INSECTICIDERESISTANCEANDRESISTANCEMANAGEMENT

Cyromazine Toxicity to Drosophila melanogaster

(Diptera: Drosophilidae) and Lack of Cross-Resistance
in Natural Population Strains

THOMAS G. WILSON

Department of Biology, Colorado State University, Fort Collins, CO 80523

J. Econ. Entomol. 90(5): 1163-1169 (1997)

ABSTRACT Cyromazine is an insect growth regulator (IGR) insecticide that is effective
a~ainst Drosophila melanogaster (Meigen). Both larvae and pupae of a standard laboratory
strain were killed over an extremely narrow range of 0.3-0.5 ppm incorporated into the diet.
Hi~her toxic doses killed larvae more rapidly, and lower toxic doses allowed pupariation but
usually blocked pupal development. These puparia were abnormally elongated, indicating a
failure of the contraction process that produces the characteristic pupal shape. When fed to
adults, cyromazine had no effect on adult survival, fecundity, or fertility, even at a 10-fold larval
LC.~olevel. In previous work, D. melanogaster strains recently derived from natural populations
Wl're found to be 25- to 100-fold resistant to at least 2 other classes of insecticides, chitin-
synthesis inhibitors and carbamates. Here I tested 9 additional strains that were recently
selected from widely separated geographical locations in the United States and found them to
have resistance to lufenuron and propoxur, but virtually no resistance to cyromazine relative to
one another or to 3 long-established laboratory strains. The results show that cross-resistance
between the IGRs lufenuron and cyromazine is not present in D. melanogaster. They further
suAAest that cyromazine control failure in pest insect populations caused by cross-resistance
may be minimal.

KEY WORDS Drosophila melanogaster, insecticide resistance, insect growth regulator,

lufenuron, propoxur

INSECT RESISTANCETO pesticides has been exten- iological process that is active in adult insects,
sively documented as a widespread phenomenon these compounds adversely influence a develop-
characteristic of a large variety of pest species mental process(es) and subsequently block devel-
(Georghiou and Saito 1983, Roush and Tabashnik opment to the adult stage (Sparks and Hammock
1990). Although it is rare that Drosophila melano- 1975, Retnakaran et a1. 1985). Resistance in pest
{!,aster (Meigen) populations are intentionally species to examples of both types of IGR insecti-
treated with insecticides, these flies have neverthe- cides has been documented (Cerf and Georghiou
less evolved resistance to a variety of commonly 1974, Grosscurt and Stoker 1991), although it is not
used insecticides, such as DDT (Crow 1954, Dap- generally widespread.
kus 1992), organophosphates (Pralavorio and We found resistance to lufenuron in strains of D.
Fournier 1992, Mutero et a1. 1994, Windelspecht et melanogaster recently established in the laboratory
al. 1995), and cyclodienes (ffrench-Constant et a1. from natural populations from 3 widely separated
1990). Presumably, resistance that evolves in these locations in the United States, but little resistance
insects is caused by insecticidal selection in a man- in strains established earlier than about 1960 (Wil-
ner similar to that in target insects. Resistance evo- son and Cryan 1996, Wilson and Cain 1997). This
lution by D. melanogaster suggests that nontarget was a surprising result because lufenuron has been
organisms may be more effected by insecticides marketed only within the past several years and has
than previously believed, and further study is war- not seen significant field usage. It was postulated
ranted to determine the extent. (Wilson and Cryan 1996) that the resistance might
In previous work I have examined resistance in be metabolic cross-resistance to a carbamate insec-
D. melano{!,aster to insecticides belonging to the ticide used previously on a widespread basis. How-
insect growth regulator (IGR) class, including ju- ever, although these strains were also resistant to
venile hormone analogs (Shemshedini and Wilson propoxur, no correlation between propoxur (a car-
1990, Minkoff and Wilson 1992) and chitin-synthe- bamate) and lufenuron resistance was found (Wil-
sis inhibitors, particularly the benzoylphenyl urea son and Cain 1997).
compound lufenuron (Wilson and Cryan 1996, Perhaps these D. melanogaster strains recently
1997). Unlike other insecticides that target a phys- derived from natural populations are resistant to a

