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A juvenile hormone analogue affects the protein pattern of the haemolymph in last-instar larvae of Locusta migratoria
J. Insect Physiol. Vol. 37, No. 2, pp. 87-93, 1991
Copyright 0
Printed in Great Britain. All rights reserved
0022-1910/91 $3.00 + 0.00 1991 Pergamon Press plc
A JUVENILE HORMONE ANALOGUE AFFECTS THE PROTEIN PATTERN OF THE HAEMOLYMPH IN LAST-INSTAR LARVAE OF LOCUSZ-” MIGRATORIA
Department
of
C. A. D. DE KORT and A. B. KO~PMANSCHAP Entomology, Agricultural University, P.O. Box 8031, 6700 EH Wageningen, The Netherlands (Received 31 August 1990)
Abstract-Pyriproxyfen, a recently developed juvenile hormone analogue, has strong morphogenetic effects when applied topically or by injection to last-instar larvae of Locusta migratoria. A single injection as low as 2 pg induced malformation of the wings and green pigmentation, whereas higher doses resulted in supernumerary-stage larvae. Pyriproxyfen repressed the synthesis of two larva-specific haemolymph proteins, but stimulated the synthesis of another high-molecular weight protein. This protein becomes a major protein of the haemolymph and remains so after the adult moult. Its molecular weight is about 500 f 13 kDa and it is composed of five subunits, with molecular weights of 110 f 8; 101 f. 6.6; 91 f 5.5; 62 + 4.0 and 53 f 3.5 kDa. This protein was independently identified as vitellogenin. Word Index: Juvenile hormone specific protein; metamorphosis
Key
analogue;
INTRODUCTION
pyriproxyfen;
haemolymph
proteins; larval
which occurs both in larvae and adults. Larval storage protein I is probably identical with our larval haemolymph protein (Wyatt, 1990). For clarity of the literature, we used their terminology throughout the rest of our paper. The temporal occurrence of larval storage protein I during the second half of the instar suggests that its rate of synthesis may be correlated with a decrease in juvenile hormone titre. This decrease is known to occur during this stage and induces metamorphosis. To study the relationship between the presence of juvenile hormone and the concentration of larval storage protein I in the haemolymph, we treated last-instar larvae with pyriproxyfen, a recently developed juvenile hormone analogue with high juvenative effects in some insect species (Hatakoshi et al., 1987, 1988; Koopmanschap et aZ., 1989), including the locust (Wyatt, 1989) and followed the concentration of larval storage protein I in the haemolymph quantitatively by rocket immuno-electrophoresis.
Haemolymph
from last-instar larvae of Locusta contains some high-molecular-weight proteins, which have been well characterized. Lipophorin, the lipid-transport protein, mainly studied in adult locusts (Beenakkers et al., 1985), also occurs in larvae and has a molecular weight of about 675 kDa (de Bruyn et al., 1986). Three other proteins of high-molecular-weight have been described, and are designated as juvenile hormone binding protein; cyanoprotein and a storage protein, which is larval specific (de Bruyn et af. 1986; de Kort and Koopmanschap, 1987). This larva-specific haemolymph protein has been isolated and some of its characteristics have been reported (de Kort and Koopmanschap, 1987). The protein possesses some striking similarities with well-known storage proteins from holometabolous insects (Levenbook, 1985), with regard to its temporal occurrence and its molecular structure. In the last larval stage, this protein becomes a major haemolymph protein after day 3 and reaches its maximum concentration in the haemolymph 1 day before the adult moult, after which it is hardly detectable in the haemolymph (de Kort and Koopmanschap, 1987). Ancsin and Wyatt (1989) recently studied the occurrence of hexaemeric proteins in the haemolymph of this locust. They characterized three proteins and suggested to use the term storage proteins. They described the characteristics of two larval storage proteins I and II and one persistent storage protein, migwtoria
MATERIALS
AND METHODS
Larvae and adults of the African migratory locust, migratoria migratorioides R and F, were reared in a culture room at 32°C under crowded conditions as described by Staal (1961), except that bran was provided together with grass. Each cage (42 x 30 x 30 cm) containing the experimental animals was provided with a continuously burning red Locusta
87
88
C. A.D. DEKORT~II~ A.B. KOOPMANSCHAP
bulb (60 W), which maintained a temperature gradient in the cage between 32-38°C. Under these conditions the last larval stage lasted 6-7 days. Larvae were selected within 16 h of the last larval moult and kept in separate cages for the rest of the experimental period, which lasted at most 10 days after the adult moult. The juvenile hormone analogue, pyriproxyfen, 2-[ 1-methyl-2-(4-phenoxyphenoxy)-ethoxylpyridine, was a 10% emulsifiable concentrate, supplied by Dr H. Oouchi from Sumitomo Chemical Co. Ltd, Osaka, Japan. Controls were treated with emulsifiable solution, without the analogue. Both solutions were kept at 4°C. For each series of experiments, fresh solutions of analogue were prepared in either acetone for topical application or olive oil for the injection experiments. Both applications were carried out with a Hamilton microsyringe, operated with a Hamilton repeating dispenser. Each dose was applied in a constant volume of 5 ~1 per animal and included 15-20 locusts. The experiments were repeated three times. Haemolymph was collected into capillary pipettes after puncturing the membrane between the thorax and the metathoracic leg. Each animal was bled only once. Collected haemolymph was immediately diluted to half in cold buffer, containing 50 mM phosphate (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.6 mM phenylmethyl sulphonyl fluoride and a few grains of phenylthiourea. Haemocytes were sedimented by centrifuging at 10,000 g for 4 min and the haemolymph was used immediately or stored at - 20°C. The haemolymph protein pattern was studied with different electrophoretic systems, native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulphate (SDS)-PAGE and agarose gel electrophoresis. Non-denaturing PAGE, using different types of polyacrylamide gradients, was essentially as described by de Bryun et al. (1986). Agarose gel electrophoresis was performed with Ciba Corning (Palo Alto, Calif.) ready-to-use universal agarose films, according to the description of the manufacturer. Samples of 1 ~1 of 1: 1 diluted haemolymph were applied by way of a microsyringe. SDS-PAGE was performed horizontally, with different types of gradient gels, in a buffer system described by Laemmli (1970). Haemolymph samples were diluted in sample buffer containing 5% mercaptoethanol and heated to 100°C for 2min, before application to the gel. The molecular weights of the proteins were estimated by comparison with standard proteins of high and low molecular weight (Pharmacia, Uppsala, Sweden), with regression analysis. After SDS-PAGE, the separated proteins were transferred electrophoretically to polyvinylidene difluoride transfer membranes (Immobilon, Millipore, Bedford, Mass.) (de Kort and Koopmanschap, 1987). The blots were stained for 2-3 min in 0.1% amido black, dissolved in 45% (v/v) methanol and 10% (v/v) acetic acid. Destaining
