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TMOF-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens.

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Archives of Insect Biochemistry and Physiology 47:169–180 (2001)
TMOF-Like Factor Controls the Biosynthesis of Serine
Proteases in the Larval Gut of Heliothis virescens
Ralf Nauen,1 Dorian Sorge,1 Andreas Sterner,2 and Dov Borovsky2*
1
Bayer AG, Agrochemicals Division, Research Insecticides, Leverkusen, Germany
2
University of Florida-IFAS, FMEL, Vero Beach
Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity
increases immediately after the molt from the 3rd to the 4th
instar larvae and declines just before the molt into the 5th
instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI)
4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosylL-phenylalanine chloromethylketone (TPCK) indicate that
protease activity is mostly trypsinlike (80%) with a small
amount of chymotrypsinlike activity (20%). Injections of late
3rd and 4th instar larval hemolymph into H. virescens larvae
inhibited tryspin biosynthesis in the larval midgut. Similar
results were obtained when highly purified 4th instar larval
hemolymph that crossreacted with Aea-TMOF antisurm using
ELISA was injected into 2nd instar larvae. Injections of AeaTMOF and its analogues into 2nd instar, and Aea-TMOF alone
into 4th instar larvae stopped trypsin biosynthesis 24 and 48
h after the injections, respectively. Injections of 4th instar H.
virescens larval hemolymph into female Aedes aegypti that
took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsinlike enzymes in the midgut of a lepidoptera is modulated with
a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch. Insect Biochem. Physiol. 47:169–180,
2001. © 2001 Wiley-Liss, Inc.
Key words: trypsin; chymotrypsin; Aea-TMOF; proteases; midgut; Lepidoptera; Heliothis virescens
INTRODUCTION
Protein digestion in Heliothis virescens is
mediated by endopeptidases and exopeptidases
secreted from the midgut epithelium cells into the
luminal fluid of the midgut (Terra, 1990). There
is considerable interest in characterizing proteolytic enzymes from agricultural pest insects in
order to develop genetically engineered insect crop
resistant plants that carry factors that modulate
serine proteases.
© 2001 Wiley-Liss, Inc.
Ralf Nauen and Dov Borovsky contributed equally to this
work.
Contract grant sponsor: NIH; Contract grant number: AI
41254, NATO CRG 940057; Contract grant sponsor: U.S. Israel Binational Science Foundation; Contract grant sponsor:
Insect BioTechnology Inc.; Contract grant sponsor: Bayer A.G.
*Correspondence to: Dov Borovsky, University of FloridaIFAS, FMEL, 200 9th St. SE, Vero Beach, FL 32962.
E-mail: dobo@gnv.ifas.ufl.edu
Received 12 December 2000; Accepted 1 April 2001
170
Nauen et al.
The use of genes encoding plant protein protease inhibitors (PI) has been tried (Hilder et al.,
1987; Johnson et al., 1989; Ryan, 1990); however,
no successful control of insects fed on these transgenic plants was achieved. An explanation to these
results is that larvae of Helicoverpa zea (corn ear
worm) and Lymantria dispar (gypsy moth) produce
large quantities of trypsin-like enzymes that are resistant to natural and genetically engineered protease inhibitors that are expressed in plants
(Broadway, 1995). These observations were also confirmed in other phytophagous insect pest species,
i.e., Spodoptera exigua (beet army worm), which
uses serine proteases as its major digestive enzyme,
and the coleopteran Leptinotarsa decemlineata
(Colorado potato beetle), which uses aspartate and
cysteine proteases (Jongsma et al., 1995; Bolter and
Jongsma, 1995).
A second approach to control agricultural pest
insects is to develop transgenic plants that produce
a bacterial toxin protein such as Bacillus thuringiensis δ-endotoxins (Vaeck et al., 1987). However,
the high production of these toxins in plants has
caused rapid resistance in insects to these proteins
(Alstad and Andow, 1996). TMOF, isolated from the
dipteran ovary, was shown to be the physiological
signal that terminates trypsin biosynthesis in mosquitoes and fleshflies (Borovsky et al., 1990, 1993,
1994, 1996; Bylemans et al., 1994). These observations indicate that insects control their digestion
with natural hormones that could be used, if identified, to control digestion in many agricultural pest
insects that use serine proteases as their main digestive enzymes (Lehane et al., 1995). In Neobellieria bullata, TMOF was shown to control trypsin
biosynthesis through a translational control of the
trypsin gene (Borovsky et al., 1996). We tested the
effect of Aea-TMOF and a highly purified TMOFlike peptide from the hemolymph of H. virescens
larvae, on trypsin biosynthesis in Heliothis and mosquitoes in order to determine if H. virescens, which
uses serine proteases as the main digestive enzymes, controls these enzymes with a TMOF-like
hormone(s).