0022-0493/97/1163-1169$02.00/0 © 1997 Entomological Society of America

1164 JOURNAL OF ECONOMIC ENTOMOLOGY
Table I. Geographical origin and year of laboratory establishment of D. me/anogaoler
Strain Country of origin
establishment
Oregon-RC USA
Samarkand Russia
Lausanne-S USA
Swedish-C USA
WCl USA (Vermont)
WC6 USA (Vermont)
WC19 USA (Vermont)
WCC96-1 USA (Colorado)
WCC96-2 USA (Colorado)
WCC96-10 USA (Colorado)
CF14 USA (Florida)
NT03 USA (Florida)
NC03 USA (Florida)
wide variety of insecticides, similar to some organ-
isms that are resistant to a wide variety of chemi-
cally unrelated compounds due to the multidrug
resistance locus (Endicott and Ling 1989). In the
current study I examined cross-resistance of these
strains to cyromazine (N-cyclopropyl-l,3,5-triazine-
2,4,6-triamine). Cyromazine is an insect growth
regulator insecticide that is effective against cer-
tain insect pests, especially dipterans such as Musca
domestica L. (EI-Oshar et al. 1985, Iseki and
Georghiou 1986) and Lucilia cuprina Weidemann
(Friedel and McDonell 1985). It is also effective
against Drosophila melanogaster (Adcock et al.
1993). Although the benzoylphenyl urea IGRs have
been clearly shown to inhibit chitin synthesis in
insects (Marks et al. 1982), the molecular target
and mode of action of cyromazine is unknown.
I was especially interested in examining cyroma-
zine cross-resistance for the following 2 reasons:
(1) a previous study in house flies (Shen and Plapp
1990) showed cross-resistance between difluben-
zuron, a benzoylphenyl urea chitin synthesis inhib-
itor, and cyromazine; and (2) the current study
would evaluate the extent of resistance to cyroma-
zine in natural populations of D. melanogaster. Af-
ter initially characterizing cyromazine toxicity to a
standard laboratory strain of D. melanogaster, I ex-
amined the resistance of both natural population
and laboratory strains. The results identified the
sensitive stages of D. melanogaster to cyromazine
toxicity and showed that cross-resistance to this
insecticide is essentially negligible.
Materials and Methods
The strains used in this study are given in Table
1 together with their origin and date of recovery
from natural populations and establishment in lab-
oratory culture. The long-established laboratory
strains were obtained from the Mid-America Dro-
sophila Center (Bowling Green, OH) and are de-
scribed in Lindsley and Grell (1968). Most strains
were collected at sites that were distant (>30 km)
from areas of high agricultural usage of insecticides
(WCl, WC6, and WC19 in Vermont; WC96-1,
Vol. 90, no. 5
strains examined
Date of
Reference
1938 Lindsley and Grell (1968)
1936 Lindsley and Grell (1968)
1938 Lindsley and Grell (1968)
1938 Lindsley and Grell (1968)
1991 Unpublished data
1991 Unpublished data
1991 Unpublished data
1996 Unpublished data
1996 Unpublished data
1996 Unpublished data
1993 Windelspecht et al. (1995)
1994 Win del specht et al. (1995)
1993 Windelspecht et al. (1995)
WC96-2, and WC96-10 in Colorado). The Florida
strains (Windelspecht et al. 1995) include 2 from
an area of intensive malathion usage (CF14, NC03)
and 1 from an area without a recent history of
insecticide usage (NT03). Recently established
strains were derived from captured single fertile
females attracted to a ripe fruit source. From each
of these females a strain was then independently
established on laboratory medium from the F 1
progeny (isofemale line). To minimize genetic
variability, each strain was additionally passed
through a population bottleneck consisting of a
single-pair mating. Strains were then maintained on
a standard yeast-agar-cornmeal-molasses diet. At
no time have any of the strains been knowingly
exposed to any insecticide subsequent to establish-
ment in laboratory culture.
Both cyromazine (N-cyclopropyl-l,3,5-triazine-
2,4,6-triamine) and lufenuron (N- [2,5-dichloro-4-
(1,l,2,3,3,3-hexafluoropropoxy) -phenylaminocar-
bonyl] -2,6-difluorobenzamide) were obtained
from Ciba (Greensboro, NC) as a >90% (cyroma-
zine) or 94% (lufenuron) pure compound. A stock
solution was prepared in either distilled H20 (cy-
romazine) or 95% ethanol (lufenuron), and dilu-
tions were made in the same solvent. Stock solu-
tions were stable at 4°C for at least several months.
Both compounds are most effective when ingested
by insects (Grosscurt and Jongsma 1987, Shen and
Plapp 1990) and thus were incorporated into Dro-
sophila Instant Food (Carolina Biological Supply,
Burlington, NC) in plastic vials (22 by 95 mm)
(Sarstedt, Newton, NC) as described previously for
lufenuron (Wilson and Cryan 1996).
Eggs were collected from each strain on standard
medium and allowed to hatch. Thirty-five newly
hatched larvae (0-4 h old) were gently picked
from the surface of the medium and transferred to
cyromazine- or lufenuron-bearing Instant Food
prepared as described above. Control vials had an
equivalent amount of95% ethanol or H20 added, as
appropriate. Larvae were raised at 25 ± 1°C at
70-80% RH. Survival was determined and ex-
pressed as the percentage of larvae that completed
