508 NATURE
,ee
hens,. but not to mice, pheasants, locusts, aphids and
flies. With rats the potentiator was active by the
oral and intra-peritoneal routes but not following
dermal administration. On demethylation of di-
methoate by reaction with salts of O,O-dimethyl
phosphorodithioic acid, O,O,S-trimethyl phosphoro-
dithioate and a potentiating material were formed.
The potentiator, also formed by the reaction of
dimethoate with lithium chloride, may be a salt of
O-methyl S-(N-methylcarbamoylmethyl) phosphoro-
thioic acid or a closely related compound. On
analogy with the potentiation of pure dimethoate by
O-ethyl O-p-nitrophenyl phenylphosphonothionate,
tri-o-cresyl phosphate and tri-o-cresyl phosphoro-
thionate10, it would appear that the potentiating
impurity in commercial dimethoate might act by
reducing the efficiency of the dimethoate detoxifica-
tion mechanism acting through hydrolysis of the
carboxyamide grouping.
Methyl and ethyl 'Cellosolve' (2-methoxy- and 2-
ethoxyethanol) have excellent solvent characteristics
for the formulation of organophosphorus insecticides,
particularly where a high degree of systemic activity
is desired after application to foliage. As a result of
routine toxicity checks on the properties of technical
dimethoate formulated in methyl 'Cellosolve', an
unexpected toxic hazard was observed. The rat oral
LD50 decreased on storage from an initial 150-250
mgm./kgm. to 30-40 mgm./kgm. after seven months
storage in England. After nine months storage under
tropical conditions the LD50 was less than 15 mgm./
kgm., and after storage at 100° C. for 63 ·hr. in the
laboratory the LD50 was 8 mgm./kgm. The mam-
malian toxicity increased several-fold before any
change in insecticidal activity was evident. Further
development of this W1Stable formulation was
immediately stopped.
Chromatography by described procedures', 9 of the
decomposed dimethoate in methyl 'Cellosolve' yielded
fourteen phosphorus-containing products. The de-
gradation involved hydrolysis at the amide and ail
the ester groupings, a marked loss of thiono sulphur,
and replacement of both the O-methyl and N-methyl-
a.mino groupings by 2-methoxyethanol. No evidence
was obtained for the formation of pyrophosphates or
thionopyrophosphates. The most toxic fraction was
purified and identified as an O,O-dialkyl S-(N-
methylcarbamoylmethyl) phosphorothiolate with
probably one, but possibly both, methyl groups of
dimethoate replaced by 2-mcthoxyethyl groupings.
This compound was less toxic to flies than dimethoatc,
but had a rat oral LD50 of about 1 mgm./kgm. and
a mouse intra-peritoneal LD50 of about O·5 mgm./
kgm. The increase in toxicity to mammals of tho
decomposed dimethoate formulation was greater
than that attributable solely to this single toxic
product. Since no other single component was toxic
enough to account for the discrepancy, it appears
that a potentiation of toxicity also occurred among
the materials in the dimethoate -methyl 'Cellosolvo'
formulation.
The relationship of stability to toxicity of di-
methoate formulated in a number of solvents was
investigated. Little if any hydrolysis or change in
mammalian toxicity occurred in non-hydroxylic
solvents. Polyhydroxylic solvents resulted in stabil-
ity during short storage without the formation of
highly toxic degradation products. Of several
monohydroxylic solvents, only methyl and ethyl
'Cellosolves' resulted in formulations highly toxic to
mammals on short-term storage at 70° C. From this
© 1961 Nature Publishing Group
February 11, 1961 voL.
work a commercial formulation was developed which
showed no significant change of toxicity after 162
days storage at 40° C.
