J. Insect Physiol. Vol. 40. No. 7. pp.

61 I-615, 1994
Copyright :ct 1994 Elsevier Science Ltd
Pergamon 0022-1910(94)EOO17-A Printed in Great Britain. All rights reserved
00x-19lOi94 $7.00 + 0.00

Lactic Acid Sensitive Receptors in the

Autogenous Mosquito Aedes atropalpus
M. F. BOWEN,*t E. E. DAVIS,* J. ROMO,* D. HAGGART*
Received 21 September 1993; revised 29 December 1993

Host seeking behavior is not observed during the first gonotrophic cycle in Aedes atropalpus. The
objectives of this study were to examine the possible role of the peripheral sensory system in this
phenomenon. The results show that the rate of maturation of the lactic acid sensitive neurons is
relatively rapid in this species so the absence of host-seeking during the first gonotrophic cycle cannot
be attributed to delayed development of the peripheral sensory system. Furthermore, lactic acid
receptors of high sensitivity are present on the antennae of females that are gravid and in the early
stages of egg development. These results support the conclusion that a humoral mechanism of
host-seeking inhibition is not operative in this species, at least during autogenous egg development,
and show that receptors for at least one host attractant are fully mature early in the first autogenous
gonotrophic cycle.

Host-seeking Olfaction Lactic acid receptors Electrophysiology

INTRODUCTION with egg development. The absence of host-responsive-

ness during adult diapause in Culex pipiens is similarly
Lactic acid is a volatile by-product of anaerobic
correlated with the absence of highly sensitive receptors
metabolism that is exploited by mosquitoes for use as a
(Bowen et al., 1988). In this species, diapause apparently
host attractant. The chemical is detected by specific
causes a delay in the maturation of the receptors respon-
olfactory receptors on the mosquito antennae that are
sible for detecting host attractants. Diapause termin-
acutely sensitive to physiological air-borne levels of
ation is accompanied by the development of high
lactic acid (Bowen, 1991). These peripheral receptors
receptor sensitivity and the appearance of host-seeking
play an important role in the control of mosquito host
behavior (Bowen, 1990).
responsiveness. Our understanding of this role is derived
Does the lactic acid receptor system play a role in the
exclusively from studies of the anautogenous mosquito
modulation of host-seeking in mosquitoes such as Aedes
Aedes aegypti in which the functional state of the lactic
atropalpus in which host-seeking is not expressed during
acid receptor system influences the expression of host-
the first autogenous gonotrophic cycle (Hudson, 1970;
seeking. Electrophysiological recordings from lactic
O’Meara and Krasnick, 1970; Bowen et al., 1994)? The
acid-sensitive receptors on the antennae of female
objectives of this study were designed to answer this
Ae. aegypti have shown that host-responsive females
question and were twofold: (1) to establish the dynamics
always have some highly sensitive lactic acid receptors.
of the receptor maturation process in Ae. atropalpus and
Likewise, non-host-responsive females never have highly
compare it with that in Ae. aegypri and (2) to determine
sensitive receptors (Davis, 1984a,b). The correlation of
if a correlation exists between host-seeking behavior
non-responsive behavior with low or no lactic acid
and receptor sensitivity in this species. We report here
sensitivity is apparent at two specific periods when
the results of our study of the sensory physiology of the
host-seeking behavior is absent: (1) at adult emergence
lactic acid receptor of Ae. atropalpus during autogenous
(Davis, 1984a) and (2) following a bloodmeal (Davis,
egg development and after oviposition of the initial
1984b). The lack of highly sensitive receptors at adult
autogenous egg clutch.
emergence is due to a delay in their maturation. In
the post-bloodmeal state the lactic acid receptors are
suppressed by a hemolymph-borne factor associated MATERIALS AND METHODS

Mosquito rearing and host -seeking behavior

*SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025,
U.S.A. The procedures were identical to those described
tTo whom all correspondence should be addressed. previously (Bowen et al., 1994).

