1471 Y. Park, Y. Kim Journal of Insect Physiology 46 2000 1469–1476
Germany under a phase contrast microscope. Three lar- vae were used at each time per treatment.
2.4. Nodulation assay At predetermined times after bacterial injection, the
test larvae were killed and kept at 270 °
C until assessed. For the nodulation assay, larvae were dissected by open-
ing the haemoceol. Melanized and dark nodules were counted under a stereomicroscope at 50-fold magnifi-
cation. Even though their sizes varied, the nodules were distinct enough to determine their locations.
To determine the time course of nodule formation, the test larvae were injected with 10
6
cells of heat-killed bacteria 60
° C for 20 min and kept at 25
° C for seven
different incubation times. Control larvae were injected with 2
µ l of sterile Ringer’s solution. Each treatment
consisted of nine test larvae. To determine the dose response of nodule formation,
the test larvae were injected with different doses of live or heat-killed bacteria and incubated for 16 h at 25
° C.
Each treatment consisted of nine test larvae. 2.5. Pharmacological treatment
Test insects were injected with 2 µ
l containing 10 µ
g of each pharmaceutical agent. Injection was conducted
with a 10 µ
l Hamilton micro-syringe through the abdominal proleg which was on the opposite side to that
of the bacterial injection. Control larvae were injected with 95 ethanol.
All test chemicals were purchased from Sigma St Louis, MO, USA. They include arachidonic acid
5,8,11,14-eicosatetraenoic acid, the specific phospho- lipase A
2
inhibitor dexamethasone 11 b,16a-9-fluoro-
11,17,21-trihydroxy-16-methylpregna-1,4-dione, the
cyclooxygenase inhibitor naproxen d-2-6-methoxy-2- naphthyl propionic acid, the lipoxygenase inhibitor
esculetin 6,7-dihydroxycoumarin and the dual cycloox- ygenase and lipoxygenase inhibitor phenidone 1-phe-
nyl-3-pyrazolidinone.
2.6. Data analysis Survival data were transformed by the square root and
arcsine method for normalization. Treatment means and variances of the transformed data were analyzed by
PROC GLM of the SAS program SAS Institute Inc., 1988.
3. Results
3.1. Pathogenicity of X. nematophilus to S. exigua The insecticidal effect of X. nematophilus was ana-
lyzed in vivo in the fifth instar larvae of S. exigua Fig.
Fig. 1. Pathogenicity of Xenorhabdus nematophilus to the fifth instar
larvae of Spodoptera exigua. The larvae injected intrahaemocoelically with different colony forming units cfu of the bacteria were kept at
25 °
C for 24 h and then assayed for mortality. Each measurement con- sisted of 36 larvae with three replications.
1. Mortalities were dependent on the bacterial doses. The slope of the dose–mortality regression was 2.6
± 0.8
and the median lethal dose of the bacteria LD
50
was estimated as 33 cfu per larva. To give 100 mortality
to the host insects, more than 325 live bacterial cells were required to be injected per larva.
Change in the total numbers of live haemocytes in fifth instar larvae of S. exigua was traced with different
time intervals after bacterial injection Fig. 2. Nonim- munized larvae had |970 000 live haemocytes per ml
of haemolymph. The bacteria had a significant cytotoxic effect
on the
haemocytes F
= 229.31;
df =
8, 18;
P =
0.0001. After 24 h post-injection, the larvae had only 55,000 live haemocytes per ml haemolymph, which rep-
resented ca. a 94 haemocyte density reduction. In con- trast, did not change the total numbers of live haemo-
cytes in the controls F =
0.43; df =
8, 18; P =
0.8903. Significant cytotoxicity t
= 17.64; df
= 4; P
= 0.0001 was
found 4 h postinjection, which was the earliest measure-
Fig. 2. Cytotoxic effect of Xenorhabdus nematophilus on the haemo-
cytes of the fifth instar larvae of Spodoptera exigua. The larvae injected haemocoelically with the bacteria were kept at 25
° C until the
haemocytes were assayed. Each measurement consisted of three larval samples. The vertical bars represent the standard deviations.
1472 Y. Park, Y. Kim Journal of Insect Physiology 46 2000 1469–1476
Fig. 3. Arachidonic acid ARA, 10
µ llarva rescues the fifth instar
larva of Spodoptera exigua infected with Xenorhabdus nematophilus at a dose of LD
80
. Controls were injected with 95 ethanol EtOH instead of arachidonic acid. Mortality was measured at 24 h after the
haemocoelic injection of the bacteria. Each treatment consisted of 20 larvae with two replications. The error bars represent the standard devi-
ations. Different letters above the error bars were significantly different at
a =
0.05.
ment time in this assay. All treated larvae had died by 16 h after the bacterial injection.
3.2. Effect of arachidonic acid on the pathogenicity of X. nematophilus
The importance of arachidonic acid was analyzed in the immunity of fifth instar larvae of S. exigua against X.
nematophilus. The insecticidal action of X. nematophilus decreased significantly by the addition of 10
µ g of arach-
idonic acid injected into bacterial-infected larvae Fig. 3.
To test the dose effect of arachidonic acid on the bac- terial insecticidal action, 10-fold dilutions of arachidonic
acid stock were prepared and compared Fig. 4. There was a dose-dependent response up to 10
µ g of arachi-
donic acid. The higher dose 100 µ
g did not, however, have a significant effect on rescuing the infected larvae.