MATERIALS AND METHODS
Insects
Larvae of H. virescens (Lepidoptera: Noctuidae) were synchronized at head-capsule slippage
from 3rd to 4th instar and raised individually in
24-well tissue culture plates (Falcon, Lincoln
Park, NJ) on an artificial diet. Under these conditions, the first and second instar stages are 2
days each, and the 4th instar stage is 4–5 days.
Chemicals
Azocoll, general protease substrate, was purchased from Calbiochem (Bad Soden/Ts., Germany),
bovine serum albumin (BSA), N-benzoyl-L-arginine p-nitroanilide (BApNA), Triton-X-100, SBTI
(Soybean Trypsin Inhibitor), BBI (Bowman-BirkInhibitor), AEBSF (4-(2-Aminoethyl)benzensulfonylfluoride) and TPCK (N-Tosyl-L-Phenylalanine
chloromethylketone) from Sigma (St. Louis, MO).
Aea-TMOF H-Tyr-Asp-Pro-Ala-(Pro)6 -COOH, Mr
1047.18, 97% pure) was obtained from Insect Biotechnology Inc. (Durham, NC). All other chemicals
were obtained from Serva (Heidelberg, Germany).
Isolation of Midguts and Extraction of Larval
Proteinases
Midguts were excised from cold-anaesthetized or etherized larvae, washed and transferred
into ice-cold 0.15 M NaCl, and homogenized in
Eppendorf tubes with a hand homogenizer. The
homogenates were centrifuged at 14,000g and the
supernatants filtered by centrifugation (Millipore
Ultrafree-MC, Millipore Corp., Bedford, MA) and
stored at –20°C until used. Protein concentrations
in the extracts were determined according to
Bradford (1976) using bovine serum albumine
(BSA) as a standard.
Enzyme Assay
Protease activity was determined using a
proteinase substrate Azocoll 1% (w/v) in 0.1 M
borate/NaOH buffer, pH 10.0. Trypsin activity was
measured with N-benzoyl-L-arginine p-nitroanilide (BApNA) (Borovsky and Schlein, 1988).
Reactions were incubated with 50 µl of midgut
extracts containing either 40 µg protein or a
known number of midgut-equivalents for 75 min
(Azocoll) or 30 min (BApNA) at 30°C. Controls
were incubated without midgut extracts, to determine nonenzymatic hydrolysis of substrates.
After incubations, reactions containing Azocoll
were chilled on ice for 5 min, centrifuged for 10
min at 16,000g in an Eppendorf microfuge, and
the absorbance of the supernatant was measured
Control of Serine Proteases in H. virescens With TMOF
at 520 nm. To determine the effect of various inhibitors on proteolytic activity, midgut-extracts
were pre-incubated for 20 min at room temperature with the inhibitors in assay-buffer before the
addition of the substrate. Controls were pre-incubated without inhibitors with water for SBTI
and AEBSF or water:ethanol (1:10) for TPCK.
Polyacrylamide Gel Electrophoresis (PAGE)
SDS PAGE was run using a vertical slab apparatus (Biometra Minigel, Biometra Gottingen,
Germany) (Laemmli, 1970) without Dithiothreitol
(DTT) and the samples were not heated to avoid
denaturation of proteases. Aliquots of midgut extracts were mixed with an equal volume of sample
buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20%
glycerol, 2 mM EDTA, 0.02% bromophenol blue)
and were run at 4°C on a 12% acrylamide gel at
14 mA. After electrophoresis, the gel was transferred to a glass plate. An indicator gel of 12%
polyacrylamide containing 0.15% BSA was laid
over it and a second glass plate was placed on
the top of the gel. The resulting gel sandwich was
held in place by two clamps and was incubated
in 0.1 M Tris-HCl, pH 8.5, buffer at room temperature for 21 h to allow diffusion of proteins
from the electrophoresis gel into the indicator gel.
After the transfer, the indicator gel was incubated
with 2.5% (w/v) Triton X-100 for 1 h at room temperature and washed two times for 10 min each
with double distilled water and incubated with
0.1 M Tris-HCl, pH 8.5, for 1 h. The gel was then
stained with 0.1% (w/v) Coomassie brilliant blue,
dissolved in ethanol:acetic acid:water (45:10:45).
Proteolytic activity appeared as clear zones on a
dark blue background of non-digested BSA substrate after destaining the gel in 25% (v/v) ethanol and 8% (v/v) acetic acid.
Larval Feeding
Food consumption and intermoult development
were observed using 4th instar larvae of known age.