occurred by several washings in 90% (v/v) methanol and 2% (v/v) acetic acid. Immunological detection of the transferred proteins was performed with colloidal gold-labelled protein A as second antibody by the method of de Kort and Koopmanschap (1987), except that a new more specific primary antiserum (No. 738) was used. Proteins were eluted from gel slices after separation by non-denaturing PAGE with the BIOR4D (Richmond, Calif.) Model 422 electro-eluter, after staining of the proteins with Coomassie R-250. After the run, native gels were stained for 30 min in 0.1% Coomassie R-250, dissolved in 10% (v/v) acetic acid, 40% (v/v) methanol. The blots were destained for 1 h in 10% (v/v) acetic acid, 40% (v/v) methanol. After the bands of interest had been cut out, the slices were eluted for 2 h according to the manufacturer’s instructions with a volatile ammonium bicarbonate buffer. The eluted protein samples were collected in about 500 /*l of the buffer, transferred to Eppendorf tubes and freeze-dried. The dried protein fractions were dissolved in 40 ~1 of SDS sample buffer by heating at 100°C for 2min. Corresponding bands from 3-5 lanes of the native gel were pooled before electro-elution. The concentrations of larval storage protein and juvenile hormone binding protein in whole haemolymph samples were measured by rocket immunoelectrophoresis as described before (de Kort and Koopmanschap, 1987), in 1% agarose plates containing antiserum 738 (larval storage protein) or antiserum 722 (juvenile hormone binding protein). The total protein concentration of the haemolymph was measured by the method of Schacterle and Pollack (1973) with bovine serum albumin as a standard. RESULTS
Morphogenetic effects of pyriproxyfen
The effects of implantation of extra corpora allata or injection of natural juvenile hormone and its analogue on morphogenesis have been extensively described for locusts (Staal, 1961; Roussel, 1977). We applied pyriproxyfen to last-instar larvae, either topically or by injection. Topical application of pyriproxyfen, 10 or 50 fig, 24-40 h or 96-l 12 h after the last larval moult interfered strongly with metamorphosis and moulting into the adult. Three distinct types of locust could be distinguished after the moult: normal adults often with green pigmentation; adultoids that look like adults but have severe malformations of the wings and usually green pigmentation or a supernumerary larval stage, with patches of adult cuiticle, particularly on the head or pronotum. Whereas control adults started oviposition after 7 days of adult life under our rearing conditions, none of the pyriproxyfen-treated animals oviposited before day 10, when the experiment was terminated.
Haemolymph
from last-instar
The observed effects were clearly dose-dependent. The proportion of supernumerary larvae was higher at the dose of 50 pg (75%), whereas green adults and adultoids were more numerous after application of 10 pg (70%). We did not observe any marked difference with time of application. The dose of 10 pg had a stronger morphogenetic effect when applied on day 4 (40% supernumerary larvae and more severe malformation of the wings), whereas 50 pg was more effective with applied on day 1. We also studied the effects of different doses of pyriproxyfen injected 24-40 h after the last larval moult. Injection was used, because in locusts this has proved to be more effective than topical application (P. Pener, personal communication). The first dose injected was 120 pg. This dose completely inhibited the moult to the adult stage and the animals died 8 days after injection. Next, doses of 2, 10 and 50 pg, dissolved in 5 ~1 of olive oil, were injected. All three doses caused morphogenetic effects, but clearly dose-dependent. The lowest dose resulted in adultoids with different degrees of malformation of the wings. Many showed olive-green pigmentation. Injection of 10 pg resulted in 40% supernumerary stage larvae and 60% adultoids. The group receiving 50 pg all moulted in supernumerary stage larvae with some adult cuticle on the pronotum. The injected animals did not behave abnormally during the instar. They fed and grew normally, resulting in the same average wet weight of 1.32 _I 0.060 g (n = 24). Apparently pyriproxyfen strongly affects metamorphosis. That it is not the only physiological effect of this juvenile hormone analogue became apparent from our studies on the protein compositon of the haemolymph. Effect on protein composition of the haemolymph Before describing the effects of pyriproxyfen on the protein composition of the haemolymph, let us explain that this treatment has a complicated effect on the duration of the instar, which is difficult to illustrate statistically. In our three independent series of experiments, moulting to the adult started about 12 h earlier than in the controls after application of the lower doses. With the higher doses, moulting was either delayed (50 pg) or prevented (120 pg). Since there is large variation in the timing of the moult within each group and since the timing is unpredictable for individual animals, we decided to study the protein composition of the haemolymph after day 4, which is well before the onset of the moult in any group. This means that the quantitative differences on day 4 are not the maximal differences between the groups but only a moment in a gradual process. Haemolymph from pyriproxyfen-treated animals was usually very green but not consistently in all animals. The haemolymph volume and pressure was high in pyriproxyfen-treated locusts at day 4 and 5, but very difficult to measure, because after punctur-
larvae
of
89
L. migratoria
ing it spurts out at relatively high pressure. Thus injection of radiolabelled inuline seems to be impossible. We also noted that haemolymph from pyriproxyfen-treated animals had a stronger tendency to clot. Collected haemolymph was therefore immediately diluted (1: 1 v/v) with EDTA-containing buffer and cooled on ice. We first measured the total protein concentration and the content of larval storage protein I and juvenile hormone binding protein in whole haemolymph. The results (Table 1) show that pyriproxyfen dramatically represses the synthesis of the storage protein, whereas it has only minor effects on the total or the binding protein concentration. If the total protein concentration was measured in locusts closer to the moult, we found values up to 200 mg/ml after treatment with pyriproxyfen. Since larval storage protein I is a major protein in the haemolymph amounting to about 40% (w/w) of total protein in controls during these experiments (see however de Kort and Koopmanschap, 1987), it is astonishing that the total protein concentration of the haemolymph is enhanced though the storage protein concentration is repressed over 95%. This can be explained if other proteins have increased in concentration or if another new protein is induced. Such possibilities were tested by electrophoresis. After native PAGE, using gradient gels of 4-20 or 5.3-20% polyacrylamide, we observed the four familiar protein bands of high-molecular-weight described in our earlier publications (de Bruyn et al., 1986; de Kort and Koopmanschap, 1987, 1988). However in haemolymph from pyriproxyfen-treated locusts, a strong band was visible with almost the same Rf as larval storage protein I. That this band was not the latter could be seen in samples from animals treated with 2 pg. The new protein had a Rf slightly different from larval storage protein I, but the difference was not significant. By comparison with high-molecular-weight standard proteins we arrived at a molecular weight for the storage protein of Table 1. Changes in protein concentration of the haemolymph after injection of different doses of pyriproxyfen in last-instar
