staining of
one to three bands at molecular weights of approximately 70,000-80,000 in the
hemolymph of late (48 h) fifth instar larvae. By comparison with other reports
of hemolymph proteins, the pattern of these proteins bore similarities in time
of appearance, abundance, and electrophoreticpattern with that of the storage
proteins characterized from T. ni. No detectable quantities of these proteins
were observed in the hemolymph from control third and fourth instar larvae.
In contrast, the protein composition of the hemolymph from both third and
fourth instar parasitized larvae did contain the characteristic banding pattern
associated with the storage proteins. The relative abundance of these proteins,
as determined by the intensity of the Coomassie blue staining of these bands,
increased with the time after parasitism in both the third and fourth instars.
Confirmation of the storage proteins was obtained by immunoblotting with
the antibodies against three storage proteins found in T. ni (Fig.2). Control third
(not shown) and fourth instar larvae did not contain detectable amounts of
storage proteins. Most notably, strong immunoreactionswith antibodies for one
acidic and two basic storage proteins were detected in the hemolymph of third
(not shown) and fourth instar parasitized larvae within 48 h after parasitism
(Fig. 2, arrows). The response signal increased in the fifth instar control larvae
with an increased time in the stadium (data not shown). A similar temporal
variability was observed in the response signal in the third (data not shown)
Control 4 t h
Parasitized 3rd
Parasit ixed 4 t h
Control 5 t h
Fig. 1. Production of storage proteins in parasitized larvae of Trichoplusia ni. Larval instar i s indicated for 3rd, 4th, and 5th instars of
control and parasitized larvae with 3rd, 4th, and3!ith, respectively. The hour of sampling after the time of last larval ecdysis is indicated
numerically. The positions and Mrvalues (x 10 ) for molecular weight standards are shown at the right.
Control 3rd
Coudron et al.
Cont ro I
24 48 24 48 72
96 120
Fig. 2. lmmunoblot of one acidic and two basic storage proteins in the hemolymph from control and
nonligated parasitized larvae of Trichoplusia ni. The hour of sampling after ecdysis to the 4th instar is
indicated numerically. Top arrows point to the acidic protein and the bottom arrows point to the basic
and fourth instar parasitized larvae, resulting in an increased signal with
additional time after parasitism.
Hemolymph Proteins in Ligated Larvae
The ligature block between the thorax and the abdomen prevented the
endogenous ecdysteroids released at approximately 28 h post-ecdysis into the
fourth stadium from reaching the abdomen of the insect 1351. This was confirmed by the absence of ecdysis of the nonparasitized isolated abdomen and
the complete apolysis into the next larval stadium of the isolated thorax.
However, neither the isolated abdomen nor the isolated thorax showed any
signs of ecdysis after parasitism.
A comparison of the electrophoreticpattern of hemolymph proteins from the
isolated thoraces and isolated abdomens in ligated control and ligated parasitized fourth instar larvae is shown in (Fig. 3). Ligation and parasitism studies of
third instar larvae were not conducted due to the minute amount of hemolymph
extracted during the bleeding process. Ligation did alter the protein composition in nonparasitized larvae. The Coomassie staining increased for several
bands in the isolated thoraces and isolated abdomens of ligated control larvae
(Fig. 3). However, the relative abundance and electrophoretic pattern of these
Storage Proteins in Envenomed larvae
tig. thorax
Pa r a sit iz ed
lip. abdomen
Iig. abdomen
lig. t h o r a x
Fig. 3. Production of storage proteins in isolated abdomens of parasitized 4th instar larvae of Trichoplusia
ni. The hour of sampling after the time of ligation and subsequent parasitism is indicated numerically.
The positions and Mrvalues (x 10-3 for molecular weight standards are shown at the right.
bands differed from that of the storage proteins observed in the nonligated
control and nonligated parasitized larvae (Fig. 1).
Examination of the hemolymph proteins in the isolated thoraces and isolated
abdomens of parasitized fourth instar larvae revealed the presence of storage
proteins, which appeared to intensify with time after parasitism but to a lesser
degree than observed in nonligated parasitized third and fourth instar larvae.