Insect growth was synchronized as follows: 3rd instar larvae showing head-capsule slippage were
weighed at the time of collection and individually
placed in 24-wells tissue culture plates, containing
artificial diet of known weight. After ecdysis to the
4th instar (usually happens within 24 h) larval
weight, developmental state, and diet weight were
monitored daily until the 5th instar molt.
171
Food was provided ad libitum and if necessary changed every 2 days to reduce microbial
contamination. To determine the time point of initial diet ingestion after ecdysis to the 4th instar,
larvae and diet were weighed 1, 3, and 6 h after
eclosion. Changes in larval weight were determined and food utilization was expressed as a percentage of weight gain vs. food intake.
Feeding of Trypsin Inhibitors and TMOF
AEBSF or SBTI (5 to 200 µg in 2 µl water
were adsorbed onto food pellets (45–60 mg each).
Control pellets were treated with 2 µl of water
without inhibitors. Food pellets were distributed
into 24-well tissue culture plates. Following eclosion, pre-weighed individual 4th instar larvae
were placed into each well. Since larvae consumed about 45–60 mg of diet every 24 h, food
was given to larvae every 24 h and weight increase of each larva was monitored at 24 and 48
h. Aea-TMOF (36 µg) in 3 µl water was applied
to food pellets (4 × 4 mm; 35–45 mg) that were
entirely consumed within 24 h. Control pellets
were adsorbed with 3 µl water and the pellets
were transferred into the wells of 24-wells tissue culture plates. Following eclosion, individual
pre-weighed 4th instar larvae were placed in
each well. New food pellets were given after 24
h and individual larvae, thus, consumed 72 µg
of TMOF within 48 h.
Injections
Aea-TMOF and its analogues. Third instar
larvae weighing 20–26 mg were injected with
TMOF and several synthetic analogues (1 µg to
1 ng) in 0.5 µl insect Ringer (86 mM NaCl, 5.4
mM KCL, 3 mM CaCl2.2H2O) into the ventral abdomen using finely drawn glass capillary tubes.
Newly molted 4th instar larvae were injected with
Aea-TMOF (36 and 72 µg) in 3 µl insect Ringer
solution with a 10-µl Hamilton-syringe into the
ventral abdomen. Controls were injected with 0.5
or 3 µl Ringer without TMOF and its analogues.
After injection, the larvae were weighed and
placed individually in 24-well trays containing
artificial diet. The weight development of each
larva was monitored after 24 and 48 h. Trypsin
activity in the midguts of 3rd and 4th instar larvae was measured at 24 and 48 h in 3 individual
guts or 3 groups of 3–4 guts.
172
Nauen et al.
Larval hemolymph. Hemolymph of 3rd instar larvae of H. virescens showing head capsule
slippage and late 4th instar larvae was collected
in chilled Eppendorf tubes by cutting off an abdominal leg. After centrifugation for 5 min at
16,300g, 5 µl hemolymph of 3rd instar larvae was
injected into newly molted 4th instar larvae using a Hamilton syringe. Controls were injected
using 5 µl insect Ringer solution. Both, hemolymph and insect Ringer contained very small
amounts (ng) of N-phenylthiourea (PTU). The
weight of injected larva was measured 24 and 48
h later. Hemolymph (0.5 µl) that was collected
from late 4th instar larvae was injected with a
finely drawn glass capillary into female Ae.
aegypti. When smaller hemolymph volumes were
used, i.e., 0.0625 to 0.25 µl appropriate dilutions
of the hemolymph were done in insect Ringer and
the final volume that was injected was 0.5 µl.
Hemolymph from late 4th instar larvae was also
highly purified using Sep Pak C18 reverse-phase
cartridge and HPLC (Borovsky et al., 1990).
TMOF active fractions were dried by Speed Vac
at 40°C, and rehydrated in insect ringer and 0.5
µl were injected into 3rd instar H. virescens lar-
vae. Twenty-four hours later, guts were dissected
out and trypsin biosynthesis was determined
(Borovsky and Schlein 1988).
Fig. 1. Comparison between larval gut protease activity,
food consumption and weight gain during the 4th instar development of H. virescens larvae. Results are expressed as
the average of 3 determinations ± S.E.M. Proteolytic activity is expressed as 100% on day 3, and as a percentage of
the maximal activity otherwise.
Determination of TMOF Concentration in the
Hemolymph
Hemolymph of late 4th instar larvae was
assayed for TMOF using ELISA (Borovsky et
al., 1992).
Statistical Analysis
Data were analyzed using the Student’s t-test.
