Page 6 - Volume 3, Issue 2, July 2010

© Benaki Phytopathological Institute
Mihou & Michaelakis 36
1.2. Bioassays with the pheromone un-
der laboratory conditions
Responses of gravid females of Cx. quin-
quefasciatus to erythro racemate and to each
enantiomer separately show that the phero-
mone is only in the (–)-(5R ,6S) form. The nat-
ural product has relatively low vapour pres-
sure, and several attempts have been made
to alter its volatility. The methods employed,
aimed at shortening the length of the car-
bon chain or opening the lactone ring of the
molecule. However, both methodologies re-
sulted in materials with no biological activi-
ty (Laurence and Pickett, 1985; Dawson et al.,
1989). Instead, when hydrogen atoms were
replaced by fluorine, the newmolecules had
biological activity as well.
More specifically, when the acetoxy
group was replaced by a trifluoroacetoxy
group, the new compound showed a high-
er activity than the natural product, proba-
bly due to its higher vapour pressure (Pickett
and Woodcock, 1996). In addition, the hep-
tadecafluoro analog, in which the n-octyl
group is replaced by perfluoroctyl, retained
high biological activity (Dawson et al., 1990).
The first bioassay employing all four ste-
reoisomers of 6-acetoxy-5-hexadecanolide
was conducted by Hwang et al. (1987). Pre-
dictably only the (–)-(5R,6S ) enantiomer was
active. The activity of the current isomer in-
creased 50-fold when it was applied direct-
ly to the water surface and 100-fold when
Cx. quinquefasciatus was employed instead
of Cx. tarsalis. Aedes aegypti and Anopheles
quadrimaculatus were not attracted to the
pheromone (Figure 4). These experiments
suggested once more that the current pher-
omone is genus-specific (Bruno and Lau-
rence, 1979; Hwang et al., 1987). In contrast
to the previous investigations, Sakakibara
et al. (1984) reported that all four stereoiso-
mers showed significant activity against Cx.
pipiens biotype molestus . According to Cle-
ments (1999), these results cannot be taken
as proof because there are no clear specifi-
cations of their synthetic procedures.
Antennae of Cx. quinquefasciatus grav-
id female mosquitoes were tested for sen-
sitivity to test materials by recording elec-
troantennograms (EAGs) during oviposition
behaviour studies (Mordue et al., 1992). The
tested materials were the oviposition pher-
omone (erythro racemate) and polluted wa-
ter (made by fermenting rabbit droppings
in distilled water). Both EAG responses and
behaviour studies to the oviposition phero-
mone increased in a dose-dependent man-
ner and the combination of the two materi-
als increased ovipositionwith the total effect
being additive. In 1985, Laurence and Pickett
showed that the dose-responses over the
concentrations were a flat curve (Laurence
and Pickett, 1985). However, in 1993, Pickett
with other co-workers, using a similar meth-
od of presentation, obtained a dose-depen-
dent response for a certain range of phero-
mone. After a critical dose, the pheromone
caused a significant reduction in oviposition
behaviour (Blackwell et al., 1993).
High pheromone concentration could
either result in confusion of the gravid fe-
males with respect to the oviposition sites
or repel gravid females of laying their eggs.
Although Millar et al. (1994) reported that
attraction was constant even to very high
pheromone concentration (1000 μg per
cage), other researchers, who synthesized
the oviposition pheromone as a mixture of
all diastereoisomers and tested it against Cx.
p. biotype molestus , obtained similar results
to Blackwell et al. (1993) (Michaelakis et al.,
2005).
Olagbemiro et al. (1999 and 2004) em-
ployed for the first time a plant-derived
Figure 4. Oviposition behaviour of Cx. quinquefasciatus gravid
females to different amounts of pheromone. Oviposition dish-
es contain the synthetic oviposition pheromone, erythro -6-ac-
etoxy-5-hexadecanolide. The broken lines represent the up-
per and lower values of the control mean±SEM (52.6±3.7%,
n=10) [for more details see Blackwell et al. (1993)].