larvae Concentration to controls
Control 2pg 1opg 50 fig
relative (%)
Total protein concentration (mg/ml)
LSP-I
JHBP
93.5 92.9 93.5 124.9
100 78 21.7 1.6
100 135 78 78
LSP-I = larval storage protein I; JHBP = juvenile hormone binding protein. Last-instar larvae were injected after day 1 and the protein concentrations were measured after day 4. Total protein was determined according to Schacterle and Pollack (1973), the concentrations of LSP-I and JHBP by rocket immuno-electrophoresis. The concentrations of LSP-I and JHBP in the controls were 36 and 2 mg/ml, respectively.
90
C. A. D.
DE KORT
and A. B. K~~PMANSCHAP
509 f 10 kDa (n = 4) and for the unknown protein of 500 + 13 kDa (n = 4). Moreover, the high intensity of this band did not agree with our quantitative measurements using rocket-electrophoresis (Table 1). To prove that these two proteins are indeed different, we used agarose-gel electrophoresis, which is suitable for studying major proteins, because the proteinbanding pattern is rather simple. The bands containing lipophorin and larval storage protein I could be identified, by combining electrophoresis with immunodiffusion. The two bands are indicated in Fig. 1. The presence of the storage protein was obvious in lanes containing haemolymph from controls (lane 1) and to a lesser extent in samples from animals treated with 2pg (lane 2). In samples from locusts treated with 10 or 50 pg pyriproxyfen, larval storage proteins I was hardly detectable by amido black staining, but a new strong band (indicated X) was present with a lower RF than the storage protein (lanes 3 and 4). Apparently, the synthesis of this new protein was stimulated by pyriproxyfen in a dose-dependent manner. To further characterize this protein, we used SDS-PAGE in combination with Western blotting. Figure 2 illustrates two blots from SDS-gels stained with amido black (A) or with our anti-larval storage protein I serum coupled with colloidal-gold-labelled protein A (B). In the sample from control locusts, two bands are prominent: apolipoprotein I (M, about 250,000) and a very thick band about 80,000, which contains a mixture of subunits from different proteins, such as apolipoprotein II, juvenile hormone binding protein, larval storage protein I and cyanoprotein (de Kort and Koopmanschap, 1987; 1988). That storage protein I is present in this band can be seen after immunological staining [Fig. 2(B)]. In the pyriproxyfen-treated haemolymph samples this band of about 80 kDa becomes weaker in a dose-dependent manner, because of disappearance of the storage protein I subunit. This is more obvious after immunological staining [Fig. 2(B)]. Concomitant with the disappearance of the above subunit, we observed a dose-dependent appearance of five new subunits. These five new proteins are not breakdown products of larval storage protein, because they do not react with our antiserum to the latter. Apparently these five protein subunits are induced by pyriproxyfen, indicating that they are derived from the native protein of 500 kDa. To prove this more convincingly, a native PAGE gel was run from samples of control and pyriproxyfen-treated animals. After staining with Coomassie Brilliant R250, the bands of molecular weight 500-510 kDa were sliced from the lanes of control and pyriproxyfen-treated samples and the stained proteins were electrophoretically eluted with a BIORAD Model 422 electro-elutor. After freeze-drying of the eluted proteins, the fractions were dissolved in 40 ~1 of SDS sample buffer by boiling for 2min and applied to SDS-gradient gel. After the run, the pro-
teins were blotted onto Immobilon and stained immunologically or with amido black. The results of the amido black staining are illustrated in Fig. 3. The lane containing the sample from controls showed only a subunit of molecular weight about 80 kDa, which responded positively to our larval storage protein I antibody. The same subunit was also present in the protein eluate (X in Fig. 3) from pyriproxyfen-treated animals, but at a much lower concentration. This eluate contained five additional bands that did not respond to the antiserum to storage protein I. The molecular weights of these five proteins were determined by comparison with standard proteins. The molecular weights were, respectively, 110 + 8 kDa (n = 4); 101.5 + 6.6 kDa (n = 4); 90.7 + 5.5 kDa (n = 3); 61.7 + 4.0 kDa (n = 3) and 56.3 f 3.5 kDa (n = 3). This experiment suggests that these five protein subunits are derived from the native protein of 500 kDa, which becomes a major protein in the haemolymph after treatment with pyriproxyfen. This protein remains a major haemolymph protein after the treated animals have moulted to the next stage (not shown). DISCUSSION
Pyriproxyfen affects the protein pattern of the haemolymph and has strong juvenilizing effects when applied to last-instar larvae of L. migratoriu. The observed morphological effects are comparable with previous experiments using implanted corpora allata (Staal, 1961), natural juvenile hormones or analogues (Roussel, 1977). Our results differ from those described by Staal (1961) and Roussel (1977), who claimed that the larvae are most sensitive during the first 3 days of the instar. We found that treatment on day 4 is still effective, though the total duration of the instar under our rearing conditions was only 6 days, even with 2pg. This makes pyriproxyfen one of the most effective juvenile hormone analogues known for locusts. Synthesis of larval storage protein I (de Kort and Koopmanschap, 1987; Ancsin and Wyatt, 1989) is dramatically repressed, but the synthesis of another high-molecular-weight protein not previously noticed by us, was stimulated in a dosedependent manner. This protein was further characterized. Its molecular weight was estimated by native PAGE and appeared to be about 500 + 13 kDa. After denaturation by boiling with sample buffer containing mercaptoethanol, the protein dissociated into five subunits with molecular weights of 110, 101, 91, 62 and 56 kDa, respectively. All five subunits were induced by pyriproxyfen in a dose-dependent manner. As shown in Fig. 2(A), the largest subunit (110 kDa) is already clearly visible at a dose of 2 pg whereas the four other subunits are still rather weak. The intensity of all the bands clearly increased after treatment with doses of 10 and 5Opg.