RESULTS
Food Consumption, Weight Gain, and
Proteolytic Activity of 4th Instar Larvae
Under the conditions of these trials, the 4th
larval instar (L4) stage of H. virescens is 4 to 5
days. The relationship between an increase in insect weight, food intake, and proteolytic activity
was studied (Fig. 1). Initial feeding started 1 h
after the molt; however, food consumption, increase in weight, and proteolytic activity at 6 h
after ecdysis were low (Fig. 1). The first fecal
Control of Serine Proteases in H. virescens With TMOF
droppings were observed 6 h after the molt in
most of the larvae that were tested (data not
shown), indicating that 5 h are needed for the
food to pass through the digestive system. Six
hours after the 3rd to 4th instar molt, the weight,
food consumption, and proteolytic enzymes activity rapidly increased and reached a peak at day
two. In preparation for the next molt, body weight
decreased from day three onward, whereas food
consumption and proteolytic enzymes activity remained high until day four (Fig. 1). The decrease
in weight that occurred between the 3rd to the
4th larval stage and the 4th to the 5th larval
stage is due mostly to the shedding of the cuticle
(Fig. 1).
Effect of Serine Protease Inhibitors
In vitro. In order to find out if the major midgut proteolytic activity of the 4th larval instar is
trypsin-like, several trypsin inhibitors SBTI, BBI,
AEBSF, and chymotrypsin inhibitor TPCK were
incubated with larval midgut extracts and general protease substrate Azocoll (Fig. 2). Proteolytic
activity of days 1–3 of 4th instar larval gut extracts was 98 and 95% inhibited by 100 µM of
trypsin inhibitors SBTI and BBI, respectively
(Fig. 2). AEBSF (100 µM) inhibited 50% of the
proteolytic activity in the larval midgut extract
(Fig. 2). To inhibit 87% (day 1) to 92% (day 2) of
total proteolytic activity, 1 mM of AEBSF was
needed (data not shown), indicating that it is not
Fig. 2. Effect of different serine protease inhibitors, in vitro,
on midgut proteases of 4th instar H. virescens larvae. Protease activity is expressed as percentage of controls incubated without the inhibitors (100 µM). Results are expressed
as an average of 3 determinations ± S.E.M.
173
as good an inhibitor as SBTI or BBI. TCPK, a
specific chymotrypsin inhibitor, had little effect
on midgut proteolytic activity activity at day 1
and 3 (22 and 14% inhibition, respectively) and
no effect at day 2 (Fig. 2). These results indicate
that the major larval proteolytic activity in the
midgut of H. virescens is trypsin-like.
In vivo. The in vitro incubations of larval gut
extracts indicated that AEBSF and SBTI were effective in inhibiting trypsin-like activity. To find
out if these inhibitors were equally effective in
vivo, increasing amounts (5–200 µg) of the inhibitors were fed with the synthetic diet to larvae.
Feeding 400 µg of AEBSF in 2 days (200 µg/day)
to individual larvae caused 80% inhibition of total proteolytic activity (Fig. 3A) with a calculated
IC50 of 60.5 µg/day. Higher quantities above 200
µg could not be tested because larvae refused to
eat the artificial diet. SBTI was more effective
earlier causing 44 and 80% inhibition of proteolytic activity within 24 h when 5 and 10 µg
were fed, respectively (Fig. 3B). Higher doses up
to 200 µg did not cause more inhibition of proteolytic activity (Fig. 3B). Feeding SBTI caused
higher inhibition of proteolytic enzymes activity
at 24 than at 48 h (Fig. 3B). These results are
different than the AEBSF feeding in which 100
and 200 µg caused higher inhibition at 48 h than
at 24 h (Fig. 3A). Feeding increasing amounts of
AEBSF proportionally reduced larval growth.
Mean weight gain of 4th instar larvae feeding on
different amounts of AEBSF treated diet was at
48 h between 42 ± 2 (mg ± S.E.M.) and 19.7 ± 1
(mg ± S.E.M.) as compared to the weight of control larvae 62.6 ± 1.5 (mg ± S.E.M.) that were fed
the untreated diet. When 5 µg of AESBF was fed
daily to individual larvae, the weight gain decreased by 67% as compared with controls. After
feeding larvae daily 20 µg of AEBSF, the weight
gain at 48 h was 70% lower than in controls (Fig.
4). The weight gain of larvae that were fed SBTI
for 48 h was 85 to 100% similar to controls that
were fed on the untreated diet (Fig. 4). These results indicate that larvae that were fed SBTI for
48 h synthesized trypsin-like enzymes that were
not affected by SBTI.
Effect of Mosquito TMOF
To find out if trypsin biosynthesis in H.
virescens is regulated by a TMOF-like factor, 2nd
174
Nauen et al.
Fig. 4. Effect of feeding trypsin inhibitors AEBSF and SBTI
for 48 h on weight gain of 4th instar H. virescens larvae.
Weight gain is expressed as percentage of controls that were
fed artifical diet without the inhibitors. Results are the average of 3 determinations ± S.E.M.