611
612 M. F. BOWEN
TABLE 1. Lactic acid-excited
Age Gonotrophic High
th) Behavior* status (%)
Cl2 NR Nulliparousj
12-24 NR Gravid
2496 NR Gravid
r96 R Parous
*NR: non-host-responsive, non-host-seeking; R: host-responsive, host-seeking.
fIncludes cells that had no spike activity, cells that had no response to any stimulus
tested and cells that displayed non-specific
$“Nulliparous” Ae. arropalpus females were defined as those between the ages of 0 and
lo-12 h of age, the time at which pre-vitellogenic
(see Clements, 1992).
Electrophysiological recording and odor delivery
Standard electrophysiological techniques were used
to record the activity of single olfactory receptor neurons
and have been described in detail elsewhere (Bowen,
1992). A cold-anesthetized female was glued to a brass
mount and the antennae held down with double-stick
tape. Under 960 x magnification, an uninsulated tung-
sten microelectrode was placed in the hemolymph space
at the tip of the antenna as the reference. A similar
recording microelectrode was inserted through the
cuticle at the base of a sensillum to detect the extra-
cellular action potentials (spikes) of the neurons. The
resulting nerve spikes were amplified, filtered, and dis-
played on an oscilloscope. The signal was also routed
to an amplitude analyzer and instantaneous frequency
converter, and the output (change in spike frequency
vs time) was plotted on a chart recorder. For each
sensillum, the mean stimulus intensity-phasic response
function was graphed. Each neuron was routinely
examined for specificity by presenting it with a variety of
olfactory stimuli (Bowen, 1992) and for sensitivity by
presenting it with a graded stimulus intensity series.
“Low” sensitivity lactic acid-excited cells were defined
as those cells that displayed a response frequency of
23 imp/s or lower at stimulus intensities of 27-32 x
lo-” mol/s (average AF = 12.2 f 1.6 imps/s, N = 18
observations). “High” lactic acid sensitivity was con-
sidered to be response frequencies above 23 imp/s
at these stimulus intensities (average AF = 45.6 + 3.8
imp/s, N = 15 observations).
RESULTS
Examination of a total of 158 antenna1 sensilla from
44 Ae. atropalpus females revealed that lactic acid-
sensitive neurons were only associated with the grooved
peg sensilla. Both lactic acid-excited (N = 44) and
-inhibited (N = 30) response patterns were observed.
The remaining grooved peg sensilla (N = 50) contained
neurons that had no spike activity, no response to any
stimulus presented, or non-specific responses to a variety
of chemical stimuli. No lactic acid-sensitive neurons
were found in any other classes of antenna1 chemo-
sensory sensilla (N = 34). See McIver (1978) for a
description of other sensilla types.
et al.
cells in Aedes alropalpus
Low Other? No. No.
(%) (%) sensilla females
0 23 77 31 11
50 11 39 18 7
31 25 44 36 14
22 44 33 9 4
responses to more than one stimulus.
development is completed at 27°C
Low sensitivity lactic acid-excited cells were present in
females younger than 12 h. High sensitivity cells first
appeared in Ae. atropalpus females between the ages of
12 and 24 h (Table 1). Cells that had no spike activity,
no response to any stimulus presented or non-specific
responses to more than one stimulus were present at all
ages, although there were significantly more of these
types of cells in females between 0 and 10 hours of age
(x * = 10.905, P < 0.01). High sensitivity lactic acid-
excited cells were observed in nulliparous, gravid and
parous females even though only parous females were
host-responsive.
The average dose-responses of all lactic acid-excited
cells from adult Ae. atropalpus females over 12 h old
(N = 24 cells) and under 10 h old (N = 8 cells) are shown
in Fig. 1. The average response frequencies are higher in
mosquitoes that are over 12 h of age, as compared with
those younger than 12 h, at all stimulus concentrations.
Also shown are the dose-response functions of lactic
acid-inhibited cells (N = 11). These cells are found in all
females regardless of age (from newly emerged to over
96 h post-emergence).
The lactic acid sensory system matures rapidly in
Ae. atropalpus (Table 2). In females less than 24 h old,
only 6% of the sensilla tested had no spike activity; in
Ae. aegypti of comparable age, spike activity was absent
in 100% of the sensilla tested (see also Davis, 1984a).
Spike activity was observed in all Ae. atropalpus females
< 24 h old and was routinely observed in newly emerged
females (between 0 and 12 h of age). Between 24 and 96 h
after emergence, the distribution of response types is
similar in both species.
DISCUSSION
During the first autogenous gonotrophic cycle gravid
Ae. atropalpus females possess olfactory receptors
that are highly sensitive to the host attractant lactic acid.
This is in contrast to the anautogenous mosquito
Ae. aegypti in which high sensitivity receptors are never
observed in gravid females (Davis and Takahashi, 1980;
Davis, 1984a). In Ae. aegypti a hemolymph-borne
factor, produced by the fat body of bloodfed females
in response to ecdysteroids from the developing
ovary, depresses the sensitivity of the peripheral host
 OLFACTION IN AN AUTOGENOUS MOSQUITO 613