The potential hazard which could have arisen is
not unique for dimethoate, since O,O-dimethyl S-
(N,N-dimethylcarba.moylmethyl) phosphorodithioate,
O,O-dimethyl S-(1,2-dicarbethoxyethyl) phosphoro-
dithioate (malathion), O-methyl O-(2,4,5-trichloro-
phenyl) phosphoramidothionate (Dow ET-15) and
O,O-dimethyl O-(2,4,5-trichlorophenyl) phosphoro-
thionate (Dow ET-57) were also found to show a
several-fold increase in mammalian toxicity after
reaction with methyl 'Cellosolve' at 70° C. for eight
days.
When 'Cellosolves' are to be used in formula.ting
phosphorothionate insecticides, the possible ester
and amide exchange reactions to yield products of
increased mammalian toxicity should be carefully
investigated. A more complete account of these
investigations will appear elsewhere.
Publication of this communication is approved by
the Director of the Wisconsin Agricultural Experi-
ment Station, and by the Directors of Fisons Pest
Control, Ltd. The investigation was supported in
part by a Haight Travel Fellowship from the Graduate
School of the University of Wisconsin to one of us
(J.E. C.), and by grants from the U.S. Public Health
Service, National Institutes of Health, and from the
U.S. Atomic Energy Commission (Contract No.
AT(ll-1)-64, project No. 14). We thank Dr. E. F.
Edson, Dr. G. S. Hartley, Mr. A. J. Lambie and Mr. P.
Carter of Fisons Pest Control, Ltd., and Mrs. Lydia
McBride of the University of Wisconsin, for their
encouragement and assistance.
JOHN E. CASIDA
Department of Entomology,
University of Wisconsin,
Madison, 6, Wisconsin.
D. M. SANDERSON
Medical Department,
Fisons Pest Control, Ltd.,
Chesterford Park Research Station,
Nr. Saffron Walden,
Essex.
1 Santi, R., and de Pietri-Tonelll, P., Nature, 183, 398 (1959); and
RichercM aul meccani-Bmo d' azwne ddla N -m<mometilammide dell'.
acido 0, O-demetilditi,o-Josforilaceti,co. Montecatini, Milano, Italy,
Bull., 29 pp. (1959).
1 Geering, Q. A., World Crops, 11, 141 (1959).
• American Cyanamid Co., Stamford, Conn., U.S.A., Dimethoate (E.I.
12,880) Bull., 86 pp. (1959).
• Hewitt, R .• Brebbla, A., and Waletzky, E., J. Econ. Entomol.,
51, 126 (1968).
'Hewitt, R., Emro, J., Entwistle, J., Pankavich, .J., Thorson, R.,
Wallace, W., and Waletzky, E., J. Eron. Entomol., 61,445 (1958).
• Fontanelli, R., and Lanforti, G. F., Rmd. ist. auper. aanili,;, 22,
82 (1969).
7 Dauterman, W. C., Casida, J.E., Knaak, J.B., and Kowalczyk, T.,
J. Agric. Food CMm., 7, 188 (1969).
• Kaplanis, J. N., Robbins, W. E., Darrow, D. I., Hopkins, D. E.,
Monroe, R. E., and Treiber, G., J. Econ. Entomol., 52, 1190 (1969).
• Dauterman, W. C., Vlado, G. B., Casida, J. E., and O'Brien, R. D.,
J. Agric. Food Chem., 8, 116 (1960).
"Seume, F. W., and O'Brien, R. D., Tozicol .. App, Pharmaccl., 2, 495
(1960).
Lysine as a Mosquito Attractant
PREVIOUS work has indicated that attractive
factors other than carbon dioxide are present in the
vapour from mammalian blood1 and body exudations•.
A distillate obtained from mammalian blood proved
highly attractive to Culex pipiens•. It has been
recently reported that a mixture of nine biological
acids and bases was attractive to 0. pipiens and
 No. 4763 February 11, 1961 NATURE
A rwpheles rnaculipennis at concentrations down to
0 ·05 per cent, although none of the compounds was
attractive when tested individually•.