Fig. 3. Effect of feeding trypsin inhibitors (A) AEBSF and
(B) SBTI on gut proteolytic activity of 4th instar H. virescens
larvae. Inhibition is expressed as percentage of controls that
were fed artificial diet without inhibitors. Results are expressed as an average of 3 determinations ± S.E.M.
and 4th instar larvae were injected with AeaTMOF (Table 1, Fig. 5). Injections of TMOF and
its analogues (1 µg to 1 ng) caused 50% inhibition of trypsin biosynthesis when 0.42 ± 0.026 (µM
± S.E.M.) TMOF was injected. When TMOF hexapeptide analogue (YDPAPP) was injected, 46 ± 2.8
(µM ± S.E.M.) caused 50% inhibition of trypsin
biosynthesis in 2nd instar H. virescens larvae.
This analogue was 109-fold less effective than the
decapeptide. The penta-peptide analogue in which
the Y was replaced with F was 1.3-fold less effective than the hexa-peptide (Table 1) and the tripeptide DPA caused 50% inhibition of trypsin
biosynthesis at a concentration of 50 ± 2 (µM ±
S.E.M.), which was similar to the effective concentrations of the hexa-peptide (Table 1). These
results indicate that H. virescens TMOF is probably very similar to mosquito TMOF, and trypsin
biosynthesis in 2nd instar larvae can be controlled
by a TMOF-like molecule. To find out if TMOF
can also inhibit trypsin biosynthesis in 4th instar larvae, TMOF (36 µg; 3 µl) was injected into
larvae and 72 µg of TMOF (36 µg every 24 h)
were fed to 3 groups of newly eclosed 4th instar
larvae. Controls were injected with insect Ringer
or were fed on a synthetic diet without TMOF.
Forty-eight hours later, midguts were removed
and analyzed for trypsin activity. In larvae that
were injected with the hormone, 70% of trypsin
activity and 61% of the weight gain were inhibited. On the other hand, in larvae that were fed
the hormone, only 30% of trypsin biosynthesis and
26% of the weight gain were inhibited (Fig. 5).
These results indicate that TMOF might be degraded in the gut, or it is not efficiently transported from the gut into the hemolymph.
Effect of Hemolymph
To find out if a TMOF-like molecule(s) is synthesized by H. virescens larvae and regulates
trypsin biosynthesis, hemolymph (0.25–0.5 µl)
from late 4th instar larvae was injected into blood
Control of Serine Proteases in H. virescens With TMOF
175
TABLE 1. Effect of TMOF and Its Analogues on
Trypsin Biosynthesis in H. virescens*
Analogue
N
YDPA(P)6
YDPAPP
FDPAP
DPAP
DPA
3
3
3
3
3
Inhibition of 50% trypsin
biosynthesis (In50)
(µM ± S.E.M.)
0.42
46
145
190
50
±
±
±
±
±
0.026
2.8
8
46
2
*Twelve groups of H. virescens (10 larvae per group) were
injected with 0.5 µl of water containing TMOF and its analogues (1 µg to 1 ng). Twenty-four hours later, guts were
removed and analyzed with BApNA for trypsin biosynthesis. Controls were injected with water and compared with
non-injected controls. Inhibition of trypsin activity was plotted against log concentrations of TMOF and its analogues
and 50% inhibition was read from this curve. Results are
expressed as an average of 3 determinations ± S.E.M.
fed females Ae. aegypti and 24 h later midguts
and ovaries were removed and analyzed for
trypsin biosynthesis and egg development (Table
2). In intact females that were injected with 0.25
µl and 0.5 µl of hemolymph, trypsin biosynthesis
was inhibited by 65 ± 18 (% ± S.E.M.) and 84 ±
10 (% ± S.E.M.), respectively, and egg development was inhibited by 44 ± 2 (% ± S.E.M.) and
100 ± 0 (% ± S.E.M.), respectively (Table 2). To
find out if the TMOF-like factor from the hemolymph acts on the gut or affects the release of
neuroendocrine factors from the brain or the thorax, female mosquitoes were fed a blood meal.
One group was immediately decapitated and a
second group was immediately ligated between
the thorax and abdomen. Injection of H. virescens
hemolymph into both groups caused 45.6 ± 14 (%
± S.E.M.) and 100 ± 0 (% ± S.E.M.) inhibition of
trypsin biosynthesis (Table 2). The enhanced inhibition of trypsin biosynthesis in ligated abdomens indicates that the Heliothis TMOF might
be structurally similar to mosquito TMOF, which
is rapidly degraded in the thorax (Borovsky et
al., 1993). To find out if the effect of the hemolymph and TMOF on trypsin biosynthesis in
Heliothis can be sustained for 3 days, 2nd instar
larvae of H. virescens (9 larvae/group) were injected with 4th instar larval hemolymph (0.25
mosquito TMOF (5 µg in 0.25 µl) and insect
Ringer. Three days later, each group was analyzed
for trypsin biosynthesis (Fig. 6). Trypsin biosynthesis 3 days after the injections of hemolymph
Fig. 5. Effect of feeding and injecting Aea-TMOF on trypsin
biosynthesis in the midgut of 4th instar H. virescens larvae.