October 1997 WILSON: CYROMAZINE
development and survived at least 1 dafter eclo-
sion.
Proproxur (2-isopropoxyphenyl methyl carba-
mate) was obtained from Chern Service (West
Chester, PA) as a 99% pure compound. A stock
solution was prepared in 95% ethanol and aliquots
incorporated into Instant Food in a manner analo-
gous to that for cyromazine and lufenuron. Unlike
these insecticides, however, proproxur is toxic to
adult insects and thus was tested on D. melano-
gaster adults. Thirty-five 2- to 5-d-old adults were
introduced into the vials following a brief cold an-
esthetization, which allowed rapid recovery and
normal activity within the vial within 1-2 min after
transfer. Vials were maintained as described above,
and survival was measured 18 h later. Most of the
adults seen at this time could be easily classified as
alive or dead, but individuals were sometimes seen
that were clearly affected but not moribund. These
individuals were classified as survivor flies if they
could quickly right themselves when perturbed.
Each strain was tested on 4- 6 concentrations of
insecticide carried out in, triplicate. LCso values
were determined using the POLO program (Rus-
sell et al. 1977).
Results
Toxicity to Oregon-RC. We first examined the
toxicity of cyromazine to Oregon-RC, a standard
laboratory strain that we have used as a susceptible
strain in previous studies with other insecticides
(Wilson and Cryan 1996,1997). The stage at which
mortality occurs with cyromazine was determined
by measuring pupal and adult survival from culture
vials containing various amounts of cyromazine
(Fig. 1). Both larvae and pupae showed sharp in-
creases in mortality between cyromazine concen-
trations of 0.3 and 0.5 ppm. Pupal survival was
essentially unaffected by cyromazine below a con-
centration of 0.3 ppm. Larval survival, however,
was a discontinuous dose response, showing 30-
40% mortality between 0.1 and 0.3 ppm, followed
by a sharp rise in mortality at higher concentra-
tions. Larval mortality at 0.05 ppm was 7%, which is
very close to that of controls (4% mortality), indi-
cating that cyromazine begins to affect larval sur-
vival at a concentration between 0.05 and 0.1 ppm.
At cyromazine concentrations above the LCgo, lar-
vae usually failed to pupariate, dying as either 1st
instars at high (>1 ppm) concentrations or in later
instars at lower (0.3-0.5 ppm) concentrations.
Therefore, the stage of development when cyroma-
zine affects D. melanogaster can vary with the dose.
Pupariation was noticeably affected in animals
raised on 0.2-0.4 ppm cyromazine. Many larvae
failed to crawl from the food in preparation for
pupariation on the walls of the vials but instead
attempted pupariation on the surface of the food,
indicating a failure of the wandering behavioral
response that lasts for 5-8 h at the end of larval
development when larvae seek an elevated site for
RESISTANCE IN Drosophila 1165
Cyromazlne (ppm)
Fig. 1. Dose-mortality curve for larvae and pupae of
the strain Oregon-RC transferred to cyromazine-contain-
ing Drosophila Instant Food as 0-4-h-old larvae. Each
point represents the mean mortality value for 6 replicate
determinations. Error bar designates standard deviation.
pupariation (Bodenstein 1935). Once pupariation
began, eversion of the anterior spiracles com-
menced but was incomplete. The characteristic
shortening of the larva that signals initiation of
pupariation failed to occur, leading to abnormally
elongate puparia (Fig. 2). At >0.3 ppm, these elon-
gate puparia usually died, but at cyromazine doses
of 0.2-0.3 ppm they often completed pupal devel-
opment. Adults that eclosed appeared normal mor-
phologically and were long lived. These females
were fecund, and both sexes were fertile. There-
fore, it appears that if larvae raised on 0.2- 0.3 ppm
cyromazine survive preadult development, they
eclose into adults having normal function.
Adults raised on noncyromazine food and trans-
ferred to 0.3-0.5 ppm cyromazine within several
hours after eclosion showed apparently normal sur-
vival, fecundity, and fertility, indicating that levels
of cyromazine toxic to larvae and pupae are not
toxic to adults. Even a higher dose of 3.0 ppm
(=lO-fold LCso concentration) did not noticeably
affect these fitness components. Because lufenuron
affects embryonic survival transovarially when fed
to adult females (Wilson and Cryan 1997), the sur-
vival of oviposited eggs was carefully examined, but
it was found to resemble that of control flies fed
noncyromazine food as adults.
Resistance in Newly Established Strains. Our
previous results showed that many strains recently
established from natural populations at widely sep-
arated locations in the United States showed con-
siderable resistance to lufenuron (Wilson and
Cryan 1996) and propoxur (Wilson and Cain 1997)
relative to laboratory strains. These strains were
not selected in the laboratory on any insecticide,
and therefore their resistance reflects that which is
1166 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 90, no. 5