Confirmation of the identity of the storage proteins was again obtained by
immunoblotting with the antibodies against the three forms of the storage
proteins found in T. ni (Fig. 4). A positive immunoreaction for both the acidic
and basic forms of the storage proteins were detected within 48 h of parasitism
for the hemolymph taken from the thoraces and the abdomens of the ligated
and parasitized larvae but not that from the ligated control larvae (Fig. 4,
arrows). The signal intensified from 48 to 96 h post-parasitism. An interesting
result observed was that the acidic protein failed to appear in the thorax section
of ligated larvae post-parasitism, whereas it did appear in abdomen section of
ligated larvae.
Coudron et al.
Cont ro I
lig. thorax
lig. abdomen
6 2 4 4 6 7 6 6 2 4 4 8 7 Z
lig. thorax
lig. abdomen
Fig. 4. lmmunoblot of one acidic and two basic storage proteins in the hemolymph from the isolated
tissues of ligated control and ligated parasitized larvae of Trichoplusia ni. The hour of sampling after
the time of ligation and subsequent parasitism is indicated numerically. Top arrows point to the acidic
protein and the bottom arrows point to the basic proteins.
It should be noted that Coomassie staining detected an abundant protein
migrating between the acidic and basic proteins at the location established for
arylphorin [39]. Also, the immunoblots showed a distinctly arched band shape
for the basic proteins, which is a diagnostic feature of high abundance of
arylphorin [39].
The expression of precocious or delayed events is common in lepidopteran
larvae parasitized by hymenopteran wasps 1221. The precocious spinning of a
cocoon reported in the penultimate instar of T. ni parasitized by Chelonus near
curvimaculatus was thought to be due to a premature decline in the juvenile
hormone level in the host 1411. The delay or suppression of metamorphosis and
the creation of a supernumerary larval stage in the lepidopteran hosts of Cotesia
and Copidosoma species was thought to be due to an abnormally high level of
juvenile hormone and altered ecdysteroid levels in the parasitized host [28,421.
A delay in larval-pupal metamorphosis reported in larvae of Anastrepha suspensa (Loew) parasitized by Biosteres Zongicaudattks was an apparent result of an
elevated level of juvenile hormone in the parasitized larvae [431. All of these
examples involve the penultimate or last larval stadium. Unique to the results
reported here is the expression of a last larval stadium event, that of the
production of storage proteins, in both the third and fourth larval stadium of a
lepidopteran host that has five larval instars.
We also report the early expression of storage proteins in isolated thoraces
and isolated abdomens. These observations suggest that the venom activates
Storage Proteins in Envenomed larvae
the expression of the storage proteins by acting directly on a particular tissue
rather than indirectly via the intact neuroendocrine center. This is similar to the
developmental arrest effect of the venom on the larvae which results in arrest
of the larval-larval ecdysis process [33]. In particular, these results demonstrate
the ability (competence) of early larval stages and isolated tissues of penultimate instar larvae to produce gene products that normally occur in the intact
last larval stadium of lepidopteran larvae.
An interesting observation is that the ligated insects in these experiments
become starved larvae following the placement of the ligature block. Thus, the
abdominal region of a fourth instar ligated larvae is in essence an isolated and
starved tissue that when parasitized demonstrated the ability to produce storage proteins. In contrast, starvation of the last instar larvae of Munduca sextu
inhibited the production of storage protein mRNA 1121. Last instar larvae of
GalZeriu that were fed for 24 h and then starved were reported to transcribe
mRNA coding for the storage proteins. However, when the larvae of Gulleviu
were starved at the onset of the last instar, there were little or no storage protein
transcripts detected. Clearly, the effect of the Euplectrus venom is different from
that of starving the larvae.
Additionally, it was observed that the venom stimulated only the abdominal
tissues to produce the acidic protein. This could indicate that the abdominal
tissues were unique in their ability to produce this gene product, or that the
expression of the protein was considerably delayed or impaired in the thoracic
tissues. There is preliminary evidence for the independent regulation of hemolymph proteins in starved larvae of G. melZoneZZu [44]. It is also plausible that the
expression of this protein was impaired in the thoracic tissue due to a high
accumulation of endogenous ecdysteroids which have been reported in parasitized larvae [351.