Larvae were fed 72 µg of TMOF (36 µg every 24 h) or were
injected with 36 µg of TMOF dissolved in insect Ringer immediately after the molt to the 4th instar. Controls were
fed artificial diet without TMOF or injected with insect
Ringer. Forty-eight hours later, guts were removed and assayed for trypsin activity using BApNA. Results are expressed as percentage inhibition as compared to controls and
are an average of 3 determinations ± S.E.M.
and TMOF, was 70 and 49% inhibited in the midguts of injected larvae, respectively, as compared
with controls that were injected with saline (Fig.
6). When hemolymph from late 3rd instar larvae
was injected into 4th instar larvae, and midgut
extracts were separated by PAGE (Fig. 7), 60 and
80% of the proteolytic activity in the midgut was
lost 24 and 40 h, respectively, after the injections.
(Fig. 7, lanes B and D) as compared with salineinjected controls (Fig. 7, lanes A and C). To find
out if the inhibition was caused by a mosquito
TMOF-like molecule, hemolymph of H. virescens
was highly purified by HPLC (Borovsky et al.,
1990). A fraction eluted between 60 to 70% acetonitrile 0.1% TFA that cross-reacted with TMOF
antiserum by ELISA (Borovsky et al., 1992), was
dried in a Speed Vac, rehydrated in insect Ringer,
and 0.5 µl (equivalent to 0.25 µl of single 4th instar larval hemolymph) was injected into second
176
Nauen et al.
TABLE 2. Effect of H. virescens Hemolymph on Trypsin Biosynthesis in Ae. aegypti*
Female Ae aegypti were
fed blood and immediately:
N
Trypsin synthesis
(ng/gut ± S.E.M.)
Inhibition
(% ± S.E.M.)
Yolk size
(µm ± S.E.M.)
Injected with
0.5 µl hemolymph
0.25 µl hemolymph
0.5 µl insect Ringer
3
3
3
93 ± 11
200 ± 55
661 ± 15
84 ± 10
65 ± 18
0
17 ± 5.6
111 ± 5.6
178 ± 6
Decapitated and injected with
0.25 µl hemolymph
0.25 µl insect Ringer
3
3
194 ± 29
425 ± 61
45.6 ± 14
0
Ligated and injected with
0.25 µl hemolymph
0.25 µl insect Ringer
3
3
0
400 ± 45
100 ± 0
0
Inhibition
(% ± S.E.M.)
100 ± 0
44 ± 2
0
*Three groups of female Ae. aegypti (10 per group) were fed a blood meal, one group was kept intact, a second group was
immediately decapitated, and a third group was immediately ligated between the thorax and abdomen. Females were
injected with Heliothis hemolymph and controls were injected with insect Ringer. Nineteen hours after injections, midguts
and ovaries were dissected out (3 per group) and analyzed for trypsin biosynthesis in midguts (Borovsky and Schlein,
1988) and yolk length in oocytes of intact females (Borovsky et al., 1990). Results are expressed as means of 3 determinations ± S.E.M.
instar larvae (Table 3). Twenty-four hours later,
midguts were assayed for trypsin biosynthesis.
Trypsin biosynthesis was significantly inhibited
54 ± 5 (% ± S.E.M.) in larvae that were injected
with purified hemolymph. When insect Ringer solution was injected, a significant increase of 2.2fold in trypsin activity was observed 24 h after
the injection as compared with a control group
that was assayed before the injections (Table 3).
On the other hand, no significant difference was
found between the control group and the hemolymph-injected larvae (Table 2). These results
strongly indicate that H. virescens larvae regulate trypsin biosynthesis in the gut with TMOFlike factor(s).
DISCUSSION
Fig. 6. Effect of injections of 4th instar larval hemolymph
and Aea-TMOF on trypsin biosynthesis in the midgut of 2nd
instar H. virescens larva. Hemolymph (0.25 µl) of 4th instar
larvae, TMOF (5 µg in 0.25 µl saline) and saline (0.25 µl)
were injected into 2nd instar larvae and 3 days later guts
were analyzed for trypsin biosynthesis (Borovsky and
Schlein, 1988). A control group prior to the injections was
also analyzed. Results are expressed as an average of 3 determinations ± S.E.M.