>lOh

<lO h
10 -
AF

10 100

[LA] x 10“’

FIGURE 1. (A) Average dose-response of all (low and high sensitivity) lactic acid-excited cells from adult Aedes atropalpus
females over 10 h old (N = 24 cells) and under IO h old (N = 8 cells). (B) Average dose-responses of lactic acid-inhibited cells
(N = 11) from females that ranged in age from newly-emerged to over 96 h. AF = change in spike frequency,
[LA] = concentration of lactic acid (mol/s).

attractant receptors during egg development; host-seek-
ing behavior is thus inhibited until the completion of
oviposition (Klowden and Lea, 1979; Klowden, 1981;
Davis, 1984a; Klowden et al., 1987; Bowen and Loess-
Perez, 1989). Although host-seeking is absent during the
first gonotrophic cycle in Ae. atropalpus, the mechanism
of this inhibition is not the same as in Ae. aegypti: there
is no evidence for humoral inhibition of host-seeking
during this time (Bowen et aE., 1994). The results re-
ported here support this conclusion: the presence of
lactic acid receptors of high sensitivity on the antennae
of females that are gravid and in the early stages of egg
development make it highly unlikely that a haemo-
lymph-borne inhibitor of receptor sensitivity is present in
vitellogenic females, at least in those undergoing their
first gonotrophic cycle.
The comparisons between Ae. atropalpus and Ae.
aeg_vpti during the first gonotrophic cycle are summar-
ized in Table 3. Gravid females of both species are
non-host-responsive; however, most of the Ae. atro-
palpus females (76%) had high sensitivity lactic acid
receptors whereas none of the Ae. aegypti females had
such receptors. Parous females of both species were
host-responsive and all had high sensitivity receptors.
Nulliparous Ae. aegypti females (those over 24 h old that
had never had a blood meal) all had receptors of high
sensitivity. None of the nulliparous Ae. atropalpus
females (those 12 h old or younger) had receptors of
high sensitivity. However, high sensitivity receptors
were found in Ae. atropalpus females over 12 h old
(Table 1).
The rate of maturation of the lactic acid-sensitive
neurons is relatively rapid in Ae. atropalpus. Spike
activity is present at emergence and low sensitivity and
inhibited cells as well as non-specific high frequency
responses to a variety of stimuli are all present in females
that are less than 12 h old. High sensitivity receptors
appear between 12 and 24 h after emergence. In contrast
receptor development in Ae. aegq’pti (reared at the same
temperature and photoperiod) is much slower: spike
activity is not observed in females that are less than
24 h old (Davis, 1984b). It is clear that the absence of
host-seeking behavior during the first gonotrophic cycle
in this species cannot be attributed to delayed develop-
ment of the peripheral sensory system.
These results provide indirect support for the hypoth-
esis that distention inhibits host-seeking during the first
gonotrophic cycle in Ae. atropalpus (Bowen et al.. 1994).
During distention inhibition in Ae. aeg_vpti, receptor
sensitivity remains high (Davis, 1984a) so the effects of

TABLE 2. Comparison of responses of lactic acid-excited cells in Ae. arropulpus

and Ae. aegypti

Spikes
Age High Low absent Other* No. No.
(h) Species (X) (%) (%) (%) XZ sensilla females

624 Ae. arropalpus 18 18 6 57 49 18

Ae. aegypti 0 0 100 0 P <O.ool 11 4
2496 Ae. atropalpus 31 25 0 44 36 14
Ae. aegypii 51 11 S 34 N.S. 65 22