LSP
LSP X
X
LP
LP 4
3
2
1
Fig. 1. Electropherogram in 1% agarose of total haemolymph of last-instar larvae of L. migratoria 3 days after injection of different doses of pyriproxyfen. Lane 1, control; lane 2,2 pg; lane 3,10 ng; lane 4,SO pg. Lp, lipophorin; LSP, larval storage protein; X, unknown protein (identified as vitellogenin in collaboration with Ancsin and Wyatt).
LMW
wo
0
2
10
50
0
2
10
50
pLs
IPI
94 67
Zig. 2. Western blots from SDS gels of total haemolymph from last-instar larvae of L. migratoria after injection of 2, 10 or 5Ong pyriproxyfen. The blots were stained with (A) amid0 black or (B) immunologically with anti-larval storage protein I serum followed by colloidally gold-labelled protein A. LMW, low-molecular-weight standard proteins. Apolp I, apolipophorin I.
91
LSP
LMW
94K
67 K
43 K
30 K 20 K 14 K
Fig. 3. Western blot from SDS gel of larval storage protein I and protein band X electro-eluted from native PAGE after staining with Coomassie R-250. The blot was stained with amido black. LMW, low-molecular-weight standard protein, LSP, larval storage protein; X, pyriproxyfen-stimulated protein band (vitellogenin, Ancsin and Wyatt, personal communication).
92
Haemolymph from last-&tar larvae of L. migratoria This protein is not identical with persistent storage protein recently discovered by Ancsin and Wyatt (1989), because the latter is composed of only two subunits of molecular weight of 74 and 77 kDa, respectively. The protein was identified by Ancsin and Wyatt, who independently obtained similar results with pyriproxyfen in locust larvae. It appeared to be vitellogenin, which is precociously induced in larvae by this juvenile hormone analogue. Dhadialla and Wyatt (1983) have previously shown that vitellogenin can be induced in larvae by methoprene, but high doses (> 100 /*g) were required to obtain this effect. Using a similar concentration range, Ancsin and Wyatt (personal communication) recently showed by rocket immuno-electrophoresis that vitellogenin becomes a major haemolymph protein after application of pyriproxyfen to last instar larvae. A number of questions still remains to be answered regarding the molecular composition and post-translational processing of this precociously induced larval vitellogenin as compared with vitellogenin from adult females. ‘Thus, pyriproxyfen represses the synthesis of larval storage protein 1 but stimulates the premature synthesis of vitellogenin, which becomes a major protein of the haemolymph in larvae and remains so after the moult to adultoid. From our native electropherograms, we could deduce that another larval protein band (storage protein II; Ancsin and Wyatt, 1989) of about M, 250 kDa (de Kort and Koopmanschap, 1987) disappears after pyriproxyfen treatment, but this protein was not studied further. Acknowledgements-The
exchange of unpublished data with Mr J. B. Ancsin and Dr G. R. Wyatt (Department of
Biology, Queens University, Kingston, Ontario) was highly appreciated. We thank Dr H. Oouchi, Sumitomo Chemical Co., Ltd, Osaka, Japan, for providing the pyriproxyfen and Mr J. C. Rigg, Pudoc, Wageningen, for checking the English. REFERENCES
Ancsin J. B. and Wyatt G. R. (1989) Purification and characterization of two hemolymph proteins from Locusta migratoria. Abstr. Int. Symp. Molecular Insect Science, Tucson, Arizona.
Beenakkers A. M. Th., van der Horst D. J. and van Marrewijk W. J. A. (1985) Insect lipids and lipoproteins,
93
and their role in physiological processes. Prog. Lipid Res. 24, 19-67.
de Bruyn S. M., Koopmanschap A. B. and de Kort C. A. D. (1986) High-molecular-weight serum proteins from Locusta migratoria: identification of a protein specifically binding juvenile hormone-III. Physiol. Entomol. 11, 7-16. Dhadialla T. S. and Wyatt G. R. (1983) Juvenile hormonedependent vitellogenin synthesis in Locusta migratoriu fat body: inducibility related to sex and stage. Devl Biol. 96, 436-444.
Hatakoshi M., Kawada H., Nishida S., Kisida H. and Nakayana I. (1987) Laboratory evaluation of 2[I-methyl-2-(4-phenoxyphenoxy)ethoxy]pyridine against larvae of mosquitoes and housefly. Jap. J. sanit. Zool. 38, 271-274.
Hatakoshi M., Nakayama I. and Riddiford L. M. (1988) The induction of an imperfect supernumerary larval moult by juvenile hormone analogues in Munduca sextu. J. Insect Physiol. 34, 373-378.
Koopmanschap A. B., Oouchi H. and de Kort C. A. D. (1989) Effects of a juvenile hormone analogue on the eggs, post-embryonic development, metamorphosis and diapause induction of the Colorado potato beetle, Leptinotarsa decemlineata. Entomologia exp. appl. 50, 255-263.
de Kort C. A. D. and Koopmanschap A. B. (1987) Isolation and characterization of a larval hemolymph protein in Locusta migratoria. Archs Insect Biochem. 191-203.