mined the lufenuron, propoxur, and cyromazine

LCso values (Table 2). As expected (Wilson and
Cryan 1996, Wilson and Cain 1997), the recently
established strains were resistant to lufenuron and
propoxur relative to the laboratory strains. Surpris-
ingly, none of these strains showed more than a
trivial amount of resistance to cyromazine. The Sa-
markand strain is unusual in having relatively high
lufenuron resistance for a long-established labora-
tory strain, but it clearly is susceptible to the other
2 insecticides. Even the strains showing the highest
resistance to lufenuron (NC03) and propoxur
(WC6) showed no significant cyromazine cross-
resistance.

Fig. 2. Appearance of Oregon-RC puparia from cul-
tures raised on (A) 0 ppm cyromazine incorporated into
Drosophila Instant Food, and (B) 0.3 ppm cyromazine.
Note the extreme elongate body form and abnormal for-
mation of the anterior end, including the poorly everted
anterior spiracles, in (B).
naturally occurring in the population instead of
resistance that is artificially enhanced through lab-
oratory selection. We selected 3 isofemale strains at
random from collections taken from natural popu-
lations at each of the different locations and deter-
Discussion
Cyromazine was found to be toxic at about the
same concentrations to both larvae and pupae
when incorporated into the diet of D. melanogaster.
However, some distinction could be made by vary-
ing the doses given to the larvae. At lower toxic
doses, larval development was completed and pu-
pation occurred, but the pupae often failed to sur-
vive to eclosion. This mortality is obviously caused
by a cumulative effect of larval feeding because no
additional cyromazine can be ingested after pupari-
ation. At higher doses, larvae were killed more
quickly, usually within 1 d of feeding. Adults raised
on regular food as larvae and then transferred to
cyromazine food readily survived high (10-fold
LCso) doses; in addition, the behavior of the adults
appeared norma\, and at least 1 developmental pro-
cess, oogenesis, was not appreciably affected.
In general, the effects of cyromazine seen on
preadult D. melanogaster are similar to those seen
with other dipteran insects (Hall and Foehse 1980,
Hart et aI. 1982, Pochon and Casida 1983, Friedel
and McDonnell 1985) . However, adult dipteran in-
sects differ in their response to cyromazine. I found
no noticeable effect of cyromazine on adult longev-
ity, fecundity, or fertility when presented either in
sublethal doses to larvae or in higher doses to

Table 2. LCso values and resistance ratios for propoxur, lufenuron, and cyromazine for both recent and long-established laboratory
strains of D. melanogmlter