*Includes cells that had no response to any stimulus tested and cells that displayed non-specific
responses to more than one stimulus.
614 M. F. BOWEN et al.
TABLE 3. A comparison of lactic acid receptor sensitivity and its
relationship to behavior and gonotrophic status in Ae. arropalpus and
Ae. aegypfi during the first gonotrophic cycle
Gonotrophic status
Nulliparous* Gravid Parous
Autogenous
Ae. atropalpus
Behavior NRt NR Rb
% females with high O(l1) 76 (21) 100 (6)
lactic acid sensitivity (N)
Anautogenous
Ae. aegypti
Behavior R NR R behavioral control is not operative in this species, at least
% females with high 100 (49) 0 (28) 100 (5)
lactic acid sensitivity (N)
*“Nulliparous” Ae. afropalpus females were defined as those between
the ages of 0 and lo-12 h of age, the time at which pre-vitello-
genie development is completed at 27°C (see Clements, 1992).
“Nulliparous” Ae. aegypti are females of various ages that have not
had a bloodmeal.
TNon-host-responsive, non-host-seeking.
$.Host-responsive, host-seeking.
distention on host-seeking behavior are implemented
centrally rather than peripherally. If distention is the
mechanism by which host-seeking is suppressed, then
the peripheral sensory receptors need not be involved
at all in host-seeking suppression. According to this
hypothesis, distention due to fat body hypertrophy
inhibits host-seeking in very young females (up to 1 day
of age); as egg development proceeds, the distended
ovary inhibits host-seeking until oviposition is com-
pleted, between 2 and 6 days after emergence (Bowen
et al., 1994). The receptors are fully mature well before
the eggs are laid, and females are thus ready to take a
bloodmeal and initiate the second gonotrophic cycle as
soon as oviposition is completed.
Because so little is known about other receptors
involved in host location in mosquitoes it must be
emphasized that distention inhibition is only one expla-
nation for the absence of host-seeking in Ae. atropalpus.
Other host-related signals such as temperature, C02, and
other (as yet unidentified) chemical stimuli are clearly
involved in mosquito host location (Bowen, 1991) but
receptors for these stimuli have not been examined in
any species with respect to a possible role in the per-
ipheral control of mosquito behavior. The possibility
that modulation of receptors other than those for lactic
acid are involved in this phenomenon cannot be ruled
out at this time.
Nonetheless, the apparent lack of humoral inhibition
of host-seeking during autogenous egg development
in Ae. atropalpus is surprising given that autogeny
is considered to have arisen secondarily from the
more primitive anautogenous (bloodfeeding) condition
(Downes, 1958, 1971). If a blood meal provides no
further increase in fecundity, it is advantageous for
any mosquito that is developing eggs (whether anauto-
genously or autogenously) to be able to -_ restrict its
_
responsiveness to host odors and to turn otf host-seeking
behavior. Ae. atropalpus develops an unusually large
autogenous egg clutch for an autogenous mosquito
(O’Meara, 1972) and host-seeking and bloodfeeding are
not necessary nor are these behaviors expressed during
autogenous egg development (Bowen et al., 1994).
Assuming that a humoral mechanism of host-seeking
inhibition existed in anautogenous bloodfeeding ances-
tors of Ae. atropalpus, it would seem reasonable that an
effective method of behavioral control that is already
in place be retained, despite the loss of the bloodfeeding
requirement. Nonetheless, a humoral mechanism of
during autogenous egg development and receptors for at
least one host attractant are fully mature early in the first
autogenous gonotrophic cycle.
REFERENCES
Bowen M. F. (1990) Post-diapause sensory responsiveness in Culex
pipiens. J. Insect Physiol. 36, 923-929.
Bowen M. F. (1991) The sensory physiology of host-seeking behavior
in mosquitoes, A. Rev. Em. 36, 139-158.
Bowen M. F. (1992) Terpene-sensitive receptors in female Culex
pipiens mosquitoes: electrophysiology and behaviour. J. Insect
Physiol. 38, 1599764.
Bowen, M. F. and Loess-Perez, S. (1989) A re-examination of the role
of ecdysteroids in the development of host-seeking inhibition
in blood-fed Aedes aegypti mosquitoes. In Host Regulated Develop-
mental Mechanisms in Vector Arthropods (Eds Borovsky D. and
Spielman A.), pp. 286291. University of Florida-IFAS, Vero Beach,
FL.
Bowen M. F., Davis E. E. and Haggart D. A. (1988) A behavioural
and sensory analysis of host-seeking behaviour in the diapausing
mosquito Culex pipiens. J. Insect Physiol. 34, 805-813.
Bowen M. F., Davis E. E. and Haggart D. A. and Romo J. (1994)
Host-seeking behavior in the autogenous mosquito Aedes atro-
palpus. J. Insect Physiol. (in press).
Clements A. N. (1992) The Biology of Mosquitoes, Volume I. Chapman
and Hall, London.
Davis E. E. (1984a) Development of lactic acid-receptor sensitivity and
host-seeking behaviour in newly emerged female Aedes aegypti
mosquitoes. J. Insect. Physiol. 30, 21 l-215.
Davies E. E. (1984b) Regulation of sensitivity in the per-
ipheral chemoreceptor systems for host-seeking behaviour by
heaemolymph-borne factor in Aedes aegypri. J. Insect Physiol. 30,
179-183.
Davis E. E. and Sokolove P. G. (1976) Lactic acid-sensitive receptors
on the antennae of the mosquito, Aedes aegypti. J. camp. Physiol.
105, 43-54.
Davis E. E. and Takahashi F. T. (1980) Humoral alteration of
chemoreceptor sensitivity in the mosquito. In Olfaction and Taste
VII. (Ed. van der Starre H.), pp. 139-142. IRL Press, London.
Downes J. A. (1958) The feeding habits of biting flies and their
significance in classification. A. Rev. Em. 3, 2499266.
Downes J. A. (1971) The ecology of blood-sucking Diptera: An
evolutionary perspective. In Ecology and Physiology of Parasites.
(Ed. Fallis, A. M.), pp. 232-258. University of Toronto Press,
Toronto.
Hudson A. (1970) Factors affecting egg maturation and oviposition
by autogenous Aedes atropalpus (Diptera: Culicidae). Can. Em. 102,
939-949.
Klowden M. J. (1981) Initiation and termination of host-seeking
inhibition in Aedes aegypti during oocyte maturation. J. Insect
Physiol. 27, 799-803.
Klowden M. J. and Lea A. 0. (1979) Humoral inhibition of host-
seeking in Aedes aegypti during oocyte maturation. J. Insect Physiol.
24, 231-235
 OLFACTION IN AN AUTOGENOUS MOSQUITO 615

