Behavioural response of Triatoma infestans (Klug) (Hemiptera: Reduviidae) to quinazolines

Article abstract of DOI:10.3390/10091190

The behavioural responses of the haematophagous bug Triatoma infestans towards some previously identified components of its faeces: 4-methylquinazoline, 2,4-dimethylquinazoline and their mixtures were evaluated using a video tracking system. Fifth instar nymphs and females but not males were significantly attracted to polyethylene glycol formulations of 4-methyl + 2,4-dimethylquinazoline (50 [μg each). Fifth instar nymphs were also attracted to 4-methylquinazoline alone (50 μg) but females were only attracted by the mixture of both methyl quinazolines (50 μg each). Syntheses of both methyl quinazolines were carried out starting from 2-aminoacetophenone by modifying the conditions of reported procedures.

Full text of DOI:10.3390/10091190

Molecules 2005, 10, 1190-1196
molecules
ISSN 1420-3049
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Behavioural Response of Triatoma infestans (Klug) (Hemiptera:
Reduviidae) to Quinazolines
R.A. Alzogaray 1, 4, †, A. Fontán 1, †, F. Camps 2, H. Masuh 1, 4, P. Santo Orihuela 1, D. Fernández 2,
A. Cork 3 and E. Zerba 1, 4, †,
*
1
Centro de Investigaciones de Plagas e Insecticidas (CIPEIN-CITEFA/CONICET), J.B. de La Salle
4397, (1603) Villa Martelli, Prov. de Buenos Aires, Argentina. Telefax: (++5411)-4709-5334.
Instituto de Investigaciones Químicas y Ambientales, CSIC Jordi Girona, 18-26, 08034-Barcelona,
Spain.
2
3
Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, United
Kingdom.
4 Escuela de Posgrado, Universidad Nacional de General San Martín, Prov. de Buenos Aires,
Argentina.
†
Members of the Carrera del Investigador Científico (CONICET).
* Author to whom correspondence should be addressed; E-mail: ezerba@citefa.gov
Received: 9 November 2004; in revised form: 29 June 2005 / Accepted: 29 June 2005 / Published:
30 September 2005
Abstract: The behavioural responses of the haematophagous bug Triatoma infestans
towards some previously identified components of its faeces: 4-methylquinazoline, 2,4-
dimethylquinazoline and their mixtures were evaluated using a video tracking system.
Fifth instar nymphs and females but not males were significantly attracted to polyethylene
glycol formulations of 4-methyl + 2,4-dimethylquinazoline (50 µg each). Fifth instar
nymphs were also attracted to 4-methylquinazoline alone (50 µg) but females were only
attracted by the mixture of both methyl quinazolines (50 µg each). Syntheses of both
methyl quinazolines were carried out starting from 2-aminoacetophenone by modifying
the conditions of reported procedures.
Keywords: 4-Methylquinazoline, 2,4-dimethylquinazoline, aggregation behaviour,
Triatoma infestans

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Introduction
The haematophagous bug Triatoma infestans (Klug) is the major vector of Chagas disease in South
America. The aggregation behaviour elicited from conspecific nymphs and adults by its feces is well
known [1,2]. This activity has been attributed to a number of nitrogenous compounds emitted by the
faeces. Ammonia elicited electrophysiological and behavioural responses in laboratory bioassays [3,4].
In concordance with the observations of other authors for haematofagous insects, we have reported a
very dose dependent response of T. infestans nymphs towards different salts used as sources of
ammonia [5].
Other nitrogenous compounds such as 4-methyl- and 2,4-dimethylquinazoline were reported to be
released from faeces of T. infestans [4]. However, the authors were unable to demonstrate that these
compounds could elicit attraction from immature stadiums when tested at doses of 0.1 – 100 ng. In the
present work, 4-methyl- and 2,4-dimethylquinazoline were synthesized and the attractant effect of these
compounds on fifth instar nymphs and adult T. infestans was assessed at different doses.
Sensitive and selective means of monitoring for T. infestans is central to any vector control
campaign [6]. However, this is currently achieved by manual surveys and the use of unbaited refuge
boxes [7,8]. Identification of bioactive compounds could be very useful for incorporation in a more
sensitive monitor or control device for this species, the principal vector of Chaga’s Disease in South
America [9].
Results
Fifth instar nymphs and females were found to be significantly attracted (P < 0.05) to blends of 50
µg of 4-methyl- plus 50 µg 2,4-dimethylquinazoline in solutions of polyethylene glycol 400 (Table 1).
No significant differences (P > 0.05) were observed when the behaviour of males was evaluated.
Table 1. Behavioural response of fifth instar nymphs and adults of T. infestans to a
4-methyl + 2,4-dimethylquinazoline blend in an arena bioassay.
4-methylquinazoline + 2,4-dimethylquinazoline
Behavioural response
Control
(5 + 5) ng
(50 + 50) µg
Fifth instar nymphs
Positive recordings (%)
30
60
0.8 ± 0.3a
100
2.2 ± 0.3b
Mean number of visits (± SE) 0.4 ± 0.2a
Females
Positive recordings (%)
Mean number of visits
Males
30
0.4 ± 0.2a
60
1.2 ± 0.4a
70
2.4 ± 0.7b
Positive recordings (%)
30
40
0.9 ± 0.5a
60
1.2 ± 0.4a
Mean number of visits (± SE) 0.7 ± 0.4 a
Values followed by the same letter were not significantly different after the Tukey-
Kramer test (P > 0.05).

