ARTICLE
L-Lactic Acid as a Mosquito (Diptera: Culicidae)
Repellent on Human and Mouse Skin
AND
MASAAKI MOROHASHI2
J. Med. Entomol. 38(1): 51Ð54 (2001)
ABSTRACT The attraction of Aedes albopictus (Skuse) to hands and forearms of human subjects
treated with several concentrations of L-LA solution were studied in a test chamber containing
proboscis-amputated mosquitoes. Fewer mosquitoes alighted on L-LA treated human skin than on
water-treated control skin. Similar results were found using normal mosquitoes following L-LA and
water treatment of mouse skin. The relative repellent effects of L-LA varied with concentration. The
minimum repellent concentration was lower than previously reported for human skin. The number
of alightments decreased at increasing concentrations of L-LA, demonstrating the absolute repellency of L-LA. Unlike previous reports suggesting that L-LA attracted mosquitoes, our studies using
human and mouse skin showed that L-LA exhibited both relative and absolute repellency.
KEY WORDS Aedes albopictus, L-lactic acid, repellency, alightment, mosquito repellent
MANY RESEARCHERS HAVE reported that L-lactic acid
(L-LA) attracts mosquitoes (Acree et al. 1968, Smith
et al. 1970, Carlson et al. 1973, Kline et al. 1990, Eiras
and Jepson 1994). Acree et al. (1968) isolated L-LA
from acetone washings of 800 human arms and demonstrated that L-LA attracted female Aedes aegypti
(L.), particularly when CO2 was added. LA is present
in human skin and sweat (Robinson and Robinson
1954, Kuno 1956, Gordon et al. 1971). However, King
(1954) reported that LA acts as a repellent to mosquitoes when tested on human skin. Smith et al. (1970)
showed that the effectiveness of L-LA declined at high
concentrations. We have described a new experimental method of assessing the response of Aedes albopictus (Skuse) to repellents (Shirai et al. 2000) and
herein compare our results with those reported previously.
Materials and Methods
Mosquitoes. Four colonies of Ae. albopictus are
maintained in our laboratory at 24 ⫾ 1⬚C, 60 Ð70% RH,
and a photoperiod of 14:10 (L:D) h. Colonies originated from Ogaki in Gifu Prefecture, Ako in Hyogo
Prefecture, Tsurumi Ryokuchi in Osaka Prefecture,
and Ishigaki in Okinawa Prefecture, Japan, and were
in generations 2Ð 6 when used for experimentation.
Previously we found that 20- to 30-d-old unfed female
Ae. albopictus bite more avidly than 3- to 5-d-old females, and these old females (20- to 30-d-old) were
used throughout.
1
Department of Biodefence Medicine, Faculty of Medicine,
Toyama Medical and Pharmaceutical University, 2630 Sugitani,
Toyama 930-0194, Japan.
2
Department of Dermatology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194,
Japan.
Effect of L-LA Treatment on One Forearm. We
adapted an aquarium (600 by 295 by 360 mm; NS-6 M,
NISSO, Tokyo, Japan) as a test chamber and introduced mosquitoes into it after amputating their proboscises (Shirai et al. 2000). L-LA (Wako Pure Chemical, Osaka, Japan) was diluted to concentrations
ranging from 1 to 10,000 ppm with distilled water. One
hand and forearm of each human subject were dipped
into the L-LA solution and then allowed to air dry. The
other hand and forearm of the same subject were used
as the control, proceeding as above with distilled water instead of L-LA. Both hands and forearms were
inserted simultaneously into the test chamber. The
number of mosquitoes that alighted on the hands and
forearms were counted every 30 s for 10 min. Attractancy or repellency was evaluated by comparing the
number of alightments on the treated arm with the
control for 10 min. Each concentration was repeated
three or six times.
Effect of L-LA Treatment on Both Forearms. Experiments comparing treated and control forearms
indicated relative attractiveness or repellency. For
determination of absolute attractiveness or repellency, both hands and forearms of each subject were
dipped into L-LA solution at the same concentration
(1,000 Ð10,000 ppm), dried, and exposed to mosquitoes
as above. Three replicates at each concentration were
examined.