Klowden M. J., Davis E. E. and Bowen M. F. (1987) Role of the fat L. P., Rey J. R. and Frank J. H.), pp. 459471. Florida Medical
body in the control of host-seeking behavior in the mosquito Aedes Entomology Laboratory, Vero Beach, FL.
aegypti. J. Insect Physiol. 33, 643646. O’Meara G. F. and Krasnick G. J. (1970) Dietary and genetic control
McIver S. B. (1974) Fine structure of antenna1 grooved-pegs of the of the expression of autogenous reproduction in Aedes afropalpus
mosquito, Aedes aegypti. Cell Tissue Res. 153, 327-337. (Coq.) (Diptera: Culicidae). J. Med. Ent. 7, 328.-334.
McIver S. (1978) Structure of sensilla trichodea of female Aedes aegypti
with comments on innervation of antenna1 sensilla. J. Insecf Physiol.
24, 383-390. Acknowledgements-The authors thank Professor George F. O’Meara
O’Meara G. F. (1972) Polygenic regulation of fecundity in autogenous (FMEL, Vero Beach Florida) and Professor George B. Craig Jr
Aedes atropalpus. Em. exp. Appl. 15, 81-89. (University of Notre Dame) for their invaluable advice and assistance
O’Meara G. F. (1985b) Ecology of Autogeny in Mosquitoes. In on this project. This work was supported by NIH Grant 21267 to
Ecology of Mosquitoes: Proceedings of a Workshop (Eds Lounibos E.E.D. and M.F.B.