Synthesis of trypsin in blood-fed insects that
take a single protein meal to produce egg yolk
proteins is cyclical. After the blood meal, trypsinlike enzymes are synthesized, reach a peak 24–
30 h after the blood meal, and then the synthesis
declines, reaching a low point 56 to 72 h later
(Borovsky, 1988). The down-regulation of trypsin
biosynthesis in mosquitoes is controlled by TMOF
(Borovsky, 1988, Borovsky et al., 1990, 1993, 1994,
1996). A similar mechanism has not been reported
in lepidopteran larvae that continuously feed.
When 4th instar larvae of H. virescens were fed
a meal, proteolytic enzymes activity increased
from a very low level immediately following
Control of Serine Proteases in H. virescens With TMOF
177
TABLE 3. Effect of Purified H. virescens Hemolymph
on Trypsin Biosynthesis†
Second instar
larvae
Injected with
purified
hemolymph
(0.5 µl) and
assayed 24 h
later
Controls
Injected with
insect Ringer
(0.5 µl) and
assayed 24 h
later
Not injected,
and assayed
immediately
N
Trypsin
(nmol/min/gut
± S.E.M.)
Inhibition
(% ± S.E.M.)
3
6.6 ± 0.6*,**
54 ± 5
3
9.4 ± 1.2*,***
0
3
4.25 ± 1.4**,***
—
†
Hemolymph of late 4th instar larvae was partially purified
on C18 Sep Pak column and HPLC (Borovsky et al., 1990),
dried in a Speed Vac, and rehydrated in insect Ringer.
Aliquots (0.5 µl) were injected into 2nd instar H. virescens
larvae weighing 20 to 25 mg each. Midguts were removed
24 h later and analyzed for trypsin activity (Borovsky and
Schlein, 1988).
*Significant difference 0.025 < P < 0.05. **Not significant
0.1 < P < 0.375. ***Significant difference 0.01 < P < 0.025.
Fig. 7. PAGE of midgut proteases of H. virescens 4th instar larvae that were injected with saline (5 µl) or 3rd instar larval hemolymph (5 µl) and analyzed 24 and 40 h later.
Each lane was loaded with 0.02 midgut equivalents and the
PAGE was analyzed for protease activity on an indicator
gel. Light color bands indicate protease activity, 24 h after
injections, (lane A) saline, (lane B) hemolymph, and 40 h
after injections, (lane C) saline and (lane D) hemolymph.
ecdysis, reached a peak in mid instar (days 1–3),
and declined at the end of the instar. The increase
and decrease in proteolytic enzymes biosynthesis
followed food consumption, which was maximal
on days 1–3 and very low before and after ecdysis
(Fig. 1), and showed a similar pattern to that observed in mosquitoes (Borovsky, 1988). Similar
results were obtained when gut extracts were incubated with the amidolytic substrate BApNA.
Although BApNA can be hydrolyzed by other proteases that are not trypsin-like, there is now evidence that most midgut enzymes that hydrolyze
BApNA are trypsin-like (Terra and Ferreira,
1994). To find out the relative amounts of trypsin
and chymotrypsin-like enzymes in the midgut of
H. virescens 4th instar larvae, trypsin inhibitors
SBTI, BBI, AEBSF, and chymotrypsin inhibitor
TPCK were incubated in vitro with a general proteinase substrate Azocoll. General proteolytic activity was almost completely inhibited by trypsin
inhibitors SBTI and BBI but only 52% by AEBSF.
Low inhibition was observed with TPCK, indicating that chymotryptic activity is low in H. virescens and the majority of the enzymes in the
larval midgut on days 1–3 of the 4th instar are
trypsin-like (Fig. 2). SBTI was reported to exhibit
a high level of inhibition in vivo on H. virescens
(Johnston et al., 1995) and on several other lepidopteran species (Johnston et al., 1991; Christeller et al., 1992; Purcell et al., 1992; Ferreira et
al., 1994; McManus and Burgess, 1995; Novillo
et al., 1999) and low level of inhibition on chymotrypsins from lepidopteran larvae (Christeller
et al., 1992). Although there are several reports
that chymotrypsins from several insect species are
not sensitive to TPCK (Johnston et al., 1991;
Gatehouse et al., 1999; Valaitis et al., 1999) and
that TPCK is more stable at neutral pH (Lee and
Anstee, 1995), our results indicate that chymotrypsin activity is no more than 20% of the total
178
Nauen et al.
trypsin activity using BApNA, trypsin specific
substrate, and BTpNA, chymotrypsin specific
substrate (Borovsky, unpublished observations).
Gene sequencing of both enzymes and Northern
analysis also confirmed that the chymotrypsin
transcript is less than 20% of the trypsin transcript (Borovsky, unpublished observations).