LC>o(95%CI)
Strain
Propoxur RR Lufenuron RR Cyromazine RR
WCl 126 (105-152) 7.9 0.97 (0.79-l.l8) 34.6 0.25 (0.21-0.28) 1.6
WC6 412 (344-499) 25.9 1.00 (0.81-1.22) 35.7 0.22 (0.18-0.25) 1.4
WC19 114 (94-138) 7.2 0.98 (0.80-1.20) 35.0 0.35 (0.30-0.40) 2.2
WCC96-1 78 (65-94) 4.9 0.61 (0.48-0.76) 21.8 0.26 (0.23-0.30) 1.6
WCC96-2 144 (121-172) 9.0 0.70 (0.56-0.86) 25.0 0.26 (0.23-0.30) 1.6
WCC96-10 156 (133-186) 9.8 0.81 (0.66-0.1.0) 28.9 0.24 (0.20-0.27) 1.5
CF14 142 (119-169) 8.9 1.03 (0.85-1.25) 36.8 0.20 (0.17-0.23) 1.2
NT03 165 (137-201) 10.3 l.l0 (0.90-1.35) 39.2 0.19 (0.15-0.22) 1.2
NC03 105 (88-125) 6.6 1.21 (1.00-1.48) 43.2 0.30 (0.27-0.34) 1.9
Lausanne 23.8 (20.4-25.4) 1.5 0.171 (0.09-0.41) 6.1 0.20 (0.16-0.24) 1.2
Swedish-C 15.9 (11.0-20.5) 1.0 0.028(0.009-0.06) 1.0 0.16 (0.13-0.19) 1.0
Samarkand 5.5 (5.1-5.9) 0.34 1.04 (0.81-l.l9) 37.1 0.24 (0.21-0.28) 1.5