Effect of L-LA Treatment on Mice. When using
human subjects, we used proboscis-amputated mosquitoes that cannot bite (Shirai et al. 2000). We compared these results to normal mosquitoes (i.e., mosquitoes with complete proboscis) and mouse skin to
ascertain whether there is any difference in behavior
between amputated and normal mosquitoes, or between human and mouse skin. Normal female mosquitoes were used as test insects, and 4-wk-old hairless
0022-2585/01/0051Ð0054$02.00/0 䉷 2001 Entomological Society of America
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YOSHIKAZU SHIRAI,1 KIYOSHI KAMIMURA,1 TAISUKE SEKI,2
52
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 38, no. 1
Table 1. Mean ⴞ SE number of alightments of female Ae.
albopictus on L-LA- or water-treated forearms during a 10-min
exposure
n
L-LA
Water
df
t-value
1
10
100
1,000
3,000
5,000
7,000
10,000
6
6
6
6
3
3
3
6
0.66 ⫾ 0.09
1.54 ⫾ 0.10
1.40 ⫾ 0.13
1.79 ⫾ 0.26
2.55 ⫾ 0.35
0.48 ⫾ 0.15
0.53 ⫾ 0.14
0.33 ⫾ 0.08
1.47 ⫾ 0.12
1.78 ⫾ 0.13
1.53 ⫾ 0.09
4.64 ⫾ 0.22
4.70 ⫾ 0.27
2.85 ⫾ 0.24
6.82 ⫾ 0.42
9.13 ⫾ 0.34
5
5
5
5
2
2
2
5
⫺2.721*
⫺0.461NS
⫺0.333NS
⫺4.586**
⫺1.106NS
⫺5.414*
⫺4.700*
⫺5.795**
*, P ⬍ 0.05; **P ⬍ 0.01; NS, P ⬎ 0.05.
mice, anesthetized by intraperitoneal administration
of chloral hydrate, were used as bait. One mouse
(except for its head) was dipped into L-LA solution
and another mouse was dipped into distilled water for
30 s and then both mice were dried with a hair dryer.
The test chamber was an aquarium (450 by 300 by 300
mm, YT-8N, Tomofuji, Saitama, Japan) similar to that
described above. A plastic vinyl chloride sheet (200 by
300 mm) and an 80-mesh nylon gauze (250 by 300
mm) having an access sleeve was attached to the open
side. Treated and control mice were inserted into the
test chamber containing 35 normal female mosquitoes,
and the number of mosquitoes biting each mouse was
counted for 10 min. Three or Þve replicates were
examined at each concentration. All experiments were
analyzed statistically using Student t-test by StatView
(1998).
Results
Effect of L-LA Treatment on One Forearm. The
relative repellency of L-LA was determined by comparing L-LA-treated and water-treated forearms.
Fewer mosquitoes alighted on the treated forearms
than on the water controls. L-LA concentrations (except 10, 100, and 3,000 ppm) showed signiÞcant differences between each set of forearms (t-test, P ⬍
0.05, Table 1). The percentage of repellency was calculated as 100 ⫻ (number on control ⫺ number on
L-LA)/(number on control). Percentage of repellency (⫽ Y) showed a signiÞcant (F ⫽ 19.7; df ⫽ 2, 5;
P ⫽ 0.004) parabolic response to the log10 of concentration of L-LA (⫽ X) (Y ⫽ 15.55X2 ⫺ 50.49X ⫹ 52.48)
and the relative Þt was very high (R2 ⫽ 0.89) (Fig. 1).
From the minimum value of Y when X ⫽ 1.62, the
minimum repellent concentration of L-LA was calculated to be 41.7 ppm.
Effect of L-LA Treatment on Both Forearms. To
determine the absolute repellency of L-LA, both forearms were treated with L-LA at various concentrations and then were exposed to mosquitoes. There
were no signiÞcant differences between the number
of alightments on the left and right forearms by t-test
at any concentration (Table 2). Furthermore, as the
concentration of L-LA increased, the sum of alightments for both forearms decreased; i.e., high concentrations of L-LA repelled the mosquitoes.