Limited information is available on the mechanisms by which the juvenile
hormones and ecdysteroids regulate gene expression of the storage proteins in
insects. Production of certain storage proteins in normally developing lepidopteran larvae occurs in the last larval instar when endogenous JH titers are low
or undetectable. In Bombyx mori, storage proteins were expressed and accumulated in the hemolymph of penultimate instar larvae that were deprived of JH
and in the last instar larvae in which the JH titer is naturally low 145,461. Storage
protein production in Spodopteru Iitura declined in larvae that were treated with
JH or a JH analog, but increased in allatectomized larvae [471. The appearance
of an acidic storage protein with a molecular weight of 76,000 in the last larval
instar of T . ni was suppressed by treatment with JH as was the concentration of
the level of its mRNA 1381. JH also suppressed mRNA abundance for the two
basic proteins [391. These results show that JH suppresses the expression of
storage proteins by regulating the level of mRNA of storage proteins. However,
a decline in level of glycolipoprotein storage proteins in the hemolymph of
Galleria larvae treated with JH was thought to be due to the induction of a
supernumerary larval molt rather than the direct action of JH on the production
and/or stability of the gene transcripts [44].
The expression of storage proteins in M. sextu [48] and in HeZiothis virescens
(Fabricius)[161appeared to be negatively regulated by 20- hydroxyecdysterone.
The ecdysteroid, 20-hydroxyecdysterone,caused a decline in the level of storage
Coudron et al.
protein mRNA transcripts in injected intact and prothorax-ligated larvae of
Galleria I441 and when applied to GaZZeria fat body in vitro 1491. Similarly, mRNA
transcripts of the storage proteins were not found in newly ecdysed last instar
larvae of Galleria when the endogenous level of 20-hydroxyecdysterone was
high 1491. Comparative results were found in in vitro studies with larval fat
body preparations from Calliphora uicina where 20-hydroxyecdysterone repressed storage protein gene expression 1501. Therefore, 20-hydroxyecdysterone appears to cause a cessation of the storage protein gene expression.
A previous study 1351has shown that ecdysteroids, including 20-hydroxyecdysterone, are absent in the hemolymph of larvae of T. ni parasitized by E.
plathypenae prior to, and during, the accumulation of the storage protein time
as reported here. Deprivation of the ecdysteroid hormone from parasitized
larvae of the early instars would eliminate the suppression of storage protein
production by this hormone. This may provide the first indication as to why the
venom alters the ecdysteroid titer of the host when the arrestment of the
larval-larval ecdysis was found to be regulated independent of 20-hydroxyecdysterone 1331. Alterations of the JH titer in parasitized larvae have not been
determined. However, a decline in the JH titer in parasitized larvae is likely,
given that the production of storage proteins follows parasitism.
Another interesting observation is that the acidic and basic storage proteins
may be independently regulated. The rate at which the basic proteins appeared
in isolated and parasitized thoracic regions was faster than the rate of appearance of the acidic protein. An inverse relation existed in the parasitized abdomen region where the rate at which the acidic proteins appeared was faster than
that of the basic proteins. Thus, the acidic and basic proteins appear to be
independently regulated and that regulatory mechanism may differ among
various tissues of the insect.
The production of storage proteins appears to occur in every larval instar
after parasitism by E. cornstockii, and the time of occurrence is approximate to
the time of the parasitoid egg hatch and larval feeding. The protein profile of
the hemolymph of the host at the time of parasitoid feeding is considerably
different than the profile at the time of oviposition. A major change includes the
concentration of the storage proteins as discussed here, as well as other proteins
of lower molecular weights. The alteration in the protein content would assure
the parasitoid of a particular protein profile in the hemolymph of the host
regardless of the larval instar of the parasitized host. The timing of the storage
protein production would imply a role of direct benefit to the developing
parasitoid as opposed to serving some function that regulates a specific stage
of host development. It is plausible that the storage proteins serve a nutritional
function for the parasitoid as they do for the last larval instar of the host.
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production, euplectrus, trichoplusia, parasitized, protein, larvae, latex, comstock, prematurely, storage
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