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The behavioural response of each methyl quinazoline was then tested only on fifth instar nymphs
and females, which were attracted by the blend. Just 4-methylquinazoline seems to be responsible for
the attractive effects of the quinazoline blend. In fact, 2,4-dimethylquinazoline did not elicit any
significant effect neither on fifth instar nymphs nor females (P > 0.05) (Tables 2 and 3). In contrast,
when the effectiveness against T. infestans females was analysed, no significant attraction (P > 0.05)
was observed for the individual components of the methylquinazoline mixture. Both compounds seem
to be necessary to elicit a significant effect on females (Tables 2 and 3).
Table 2. Behavioural response of fifth stadium nymphs of T. infestans to 4-methyl
quinazoline and 2,4-dimethyl quinazoline in an arena bioassay
4-methyl
quinazoline
(50 µg)
2,4-dimethyl
quinazoline
(50 µg)
Behavioural response
Control
Positive recordings (%)
25
65
35
Mean number of visits (± SE)
0.2 ± 0.1 a
1.5 ± 0.2 b
0.7 ± 0.3 a
Values followed by the same letter were not significantly different after the Tukey-
Kramer test (P > 0.05).
Table 3. Behavioural response of females of T. infestans to 4-methyl quinazoline and 2,4-
dimethylquinazoline in an arena bioassay
4-methyl
quinazoline
(50 µg)
2,4-dimethyl
quinazoline
(50 µg)
Behavioural response
Control
Positive recordings (%)
30
70
80
Mean number of visits (± SE)
0.2 ± 0.1 a
1.0 ± 0.3 a
0.9 ± 0.2 a
Values followed by the same letter were not significantly different after the Tukey-
Kramer test (P > 0.05).
Discussion
The preparation of 4-methylquinazoline was carried out by condensation of aminoacetophenone
with formamide in the presence of boron trifluoride etherate as catalyst [10], as shown in Scheme 1.
The reaction yield has been increased from 55% to 75% by increasing the amount of catalyst used and
final purification by flash silica chromatography. Likewise, synthesis of 2,4-dimethylquinazoline was
performed according to [11], as shown in Scheme 2. The corresponding N-oxide was prepared by
condensation of the oxime of N-acetylaminoacetophenone with hydroxylamine hydrochloride, followed
by reduction of this compound with phosphorus oxychloride or iron powder. In this case, the yields
obtained in the different steps were similar to those previously reported [11].