These results contradict an earlier report that
chymotrypsin activity accounts for 50% of total
proteolytic activity in H. virescens (Johnston et
al., 1995). In Helicoverpa armigera it is thought
that trypsin and chymotrypsin activities play an
equal role in proteolysis (Bown et al., 1997),
whereas the chymotryptic activity of the tomato
moth, Lacanobia oleracea, accounted for 30% of
the total proteolytic activity (Gatehouse et al.,
1999). The data presented by Johnston et al.
(1995) are not comparable to our results because
the larvae in the previous studies were not standardized as to age and phase within an instar.
It is possible that enzyme levels may vary in different phases of larval development (Christeller
et al., 1992; Keller et al., 1996; Novillo et al.,
1999). It has been shown that several herbivorous insects adjust to the presence of protease
inhibitors in their diet by producing novel inhibitor resistant trypsins in their midguts (Broadway, 1995; Jongsma et al., 1995; Wu et al., 1997;
Bown et al., 1997; Gatehouse et al., 1997; Giri
et al., 1998). Feeding of SBTI in a diet for 30
min stimulated the synthesis of SBTI resistant
enzymes in newly molted 5th instar larvae of H.
zea and Agrotis ipsilon and the enzymes persisted
even if the insects were not fed proteinase inhibitor (Broadway, 1997). Our results from feeding larvae with a SBTI-containing diet suggest
that a similar mechanism exists in H. virescens
(Figs. 3,4). The higher levels of proteolytic activity observed 48 h after feeding SBTI are due to
the induction of SBTI-insensitive serine proteases. This also explains why the increase in
larval weight was almost unaffected by SBTI
(Fig. 4). Failure to affect H. virescens larval
growth was reported by Gatehouse et al. (1994)
after feeding transgenic tobacco plants expressing SBTI, despite the fact that larval tryspsin
was completely inhibited by SBTI in vitro. Bown
et al. (1997) showed that feeding SBTI to H.
armigera larvae caused preferential induction of
trypsin mRNA transcripts that encoded trypsin
insensitive SBTI. Thus, these results suggest
that inhibitors that arrest enzyme activity might
also stimulate over-production of the same enzymes or stimulate over-production of mRNA
transcripts that are usually rare but, in the presence of an inhibitor, become the preferred transcripts. Borovsky (1988) showed that trypsin
synthesis in mosquitoes is regulated by a factor
that is synthesized by the mosquito ovary. A
decapeptide was later purified and sequenced
from the mosquito ovaries and was named TMOF
(Borovsky et al., 1990, 1991). The hormone terminates the biosynthesis of trypsin-like enzymes
in mosquitoes and several other insects (Borovsky et al., 1990, 1992). In the fleshfly, a
hexapeptide named Neb-TMOF was isolated, sequenced, and shown to have a translational control of trypsin mRNA (Bylemans et al., 1994;
Borovsky et al., 1996). Since the majority of the
proteases in the midgut of H. virescens are
trypsin-like, our results show that trypsin biosynthesis in Lepidoptera is regulated by TMOF
like factor(s) circulating in the hemolymph. When
Aea-TMOF and several of its analogues were injected into 2nd instar larvae, trypsin biosynthesis was inhibited 50% by 0.42 µM TMOF, whereas
other analogues of shorter chain length were 100to 452-fold less effective, indicating that H.
virescens TMOF might be structurally similar to
Aea-TMOF. Indeed, injections of 4th instar larval
hemolymph and highly purified hemolymph into
blood-fed female A. aegypti stopped trypsin biosynthesis and egg development (Table 2). H.
virescens hemolymph factor, like mosquito TMOF,
acts directly on the midgut; ligation and decapitation did not prevent the factor from directly inhibiting trypsin biosynthesis in the midgut and
indirectly inhibiting egg development (Borovsky et
al., 1990, 1993). To prevent the possibility that the
hemolymph carries toxic substances that indirectly
may cause these effects, a highly purified hemolymph fraction that exhibited crossreactivity with
Aedes TMOF antibodies by ELISA (Borovsky et al.,
1992) was injected and significantly inhibited
trypsin biosynthesis in 2nd instar larvae of H.
virescens (Table 3). This is the first report that
shows that A. aegypti TMOF like factor is circulating in the hemolymph of late 4th and 3rd instar
H. virescens larvae and it has a role in regulating
trypsin biosynthesis in the larval gut.
Control of Serine Proteases in H. virescens With TMOF
ACKNOWLEDGMENTS
This work was partially supported by NIH AI
41254, NATO CRG 940057, U.S. Israel Binational
Science Foundation, Insect BioTechnology Inc.,
Durham, N.C. and Bayer A.G. grants to professor
Dov Borovsky. This paper is part of University of
Florida, Florida Agricultural Experimental Station
Journal Series No. R-07903
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