Fig. 1. Relationship between percent repellency against
alighting on human skin and the logarithm of L-LA concentration (ppm). Percentage of repellency was calculated by
comparing the number of alightments on L-LA-treated forearms with those on water-treated forearms. Six (1, 10, 100,
1,000, and 10,000 ppm) or three (3,000, 5,000, and 7,000 ppm)
replicates were done. In this test, proboscis-amputated mosquitoes were used.
Effect of L-LA Treatment on Mouse Skin. Similar to
the above tests, mice treated with L-LA showed repellency. When normal mosquitoes and mice were
used instead of mosquitoes lacking proboscises and
human forearms, L-LA acted as a repellent (Tables 3;
Fig. 2). Again, L-LA concentrations (except 10, 100,
and 1,000 ppm) showed signiÞcant differences between L-LA-treated and water-treated skin by t-test.
From the signiÞcant (F ⫽ 10.1; df ⫽ 2, 5; P ⫽ 0.017)
parabolic curve (Y ⫽ 17.39X2 ⫺ 61.15X ⫹ 65.03; Y ⫽
percent repellency on mouse skin, X ⫽ log10 of concentration for L-LA) with a high relative Þt (R2 ⫽
0.80), the minimum repellency concentration was calculated to be 61.7 ppm when X ⫽ 1.79.
Discussion
Smith et al. (1970) suggested that under some conditions LA could act as a repellent, whereas under
others it acted as a true attractant. Carlson et al. (1973)
showed that 50 ␮g of L-LA was the most attractive
amount tested, and these results were similar to data
Table 2. Mean ⴞ SE number of alightments of female Ae.
albopictus on similarly L-LA-treated forearms during a 10-min
exposure
Concn of
L-LA, ppm
n
L-LA
L-LA
df
t-value
1,000
3,000
5,000
7,000
10,000
3
3
3
3
3
3.58 ⫾ 0.25
2.95 ⫾ 0.31
1.72 ⫾ 0.23
1.18 ⫾ 0.20
1.15 ⫾ 0.15
1.85 ⫾ 0.29
3.55 ⫾ 0.36
2.35 ⫾ 0.22
1.40 ⫾ 0.22
0.97 ⫾ 0.17
2
2
2
2
2
2.164NS
3.328NS
⫺1.226NS
⫺0.983NS
⫺0.792NS
NS, P ⬎ 0.05.
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Concn of
L-LA, ppm
January 2001
SHIRAI ET AL.: L-LACTIC ACID AS A MOSQUITO REPELLENT
Table 3. Mean ⴞ SE number of bites by female Ae. albopictus
on L-LA- or water-treated mouse skin during a 10-min exposure
n
L-LA
Water
df
t-value
1
10
100
1,000
3,000
5,000
7,000
10,000
3
5
3
5
5
5
5
5
4.3 ⫾ 0.7
12.4 ⫾ 2.0
8.7 ⫾ 2.2
9.4 ⫾ 3.4
4.4 ⫾ 1.5
2.4 ⫾ 0.8
2.2 ⫾ 1.0
0.0
18.3 ⫾ 2.9
11.4 ⫾ 1.2
13.0 ⫾ 4.0
17.0 ⫾ 2.8
9.6 ⫾ 1.5
12.8 ⫾ 0.6
11.2 ⫾ 1.1
9.2 ⫾ 2.0
2
4
2
4
4
4
4
4
⫺4.583*
0.373NS
⫺0.476NS
⫺1.224NS
⫺3.399*
⫺8.072**
⫺5.285**
⫺4.695**
*, P ⬍ 0.05; **, P ⬍ 0.01; NS, P ⬎ 0.05.
obtained using L-LA ranging from 5 to 600 ␮g by Smith
et al. (1970). Our result showing an optimal concentration of L-LA that does not repel mosquitoes is
consistent with Smith et al. (1970) and Carlson et al.
(1973). Our experiments showed that high and low
concentrations repelled mosquitoes. Repellency at
the high range was consistent with reports by Brown
et al. (1951), but repellency at the low range conßicted with previous reports. Results that all concentrations displayed repellency are similar to Rudolfs
(1922) and Skinner et al. (1968). King (1954) described that LA acted as a repellent to mosquitoes
when applied to human skin, consistent with our results. In tests of both L-LA treated forearms, repellency increased proportionally to the concentration.