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Scheme 1
O
CH3
CH3
N
CHO-NH2
Et2O
heat
BF3
NH2
N
Scheme 2
CH3
CH3
O
O-
N+
NH2OHHCl
NOH
NH2OHHCl
CH3
N
CH3
NHAc
NHAc
CH3
N
POCl3
HCCl3
CH3
N
Lorenzo et al. [1] demonstrated that every stadium of T. infestans was attracted to and arrested by
their own faeces. In addition, it has been reported that the faeces release ammonia which elicited
electrophysiological responses from the antennae of T. infestans [12] and attracted nymphs in a
servosphere bioassay [3].
In this work the attractiveness of blends of the previously identified fecal components 4-methyl-
and 2,4-dimethylquinazoline on fifth instar nymphs and females was established. 4-methylquinazoline
seems to be responsible for the effects observed in nymphs. In contrast, for females and according to
the test conditions, both compounds seem to be necessary to elicit this behaviour.
In concordance with the results previously reported by López and Morgan [4] we could not
establish an attractant effect when these compounds were assessed at low doses. However, in that work
[4], mature and the closer to maturity nymphal instars were studied. Differential responsiveness could
be hypothesised to be a consequence of the physiological function of these compounds. Faeces have
been pointed out as markers of T. infestans refuges [13]. These kinds of signals should be more relevant
for more mature stadiums, notably females responsible for the colonisation process.
Synthetic 4-methyl and 2,4-dimethylquinazoline appear as promising compounds to be included in
monitor and control devices currently under development for T. infestans, the most important vector of
Chagas disease in Argentina and much of South America [9].

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Acknowledgements
This investigation received financial support from the Agencia Nacional de Promoción de Ciencia y
Técnica of Argentina, the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina
(CONICET) and the European Communities Programme on Scientific and Technical Cooperation with
Developing Countries. The authors also thank Dra. Delmi Canale (Insectario del Servicio Nacional de
Chagas, Córdoba, Argentina) for supplying the insects; the Editor and two anonymous reviewers for
their suggestions and comments, that helped us to improve this article.
Experimental
General
All chemicals were analytical grade from Aldrich Co., Saint Louis, MO (USA) and used without
further purification. NMR spectra were recorded on a Varian Gemini 200 spectrometer (USA).
Syntheses of 4-methylquinazoline and 2,4-dimethylquinazoline.
For the preparation of 4-methylquinazoline a modification of the reported method [10] was used
(Scheme1). Thus, a solution of 2-aminoacetophenone (3 g) in freshly distilled formamide (60 mL)
containing boron trifluoride etherate (1 mL) was heated at 150ºC for 6 hr, while the complete
disappearance of the starting product was followed by TLC on silica gel eluting with a mixture of
CH2Cl2-MeOH (95/5). The reaction mixture was cooled to room temperature, extracted with benzene
(3 x 75 mL), dried (Na2SO4) and evaporated. The residue was purified by column chromatography on
flash silica gel (35-75 mesh), eluting the reaction products first with CH2Cl2 and with 95:5 CH2Cl2-
MeOH to give a yellow oil (2.4 g) with the expected spectral features of 4-methylquinazoline. Bulb-to-
bulb distillation of this oil at 50-60 ºC/0.5 torr. (lit. [4] 85 ºC/1 torr) afforded a colourless oil which
1
crystallised on standing in the refrigerator without solvent. The H-NMR spectrum agreed with the
structure of the expected product: δ4 (CDCl3) 9.18 (1H, s), 8.12 (1H, d) 8.04 (1H, d), 7.9 (1H, t) 7.66
(1H, t) and 2.97 (3H, s).
2,4-Dimethylquinazoline was prepared according to [11] (Scheme 2). Thus, a solution of
2-aminoacetophenone (3 g) in acetic anhydride (30 mL) was heated at 50 ºC for 30 min. Evaporation
under reduced pressure afforded a solid residue which was dissolved in methylene chloride and washed
three times with a saturated solution of sodium bicarbonate. The organic layer was dried (Na2SO4) and
evaporated under reduced pressure to give 3.9 g of the expected acetate. Treatment as reported [11] of
this acetate (8.4 g) with hydroxylamine hydrochloride afforded a 2:1 mixture of oxime and N-oxide, as
deduced by 1H-NMR analyses. A mixture of this product (6.5 g) and hydroxylamine hydrochloride (2,4
g) in ethanol (50 mL) was refluxed for 3 hr, while the complete disappearance of the initially formed
oxime was monitored by TLC on silica gel (eluting with a 98:2 mixture of CH2Cl2-MeOH). The solvent
was removed under reduced pressure and the residue was filtered through a silica column (6 x 4 cm)
eluting with 98:2 CH2Cl2-MeOH. After evaporation of the solvents the N-oxide of 2,4-dimethyl-
1
quinazoline was obtained (4.4 g, 75% yield, mp 96-7 ºC, lit. [11] 103 ºC); H-NMR: δ (CDCl3) 7.92