Physiol. 4,
de Kort C. A. D. and Koopmanschap A. B. (1988) Isolation and characterization of a high molecular weight JH-III transport protein in the hemolymph of Locusta migratoria. Archs Insect B&hem.
Physiol. 7, 105-l 18.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Levenbook L. (1985) Insect storage proteins. In Comprehensive Insect Physiology, Biochemistry and Pharmacology
(Eds Kerkut G. A. and Gilbert L. I.). Pergamon Press, Oxford. Roussel J. P. (1977) L’action differentielle des hormones juveniles sur la metamorphose chez Locusta migratoriu. 3. Insect Physiol. 23, 1143-I 150. Schacterle G. R. and Pollack R. L. (1973) A simolified method for the quantitative assay ok small amounts of protein in biological material. Analyt. Biochem. 51, 654-655. Staal G. B. (1961) Studies on the physiology of phase induction in Locusta migratoria migratorioides. R & F
Thesis Wageningen, The Netherlands. Wyatt G. R. (1989) How do juvenile hormone and juvenoid IGR’s work at the molecular level? Abstr. 5th Int. Congr. Invertebrate Reproduction, Nagoya, Japan. Wyatt G. R. (1990) Locust hemolymph proteins and effects of juvenile .hor&one. In Inset; koIe&ar Science (Eds Hagedorn H. H., Hildebrand J. G., Kidwell M. G. and Law J. H.). Plenum Press, New York.
Copyright 0
Printed in Great Britain. All rights reserved
0022-1910/91 $3.00 + 0.00 1991 Pergamon Press plc
A JUVENILE HORMONE ANALOGUE AFFECTS THE PROTEIN PATTERN OF THE HAEMOLYMPH IN LAST-INSTAR LARVAE OF LOCUSZ-” MIGRATORIA
Department
of
C. A. D. DE KORT and A. B. KO~PMANSCHAP Entomology, Agricultural University, P.O. Box 8031, 6700 EH Wageningen, The Netherlands (Received 31 August 1990)
Abstract-Pyriproxyfen, a recently developed juvenile hormone analogue, has strong morphogenetic effects when applied topically or by injection to last-instar larvae of Locusta migratoria. A single injection as low as 2 pg induced malformation of the wings and green pigmentation, whereas higher doses resulted in supernumerary-stage larvae. Pyriproxyfen repressed the synthesis of two larva-specific haemolymph proteins, but stimulated the synthesis of another high-molecular weight protein. This protein becomes a major protein of the haemolymph and remains so after the adult moult. Its molecular weight is about 500 f 13 kDa and it is composed of five subunits, with molecular weights of 110 f 8; 101 f. 6.6; 91 f 5.5; 62 + 4.0 and 53 f 3.5 kDa. This protein was independently identified as vitellogenin. Word Index: Juvenile hormone specific protein; metamorphosis
Key
analogue;
INTRODUCTION
pyriproxyfen;
haemolymph
proteins; larval
which occurs both in larvae and adults. Larval storage protein I is probably identical with our larval haemolymph protein (Wyatt, 1990). For clarity of the literature, we used their terminology throughout the rest of our paper. The temporal occurrence of larval storage protein I during the second half of the instar suggests that its rate of synthesis may be correlated with a decrease in juvenile hormone titre. This decrease is known to occur during this stage and induces metamorphosis. To study the relationship between the presence of juvenile hormone and the concentration of larval storage protein I in the haemolymph, we treated last-instar larvae with pyriproxyfen, a recently developed juvenile hormone analogue with high juvenative effects in some insect species (Hatakoshi et al., 1987, 1988; Koopmanschap et aZ., 1989), including the locust (Wyatt, 1989) and followed the concentration of larval storage protein I in the haemolymph quantitatively by rocket immuno-electrophoresis.
Haemolymph
from last-instar larvae of Locusta contains some high-molecular-weight proteins, which have been well characterized. Lipophorin, the lipid-transport protein, mainly studied in adult locusts (Beenakkers et al., 1985), also occurs in larvae and has a molecular weight of about 675 kDa (de Bruyn et al., 1986). Three other proteins of high-molecular-weight have been described, and are designated as juvenile hormone binding protein; cyanoprotein and a storage protein, which is larval specific (de Bruyn et af. 1986; de Kort and Koopmanschap, 1987). This larva-specific haemolymph protein has been isolated and some of its characteristics have been reported (de Kort and Koopmanschap, 1987). The protein possesses some striking similarities with well-known storage proteins from holometabolous insects (Levenbook, 1985), with regard to its temporal occurrence and its molecular structure. In the last larval stage, this protein becomes a major haemolymph protein after day 3 and reaches its maximum concentration in the haemolymph 1 day before the adult moult, after which it is hardly detectable in the haemolymph (de Kort and Koopmanschap, 1987). Ancsin and Wyatt (1989) recently studied the occurrence of hexaemeric proteins in the haemolymph of this locust. They characterized three proteins and suggested to use the term storage proteins. They described the characteristics of two larval storage proteins I and II and one persistent storage protein, migwtoria
MATERIALS
AND METHODS
Larvae and adults of the African migratory locust, migratoria migratorioides R and F, were reared in a culture room at 32°C under crowded conditions as described by Staal (1961), except that bran was provided together with grass. Each cage (42 x 30 x 30 cm) containing the experimental animals was provided with a continuously burning red Locusta
87
88
C. A.D. DEKORT~II~ A.B. KOOPMANSCHAP