LC>oin ppm incorporated in Drosophila Instant Food. 95%CI values are given in parentheses. RR, resistance ratio given as LC>o
strainlLC>oSwedish-Cstrain.
October 1997 WILSON: CYROMAZINE RESISTANCE IN
adults. Studies on the face fly, Musca autumnalis De
Geer (Hall and Foehse 1980) and the house fly,
Musca domestica L. (Hall and Foehse 1980, Pochon
and Casida 1983) similarly found no effect on ovi-
position or egg hatch for adults fed cyromazine.
Two studies on the blow fly Lucilia cuprina Wiede-
mann found differing effects on adults. Friedel and
McDonell (1985) showed fecundity to be affected
following administration to adults, whereas Kotze
(1992) could find no effect on fecundity or egg
hatch. Work that examined the results of feeding
5% cyromazine to adult Mexican fruit flies, Anas-
trepha ludens (Loew), showed both egg production
and survival to be reduced (Martinez and Moreno
1991, Moreno et al. 1994). Finally, Stark et al.
(1992) found reduced fecundity and fertility of the
melon fly, Bactrocera cucurbitae Coquillett, but not
the oriental fruit fly, Bactrocera dorsalis Hendel,
following exposure of late 3rd instars and pupae to
cyromazine. It is clear that the response of adults to
cyromazine is species-specific.
Although both cyromazine and benzoylphenyl
urea chitin synthesis inhibitor insecticides are clas-
sified as IGRs, the characteristics of cyromazine
poisoning of D. melanogaster are different from
those found for the IGR lufenuron (Wilson and
Cryan 1997). Oregon-RC 1st and 2nd instars fed
toxic doses of lufenuron completed development
within that instar but died without completing ec-
dysis to the next instar, presumably because of in-
adequate cuticle synthesis; the time of death for
cyromazine-fed larvae was variable, depending on
the dose. Lufenuron-fed larvae never exhibited the
wandering behavior peculiarity seen for 3rd instars
fed cyromazine. The puparia of lufenuron-fed lar-
vae were also abnormal, but with an appearance
almost opposite that for cyromazine: puparia from
lufenuron-fed larvae were abnormally contracted
instead of extended. Adults were also unaffected by
lufenuron as by cyromazine, but fertilized eggs laid
by lufenuron-fed females failed to hatch, although
embryonic development was completed (Wilson
and Cryan 1997). Finally, the cyromazine-treated
flies never displayed any of the lufenuron-associ-
ated exoskeletal defects, such as twisted legs and
loss of flight ability (Wilson and Cryan 1997).
The cyromazine LCso values obtained for the 12
strains examined were remarkably similar. There-
fore, although natural populations of D. melana-
gaster have shown resistance to a variety of insec-
ticides with different molecular targets, this
resistance does not extend to cyromazine.
In a previous study (Wilson and Cain 1997) that
tracked. resistance chronologically, it was found
that lufenuron and propoxur resistance increased
dramatically in D. melanogaster strains taken from
natural populations and established in laboratory
culture within the past 5-7 yr. Wilson and Cain's
interpretation was that the resistance seen relative
to older laboratory strains was the result of agricul-
tural chemical usage and that D. melanogaster had
been indirectly affected. Another interpretation
Drosophila 1167
was also possible: that the susceptibility found in
older laboratory strains reflects the loss of natural
resistance to xenobiotic compounds after years of
laboratory culture. The current results demonstrat-
ing complete susceptibility to cyromazine (but re-
sistance to the other 2 insecticides) in recently
sampled natural populations argues against the loss
of resistance hypothesis and suggests that D. mela-
nogaster is susceptible to cyromazine because this
insect has not experienced cyromazine or other
chemically related compound as a basis for resis-
tance to evolve.
In laboratory experiments, cyromazine resis-
tance has been induced with the chemical mutagen
ethyl methanesulfonate in both D. melanogaster
(Adcock et al. 1993) and in L. cuprina (Yen et al.
1996). For both sets of resistant mutants, low «5-
fold) resistance was found. Usually, this level of
resistance would be considered within the limits of
genetic variability, but considering the lack of ge-
netic variability for resistance seen in the present
study, 5-fold resistance now seems more significant.
Interestingly, 1 of the 2 D. melanogaster cyroma-
zine-resistant mutants described by Adcock et al.
(1993) mapped to 64 on chromosome II, where
other insecticide-resistant genes have mapped
(Kikkawa 1964). We have mapped the propoxur
resistance in one of the WC strains (WC2) to the
same or closely linked location (T.G.W., unpub-
lished data), but this strain was similar to the other
recently established strains in being susceptible to
cyromazine. Therefore, there may be > 1 resistance
gene at this map location, one identified by Adcock
et al. (1993) and another nearby one that confers
resistance to other insecticides, including pro-
poxur, but not to cyromazine. In a study of cyroma-
zine-resistant house flies, Shen and Plapp (1990)
found apparent cross-resistance between cyroma-
zine and the benzoylphenyl urea chitin-synthesis
inhibitor diflubenzuron. These results suggested to
them that a single resistance gene may be common
to both modes of resistance; if so, it is different
from the lufenuron resistance gene in D. melano-
gaster, which clearly shows no cross-resistance to
cyromazine.
Other studies have shown that laboratory strains
of pest insects that are resistant to conventional
insecticides show little cross-resistance to cyroma-
zine (e.g., EI-Oshar et al. 1985, Iseki and Georghiou
1986, Kelly et al. 1987, Shen and Plapp 1990 [for
strains other than the diflubenzuron cross-resistant
strain], Keiding et al. 1991). These results suggest
that cross-resistance in pest species will be minimal
and that cyromazine should be effective to control
pest insects that are resistant to other insecticides.
However, it is unclear whether cyromazine will
maintain this initial advantage. Cyromazine resis-
tance at serious levels (25- to 45-fold) have been
either selected in the laboratory (Bloomcamp et al.
1987) or identified after cyromazine control failure
in poultry houses (Sheppard et al. 1992). Because
cyromazine cross-resistance to other groups of in-
1168 JOURNAL OF ECONOMIC
secticides is proving to be almost negligible, the
chemistry and action of this insecticide must be
very different from conventional insecticides. To
this end the identification of the target molecule
will prove interesting and useful, although using a
biochemical approach in this research can prove
difficult. Perhaps through a mutagenesis analysis
similar to that of Adcock et al. (1990) in a geneti-
cally amenable insect such as D. melanogaster (Wil-
son 1988), a target-site locus can be identified,
which may upon molecular analysis give clues as to
the identity of the target molecule.
Acknowledgments
We thank Ciba-Geigy for supplying both lufenuron and
cyromazine, and Michael Windelspecht for supplying the
Florida strains. Undergraduate students Tammy Osborne
and Stephan Frezel assisted in some of the LC50 determi-
nations.
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Received for publication 19 February 1997; accepted 1
July 1997.