Fig. 4. Dose effect of arachidonic acid on the pathogenicity of Xenor-
habdus nematophilus to the fifth instar larvae of Spodoptera exigua. All larvae were injected with an LD
80
dose of bacteria after arachidonic acid treatments. Mortality was measured 24 h after the bacterial injec-
tion. Each measurement consisted of 20 larvae with two replications. The error bars represent the standard deviations. Different letters above
the error bars were significantly different at a
= 0.05.
Fig. 5. Synergistic effect of dexamethasone DEX, a specific inhibi-
tor of phospholipase A
2
, on the pathogenicity of Xenorhabdus nemato- philus to the fifth instar larvae of Spodoptera exigua. All larvae were
injected with an LD
20
dose of bacteria after being treated with different inhibitors. Mortality was measured 24 h after the bacterial injection.
Each measurement consisted of 20 larvae with two replications. The error bars represent the standard deviations. Different letters above the
error bars were significantly different at a
= 0.05.
3.3. Effect of eicosanoid biosynthesis inhibitors on the pathogenicity of X. nematophilus
The effects of eicosanoid biosynthesis inhibitors were examined to test the importance of eicosanoids in insect
immunity and to determine whether the insecticidal action of X. nematophilus inhibits the eicosanoid path-
way.
Dexamethasone DEX, a specific inhibitor of phos- pholipase A
2
, enhanced the insecticidal effect of X. nem- atophilus on the fifth instar larvae of S. exigua Fig. 5.
This synergistic DEX effect on X. nematophilus was dose-dependent, but did not change above 10
µ g.
All the other eicosanoid biosynthesis inhibitors tested in this study also increased the lethal effect of X. nema-
tophilus Fig. 6. There were no significant differences among the effects of the individual inhibitors such as
esculetin, phenidone, and naproxen. The effects of all
Fig. 6. Effect of other eicosanoid biosynthesis inhibitors all 10
µ glarva on the pathogenicity of Xenorhabdus nematophilus to the
fifth instar larvae of Spodoptera exigua. All larvae were injected with an LD
20
dose of bacteria after being treated with different inhibitors. Mortality was measured at 24 h after the bacterial injection. Each
measurement consisted of 20 larvae with two replications. The error bars represent the standard deviations. Different letters above the error
bars were significantly different at a
= 0.05. Abbreviations: esculetin,
ESC; phenidone, PHE; naproxen, NAP.
1473 Y. Park, Y. Kim Journal of Insect Physiology 46 2000 1469–1476
Fig. 7. Time-course of nodule formation in the fifth instar larvae of
Spodoptera exigua in response to haemocoelic injection of heat-killed Xenorhabdus nematophilus 10
6
cellslarva. Controls were injected with sterile Ringer’s solution. Each measurement consisted of five lar-
vae. The vertical bars represent the standard deviations. Different let- ters above the error bars were significantly different at
a =
0.05.
other inhibitors were lower than that of DEX on the lar- vae at the same pharmaceutical dose 10
µ g.
3.4. Immunodepressive effect caused by X. nematophilus
The lethal effect of X. nematophilus on the larvae of S. exigua was further analyzed by measuring a cellular
immune response, nodule formation, since the bacteria had significant cytotoxic effect on the haemocytes,
immunocompetent cells.
The fifth instar larvae of S. exigua were able to form nodules in response to bacterial infection temporally and
quantitatively Figs. 7 and 8. When the larvae were injected with 10
6
cells of heat-killed bacteria, they began to form nodules t
= 2.89; df
= 9; P
= 0.0203 1 h postinjec-
tion, the earliest time measured Fig. 7. They formed nodules in a time-dependent manner and had maximal
capacity |33 nodules 16 h postinjection. We used the 16 h incubation period for the subsequent nodule assays.
Fig. 8. Inhibitory effect of Xenorhabdus nematophilus on nodule for-
mation of the fifth instar larvae of Spodoptera exigua. Bacteria-injected larvae were incubated for 16 h at 25
° C. Each measurement consisted
of five larvae. The vertical bars represent the standard deviations.
All treated larvae injected with the heat-killed bacteria were alive and pupated successfully.
The number of nodules formed by the larvae increased with the bacterial doses for both live and heat-killed bac-
teria Fig. 8. The bacterial concentrations above 10
5
cells did not, however, change nodule formation in both treatments. The maximum number of nodules was 12
and 31 for live and heat-killed bacterial injections, respectively. There was a significant reduction in nodule
formation for live bacterial injections at all doses F
= 78.45; df
= 1, 13; P
= 0.0001.
3.5. Comparison of the effects of arachidonic acid and eicosanoid biosynthesis inhibitors on the action of
X. nematophilus in nodule formation
The pharmaceutical effects on the lethal action of X. nematophilus were analyzed by nodule assays. Appli-
cation of arachidonic acid significantly increased nodule formation in larvae which were injected with 10
6
cells of live bacteria t
= 22.95; df
= 5; P
= 0.0087. The infected
larvae treated with arachidonic acid had fewer nodules 8.4
± 4.5 per larva than the control infected larvae
26.0 ±
4.5 per larva. In comparison, DEX and other eicosanoid biosynthesis inhibitors reduced the nodule
formation of larvae injected with heat-killed bacteria 10
6
cells Fig. 9. There was no significant variation among the inhibitors in their inhibitory action on nod-
ule formation.
4. Discussion