bulb (60 W), which maintained a temperature gradient in the cage between 32-38°C. Under these conditions the last larval stage lasted 6-7 days. Larvae were selected within 16 h of the last larval moult and kept in separate cages for the rest of the experimental period, which lasted at most 10 days after the adult moult. The juvenile hormone analogue, pyriproxyfen, 2-[ 1-methyl-2-(4-phenoxyphenoxy)-ethoxylpyridine, was a 10% emulsifiable concentrate, supplied by Dr H. Oouchi from Sumitomo Chemical Co. Ltd, Osaka, Japan. Controls were treated with emulsifiable solution, without the analogue. Both solutions were kept at 4°C. For each series of experiments, fresh solutions of analogue were prepared in either acetone for topical application or olive oil for the injection experiments. Both applications were carried out with a Hamilton microsyringe, operated with a Hamilton repeating dispenser. Each dose was applied in a constant volume of 5 ~1 per animal and included 15-20 locusts. The experiments were repeated three times. Haemolymph was collected into capillary pipettes after puncturing the membrane between the thorax and the metathoracic leg. Each animal was bled only once. Collected haemolymph was immediately diluted to half in cold buffer, containing 50 mM phosphate (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.6 mM phenylmethyl sulphonyl fluoride and a few grains of phenylthiourea. Haemocytes were sedimented by centrifuging at 10,000 g for 4 min and the haemolymph was used immediately or stored at - 20°C. The haemolymph protein pattern was studied with different electrophoretic systems, native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulphate (SDS)-PAGE and agarose gel electrophoresis. Non-denaturing PAGE, using different types of polyacrylamide gradients, was essentially as described by de Bryun et al. (1986). Agarose gel electrophoresis was performed with Ciba Corning (Palo Alto, Calif.) ready-to-use universal agarose films, according to the description of the manufacturer. Samples of 1 ~1 of 1: 1 diluted haemolymph were applied by way of a microsyringe. SDS-PAGE was performed horizontally, with different types of gradient gels, in a buffer system described by Laemmli (1970). Haemolymph samples were diluted in sample buffer containing 5% mercaptoethanol and heated to 100°C for 2min, before application to the gel. The molecular weights of the proteins were estimated by comparison with standard proteins of high and low molecular weight (Pharmacia, Uppsala, Sweden), with regression analysis. After SDS-PAGE, the separated proteins were transferred electrophoretically to polyvinylidene difluoride transfer membranes (Immobilon, Millipore, Bedford, Mass.) (de Kort and Koopmanschap, 1987). The blots were stained for 2-3 min in 0.1% amido black, dissolved in 45% (v/v) methanol and 10% (v/v) acetic acid. Destaining
occurred by several washings in 90% (v/v) methanol and 2% (v/v) acetic acid. Immunological detection of the transferred proteins was performed with colloidal gold-labelled protein A as second antibody by the method of de Kort and Koopmanschap (1987), except that a new more specific primary antiserum (No. 738) was used. Proteins were eluted from gel slices after separation by non-denaturing PAGE with the BIOR4D (Richmond, Calif.) Model 422 electro-eluter, after staining of the proteins with Coomassie R-250. After the run, native gels were stained for 30 min in 0.1% Coomassie R-250, dissolved in 10% (v/v) acetic acid, 40% (v/v) methanol. The blots were destained for 1 h in 10% (v/v) acetic acid, 40% (v/v) methanol. After the bands of interest had been cut out, the slices were eluted for 2 h according to the manufacturer’s instructions with a volatile ammonium bicarbonate buffer. The eluted protein samples were collected in about 500 /*l of the buffer, transferred to Eppendorf tubes and freeze-dried. The dried protein fractions were dissolved in 40 ~1 of SDS sample buffer by heating at 100°C for 2min. Corresponding bands from 3-5 lanes of the native gel were pooled before electro-elution. The concentrations of larval storage protein and juvenile hormone binding protein in whole haemolymph samples were measured by rocket immunoelectrophoresis as described before (de Kort and Koopmanschap, 1987), in 1% agarose plates containing antiserum 738 (larval storage protein) or antiserum 722 (juvenile hormone binding protein). The total protein concentration of the haemolymph was measured by the method of Schacterle and Pollack (1973) with bovine serum albumin as a standard. RESULTS
Morphogenetic effects of pyriproxyfen
The effects of implantation of extra corpora allata or injection of natural juvenile hormone and its analogue on morphogenesis have been extensively described for locusts (Staal, 1961; Roussel, 1977). We applied pyriproxyfen to last-instar larvae, either topically or by injection. Topical application of pyriproxyfen, 10 or 50 fig, 24-40 h or 96-l 12 h after the last larval moult interfered strongly with metamorphosis and moulting into the adult. Three distinct types of locust could be distinguished after the moult: normal adults often with green pigmentation; adultoids that look like adults but have severe malformations of the wings and usually green pigmentation or a supernumerary larval stage, with patches of adult cuiticle, particularly on the head or pronotum. Whereas control adults started oviposition after 7 days of adult life under our rearing conditions, none of the pyriproxyfen-treated animals oviposited before day 10, when the experiment was terminated.