However, previous reports concluded that L-LA acted
as an attractant regardless of concentration. Naturally,
LA exists on human skin in sweat. Kuno (1956)
showed that sweat contains 35 mg % LA, and Robinson
and Robinson (1954) showed that the concentration
of LA ranges from 4 to 40 mEq. per liter of sweat. Both
reports showed similar concentrations of LA in sweat;
Fig. 2. Relationship between percent repellency against
mouse skin and the logarithm of L-LA concentration (ppm).
Percentage of repellency was calculated by comparing the
number of bites on L-LA-treated mouse skin with those on
water-treated mouse skin. Five (10, 1,000, 3,000, 5,000, 7,000,
and 10,000 ppm) or three (100 and 1,000 ppm) replicates
were done. In this test, normal mosquitoes were used.
350 Ð3,600 ppm, and this same range was used in our
experiments. Also, Thurmon and Ottenstein (1952)
discussed how the LA concentration in sweat might
differ between genders, being greater in males than in
females.
The minimum repellency concentration of L-LA in
our experiments was ⬇41.7 ppm, lower than that
present on human skin. Furthermore, Dill et al. (1938)
and van Heyningen and Weiner (1952) found that the
amount of LA in arm sweat was 1.5- to threefold
greater than in total body sweat. Therefore, the concentration of L-LA applied to the skin in our experiments might actually be higher when added to the
natural amount of LA on forearms. However, because
our experiments were done at 23⬚C, a temperature at
which measurable perspiration usually does not occur,
interference by natural LA appeared to be minimal. In
addition, we obtained similar results in tests with mice
that do not sweat.
Mosquitoes such as Ae. aegypti have LA-excited
neurons of the antennal grooved-peg sensilla, and the
LA sensitivity is inßuenced by sucrose-feeding, bloodfeeding, and oviposition (Davis 1984a, 1984b). Moreover, there is a pair of neurons sensitive to one chemoreceptor responding to LA in the grooved-peg
(A3) sensilla. One of them responds to LA with an
increase in spike frequency, whereas the other chemoreceptor exhibits a decrease when presented with
LA (Davis and Sokolove 1976). The different function
of these two chemoreceptors may explain both the
attractiveness and the repellency of Aedes mosquitoes
to LA.
The attraction of mosquitoes to human sweat has
been reported previously (Rudolfs 1922, Khan et al.
1969), whereas others have reported on its repellency
(Smart and Brown 1956). This controversy was discussed by Skinner et al. (1965). Brown et al. (1951)
showed that sweat was signiÞcantly attractive at a low
vapor concentration, but was signiÞcantly repellent at
a high concentration. van Heyningen and Weiner
(1952) assumed that the high lactate concentration in
sweat was not derived from plasma but was from the
metabolism of the sweat gland itself. However, Gordon et al. (1971) reported that sweat lactate in humans
was derived from blood glucose. On the other hand,
Sato (1977) described that at low sweat rates the
lactate concentration was as high as 30 Ð 40 mM, but
rapidly dropped to a plateau at around 10 Ð15 mM as
the sweat rate increased. Therefore, the sweat lactate
concentration seems to be related to the sweat rate.
The divergence of results among researchers on the
attraction or repellency of sweat to mosquitoes might
depend on the concentration of LA in sweat.
Lactic acid also is related to physiological condition
and disease. For example, lactic acidosis is a human
sickness in which blood lactate concentrations become abnormally high (Huckabee 1961, Kreisberg
1980). Similarly, accumulation of large amounts of LA
in blood occasionally causes problems in the human
body. From our observations, mosquitoes might avoid
large amounts of LA on human skin, so as to prevent
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Concn of
L-LA, ppm
53
54
JOURNAL OF MEDICAL ENTOMOLOGY
imbibing LA-abundant blood. Currently, the effect of
LA-rich blood in mosquito blood meals is unknown.
Acknowledgments
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Received for publication 2 March 2000; accepted 19 August
2000.
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We thank several students at the Toyama Medical and
Pharmaceutical University for their help in our experiment.
We also thank Ines Tomoco Matsuse for reviewing the manuscript and Hisashi Funada for valuable suggestions.
Vol. 38, no. 1