Molecules 2005, 10
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(1H, d) 7.86 (1H, d) 7.72 (1H, t), 7.63 (1H, t) and 2.9 (s, 6H). For reduction of this N-oxide to 2,4-
dimethylquinazoline, two different methods were used:
Method A: A mixture of N-oxide (1 g) and phosphorus oxychloride (1.8 mL) in chloroform (8 mL)
was heated at reflux for 15 min. The deep red reaction mixture was allowed to cool and diluted with
H2O (8 mL). After treatment with 20% aqueous NaOH solution to a basic pH and repeated extraction
with chloroform, the combined organic extracts were washed with H2O, dried (Na2SO4) and evaporated
under reduced pressure to yield a red oil (850 mg). TLC analysis of this oil revealed the absence of
starting N-oxide and the presence of three products. Column chromatography (3 x 10 cm) with flash
1
silica gel eluting with methylene chloride afforded a pure product (160 mg, 18%), identified by H-
NMR as the desired 2,4-dimethylquinazoline (see below).
Method B. To a solution of N-oxide (4.5 g) in AcOH (27 mL) and H2O (4.5 mL) iron powder (1.4
g) was added and the mixture was stirred at 80ºC for 20 h. The solvent was removed under reduced
pressure and the residue was washed with aqueous sodium bicarbonate solution. The aqueous phase
was repeatedly extracted with CH2Cl2 (5 x 20 mL). The combined organic extracts were dried (Na2SO4)
and evaporated. The oily residue (4 g) was purified by column chromatography (4 x 15 cm) with flash
silica gel, eluting with 9:1 CH2Cl2-n-hexane to give 2,4-dimethylquinazoline (2.5 g, 65%). Bulb to bulb
microdistillation afforded a colourless oil which crystallized on standing in the refrigerator (m.p. 70 ºC,
lit. [10] 72 ºC). 1H-NMR δ (CDCl3). 8.1 (1H, d) 7.95 (1H, d) 7.85 (1H, t), 7.58 (1H, t), 2.94(3H, s) and
2.86 (3H, s).
Biological material
Fifth instar nymphs, females and males of T. infestans were kept at the Centro de Investigaciones de
o
Plagas e Insecticidas (Buenos Aires, Argentina) in Perspex® containers (25 x 25 x 25 cm) at 27 C,
70% RH, and with a 12:12 (L:D) h photoperiod. The insects were fed on pigeon blood every seven
days. They were starved for seven days before being utilised for bioassays.
Bioassay
The behavioral response of T. infestans to the synthetic compounds was tested using an open arena
bioassay. Individual T. infestans were placed in a circular polypropylene arena (18 cm diameter, 15 cm
high) with polished walls. The floor of the arena was covered with filter paper to allow unimpeded
insect movement. Each arena was surrounded with matte black cardboard (20 cm high) to reduce the
effect of extraneous light interfering with insect behaviour. The temperature in the arenas was
maintained at 26°C and diffuse lighting was provided by a 40 W fluorescent tube placed below the
arena.
An insect was placed in the arena and allowed to acclimatise for 30 minutes before the bioassay. In
this time the insects typically adopted a thigmotactic behaviour, coming to rest in contact with the arena
walls. Ten µL of polyethylene glycol containing the test compound(s) was then placed in the middle of
the arena and the insect response monitored for 30 min. Ten µL of polyethylene glycol alone were used
as control. Each test was replicated at least 12 times using a different insect in each replicate.

Molecules 2005, 10
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The insect behaviour was monitored during 30 min using a Videomex-V (Columbus, OH, USA)
video recording system. Each arena was divided into a circular central zone (4 cm in diameter) and
outer zones using Multiple Zone Motion Monitor software (Columbus, OH, USA).
The presence of the insect was identified as a white image over a black background (pixels ‘on’).
To evaluate the approach of the insect to the source of compound, two parameters were analysed: (a)
percentage of positive recordings, defined as the percent of independent replicates when the area
occupied in the central zone during the bioassay was different from zero; (b) mean number of visits to
the source, the total number of visits to the central zone divided by the number of replicates.
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