Haemolymph
from last-instar
The observed effects were clearly dose-dependent. The proportion of supernumerary larvae was higher at the dose of 50 pg (75%), whereas green adults and adultoids were more numerous after application of 10 pg (70%). We did not observe any marked difference with time of application. The dose of 10 pg had a stronger morphogenetic effect when applied on day 4 (40% supernumerary larvae and more severe malformation of the wings), whereas 50 pg was more effective with applied on day 1. We also studied the effects of different doses of pyriproxyfen injected 24-40 h after the last larval moult. Injection was used, because in locusts this has proved to be more effective than topical application (P. Pener, personal communication). The first dose injected was 120 pg. This dose completely inhibited the moult to the adult stage and the animals died 8 days after injection. Next, doses of 2, 10 and 50 pg, dissolved in 5 ~1 of olive oil, were injected. All three doses caused morphogenetic effects, but clearly dose-dependent. The lowest dose resulted in adultoids with different degrees of malformation of the wings. Many showed olive-green pigmentation. Injection of 10 pg resulted in 40% supernumerary stage larvae and 60% adultoids. The group receiving 50 pg all moulted in supernumerary stage larvae with some adult cuticle on the pronotum. The injected animals did not behave abnormally during the instar. They fed and grew normally, resulting in the same average wet weight of 1.32 _I 0.060 g (n = 24). Apparently pyriproxyfen strongly affects metamorphosis. That it is not the only physiological effect of this juvenile hormone analogue became apparent from our studies on the protein compositon of the haemolymph. Effect on protein composition of the haemolymph Before describing the effects of pyriproxyfen on the protein composition of the haemolymph, let us explain that this treatment has a complicated effect on the duration of the instar, which is difficult to illustrate statistically. In our three independent series of experiments, moulting to the adult started about 12 h earlier than in the controls after application of the lower doses. With the higher doses, moulting was either delayed (50 pg) or prevented (120 pg). Since there is large variation in the timing of the moult within each group and since the timing is unpredictable for individual animals, we decided to study the protein composition of the haemolymph after day 4, which is well before the onset of the moult in any group. This means that the quantitative differences on day 4 are not the maximal differences between the groups but only a moment in a gradual process. Haemolymph from pyriproxyfen-treated animals was usually very green but not consistently in all animals. The haemolymph volume and pressure was high in pyriproxyfen-treated locusts at day 4 and 5, but very difficult to measure, because after punctur-
larvae
of
89
L. migratoria
ing it spurts out at relatively high pressure. Thus injection of radiolabelled inuline seems to be impossible. We also noted that haemolymph from pyriproxyfen-treated animals had a stronger tendency to clot. Collected haemolymph was therefore immediately diluted (1: 1 v/v) with EDTA-containing buffer and cooled on ice. We first measured the total protein concentration and the content of larval storage protein I and juvenile hormone binding protein in whole haemolymph. The results (Table 1) show that pyriproxyfen dramatically represses the synthesis of the storage protein, whereas it has only minor effects on the total or the binding protein concentration. If the total protein concentration was measured in locusts closer to the moult, we found values up to 200 mg/ml after treatment with pyriproxyfen. Since larval storage protein I is a major protein in the haemolymph amounting to about 40% (w/w) of total protein in controls during these experiments (see however de Kort and Koopmanschap, 1987), it is astonishing that the total protein concentration of the haemolymph is enhanced though the storage protein concentration is repressed over 95%. This can be explained if other proteins have increased in concentration or if another new protein is induced. Such possibilities were tested by electrophoresis. After native PAGE, using gradient gels of 4-20 or 5.3-20% polyacrylamide, we observed the four familiar protein bands of high-molecular-weight described in our earlier publications (de Bruyn et al., 1986; de Kort and Koopmanschap, 1987, 1988). However in haemolymph from pyriproxyfen-treated locusts, a strong band was visible with almost the same Rf as larval storage protein I. That this band was not the latter could be seen in samples from animals treated with 2 pg. The new protein had a Rf slightly different from larval storage protein I, but the difference was not significant. By comparison with high-molecular-weight standard proteins we arrived at a molecular weight for the storage protein of Table 1. Changes in protein concentration of the haemolymph after injection of different doses of pyriproxyfen in last-instar
larvae Concentration to controls
Control 2pg 1opg 50 fig
relative (%)
Total protein concentration (mg/ml)
LSP-I
JHBP
93.5 92.9 93.5 124.9
100 78 21.7 1.6
100 135 78 78
LSP-I = larval storage protein I; JHBP = juvenile hormone binding protein. Last-instar larvae were injected after day 1 and the protein concentrations were measured after day 4. Total protein was determined according to Schacterle and Pollack (1973), the concentrations of LSP-I and JHBP by rocket immuno-electrophoresis. The concentrations of LSP-I and JHBP in the controls were 36 and 2 mg/ml, respectively.
90
C. A. D.
DE KORT
and A. B. K~~PMANSCHAP
509 f 10 kDa (n = 4) and for the unknown protein of 500 + 13 kDa (n = 4). Moreover, the high intensity of this band did not agree with our quantitative measurements using rocket-electrophoresis (Table 1). To prove that these two proteins are indeed different, we used agarose-gel electrophoresis, which is suitable for studying major proteins, because the proteinbanding pattern is rather simple. The bands containing lipophorin and larval storage protein I could be identified, by combining electrophoresis with immunodiffusion. The two bands are indicated in Fig. 1. The presence of the storage protein was obvious in lanes containing haemolymph from controls (lane 1) and to a lesser extent in samples from animals treated with 2pg (lane 2). In samples from locusts treated with 10 or 50 pg pyriproxyfen, larval storage proteins I was hardly detectable by amido black staining, but a new strong band (indicated X) was present with a lower RF than the storage protein (lanes 3 and 4). Apparently, the synthesis of this new protein was stimulated by pyriproxyfen in a dose-dependent manner. To further characterize this protein, we used SDS-PAGE in combination with Western blotting. Figure 2 illustrates two blots from SDS-gels stained with amido black (A) or with our anti-larval storage protein I serum coupled with colloidal-gold-labelled protein A (B). In the sample from control locusts, two bands are prominent: apolipoprotein I (M, about 250,000) and a very thick band about 80,000, which contains a mixture of subunits from different proteins, such as apolipoprotein II, juvenile hormone binding protein, larval storage protein I and cyanoprotein (de Kort and Koopmanschap, 1987; 1988). That storage protein I is present in this band can be seen after immunological staining [Fig. 2(B)]. In the pyriproxyfen-treated haemolymph samples this band of about 80 kDa becomes weaker in a dose-dependent manner, because of disappearance of the storage protein I subunit. This is more obvious after immunological staining [Fig. 2(B)]. Concomitant with the disappearance of the above subunit, we observed a dose-dependent appearance of five new subunits. These five new proteins are not breakdown products of larval storage protein, because they do not react with our antiserum to the latter. Apparently these five protein subunits are induced by pyriproxyfen, indicating that they are derived from the native protein of 500 kDa. To prove this more convincingly, a native PAGE gel was run from samples of control and pyriproxyfen-treated animals. After staining with Coomassie Brilliant R250, the bands of molecular weight 500-510 kDa were sliced from the lanes of control and pyriproxyfen-treated samples and the stained proteins were electrophoretically eluted with a BIORAD Model 422 electro-elutor. After freeze-drying of the eluted proteins, the fractions were dissolved in 40 ~1 of SDS sample buffer by boiling for 2min and applied to SDS-gradient gel. After the run, the pro-
teins were blotted onto Immobilon and stained immunologically or with amido black. The results of the amido black staining are illustrated in Fig. 3. The lane containing the sample from controls showed only a subunit of molecular weight about 80 kDa, which responded positively to our larval storage protein I antibody. The same subunit was also present in the protein eluate (X in Fig. 3) from pyriproxyfen-treated animals, but at a much lower concentration. This eluate contained five additional bands that did not respond to the antiserum to storage protein I. The molecular weights of these five proteins were determined by comparison with standard proteins. The molecular weights were, respectively, 110 + 8 kDa (n = 4); 101.5 + 6.6 kDa (n = 4); 90.7 + 5.5 kDa (n = 3); 61.7 + 4.0 kDa (n = 3) and 56.3 f 3.5 kDa (n = 3). This experiment suggests that these five protein subunits are derived from the native protein of 500 kDa, which becomes a major protein in the haemolymph after treatment with pyriproxyfen. This protein remains a major haemolymph protein after the treated animals have moulted to the next stage (not shown). DISCUSSION
Pyriproxyfen affects the protein pattern of the haemolymph and has strong juvenilizing effects when applied to last-instar larvae of L. migratoriu. The observed morphological effects are comparable with previous experiments using implanted corpora allata (Staal, 1961), natural juvenile hormones or analogues (Roussel, 1977). Our results differ from those described by Staal (1961) and Roussel (1977), who claimed that the larvae are most sensitive during the first 3 days of the instar. We found that treatment on day 4 is still effective, though the total duration of the instar under our rearing conditions was only 6 days, even with 2pg. This makes pyriproxyfen one of the most effective juvenile hormone analogues known for locusts. Synthesis of larval storage protein I (de Kort and Koopmanschap, 1987; Ancsin and Wyatt, 1989) is dramatically repressed, but the synthesis of another high-molecular-weight protein not previously noticed by us, was stimulated in a dosedependent manner. This protein was further characterized. Its molecular weight was estimated by native PAGE and appeared to be about 500 + 13 kDa. After denaturation by boiling with sample buffer containing mercaptoethanol, the protein dissociated into five subunits with molecular weights of 110, 101, 91, 62 and 56 kDa, respectively. All five subunits were induced by pyriproxyfen in a dose-dependent manner. As shown in Fig. 2(A), the largest subunit (110 kDa) is already clearly visible at a dose of 2 pg whereas the four other subunits are still rather weak. The intensity of all the bands clearly increased after treatment with doses of 10 and 5Opg.
LSP
LSP X
X
LP
LP 4
3
2
1
Fig. 1. Electropherogram in 1% agarose of total haemolymph of last-instar larvae of L. migratoria 3 days after injection of different doses of pyriproxyfen. Lane 1, control; lane 2,2 pg; lane 3,10 ng; lane 4,SO pg. Lp, lipophorin; LSP, larval storage protein; X, unknown protein (identified as vitellogenin in collaboration with Ancsin and Wyatt).
LMW
wo
0
2
10
50
0
2
10
50
pLs
IPI
94 67
Zig. 2. Western blots from SDS gels of total haemolymph from last-instar larvae of L. migratoria after injection of 2, 10 or 5Ong pyriproxyfen. The blots were stained with (A) amid0 black or (B) immunologically with anti-larval storage protein I serum followed by colloidally gold-labelled protein A. LMW, low-molecular-weight standard proteins. Apolp I, apolipophorin I.
91
LSP
LMW
94K
67 K
43 K
30 K 20 K 14 K
Fig. 3. Western blot from SDS gel of larval storage protein I and protein band X electro-eluted from native PAGE after staining with Coomassie R-250. The blot was stained with amido black. LMW, low-molecular-weight standard protein, LSP, larval storage protein; X, pyriproxyfen-stimulated protein band (vitellogenin, Ancsin and Wyatt, personal communication).
92
Haemolymph from last-&tar larvae of L. migratoria This protein is not identical with persistent storage protein recently discovered by Ancsin and Wyatt (1989), because the latter is composed of only two subunits of molecular weight of 74 and 77 kDa, respectively. The protein was identified by Ancsin and Wyatt, who independently obtained similar results with pyriproxyfen in locust larvae. It appeared to be vitellogenin, which is precociously induced in larvae by this juvenile hormone analogue. Dhadialla and Wyatt (1983) have previously shown that vitellogenin can be induced in larvae by methoprene, but high doses (> 100 /*g) were required to obtain this effect. Using a similar concentration range, Ancsin and Wyatt (personal communication) recently showed by rocket immuno-electrophoresis that vitellogenin becomes a major haemolymph protein after application of pyriproxyfen to last instar larvae. A number of questions still remains to be answered regarding the molecular composition and post-translational processing of this precociously induced larval vitellogenin as compared with vitellogenin from adult females. ‘Thus, pyriproxyfen represses the synthesis of larval storage protein 1 but stimulates the premature synthesis of vitellogenin, which becomes a major protein of the haemolymph in larvae and remains so after the moult to adultoid. From our native electropherograms, we could deduce that another larval protein band (storage protein II; Ancsin and Wyatt, 1989) of about M, 250 kDa (de Kort and Koopmanschap, 1987) disappears after pyriproxyfen treatment, but this protein was not studied further. Acknowledgements-The
exchange of unpublished data with Mr J. B. Ancsin and Dr G. R. Wyatt (Department of
Biology, Queens University, Kingston, Ontario) was highly appreciated. We thank Dr H. Oouchi, Sumitomo Chemical Co., Ltd, Osaka, Japan, for providing the pyriproxyfen and Mr J. C. Rigg, Pudoc, Wageningen, for checking the English. REFERENCES
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Thesis Wageningen, The Netherlands. Wyatt G. R. (1989) How do juvenile hormone and juvenoid IGR’s work at the molecular level? Abstr. 5th Int. Congr. Invertebrate Reproduction, Nagoya, Japan. Wyatt G. R. (1990) Locust hemolymph proteins and effects of juvenile .hor&one. In Inset; koIe&ar Science (Eds Hagedorn H. H., Hildebrand J. G., Kidwell M. G. and Law J. H.). Plenum Press, New York.