Our experiments on Aedes aegypti were guided by
an old report 5 that peptone and certain amino-acids
had shown attractiveness for A. sollicitans, and by
the fact that protein hydrolysates are employed
successfully as baits for fruit flies. Control and
experimental solutions were exposed on filter-paper
in wann Petri dishes' in a cage containing mosquitoes
maintained only on sugar-water ; the number of
approaches to the experimental solution, as compared
to that to the control, was expressed as the attrac-
tiveness ratio. Of 16 different protein hydrolysates
commercially available ahd tested in 10 per cent
solution, 11 proved to be attractive. Of these, 8
were more attractive than ox blood plasma ; on
distilling them to dryness at 7 mm. mercury pressure
and 37° C., the distillates of five of them proved
significantly attractive.
Individual amino-acids were then tested, dissolved
in distilled water in the relative amounts in which
they occur in 10 per cent lactalbumin hydrolysate.
Of the 17 tested, the only amino-acids to be signific-
antly attractive were (with their attractiveness
ratios) : L-arginine 1 ·24, L-alanine l ·32, and L-lysine
3 ·84. A mixture of all the amino-acids except
lysine showed an attractiveness ratio of l · 14 ;
addition of lysine raised the attractiveness ratio to
2 ·80. At the buffered pH of 7 ·4, lysine showed an
attractiveness ratio of 3 · 13, and raised the attractive-
ness ratio of the amino-acid mixture from 1 ·28 to
6·13.
L-Lysine hydrochloride was not attractive in acid
solution, but became highly attractive in alkali
(attractiveness ratio 2 ·59 at pH 7 ·4) ; L-lysine free
base was attractive also in acid solution (attractive-
ness ratio 5 ·62 at pH 4 ·4). The hydrochloride of the
unnatural enantiomorph D-lysine showed an attrac-
tiveness of l ·20 at pH 6 ·5 and of 3 ·70 at pH 11 ·6.
The potency of L-lysine at pH 10 was such that in
0 ·001 per cent and O·0001 per cent (1 p.p.m.) solutions
its attractiveness ratios were respectively 5 ·95 and
2 ·86. When a O·7 per cent solution of L-lysine was
distilled at 7 mm. mercury pressure and 37° C.,
the distillate showed an attractiveness ratio of
7 ·73 and gave a positive ninhydrin test.
When a mixture of O·88 per cent L-lysine and O·24
per cent L-alanine was chromatographed through a
50 x l ·2 cm. column of 'Dowex 50W-X4' resin 6
and the successive eluate fractions tested, the attrac-
tiveness of alanine appeared at 40-50 ml. and that of
lysine at 75-88 ml. after the addition of the sample
to the column. When deproteinized lactalbumin
hydrolysate (10 and O·l per cent solutions assayed)
was similarly fractionated, the greatest attraction
was at 66-90 ml., with a minor peak at 53 ml. The
fraction at 87 ml. was tested by paper chromatography
and found to contain L-lysine. When deproteinized
ox blood plasma was chromatographed, the main
peak of attractiveness was around 90 ml., with a
smaller peak at 30-50 ml. ; the amino-acid present
in the 93- and 88-ml. fractions was identified as
L-lysine. When deproteinized human blood plasma
was fractionated, the main peaks were at 40-50 ml.
and around 80 ml.; the fractions at 44 and 81 ml.
were found to contain L-alanine and L-lysine respect-
ively. In all the fractionations, a minor peak was
found at about 120 ml., possibly due to L-arginine.
The results thus indicate that the most attractive
principle in the protein hydrolysate and blood
© 1961 Nature Publishing Group
509
plasmas tested was L-lysine. Of possible contamin-
ants, ornithine and asparagine showed no attractive-
ness, whereas ammonia, putrescine and cadaverine
were strongly repellent at high concentrations and
never became attractive on progressive dilution.
L-Lysine proved attractive not only to the Orlando
strain of Aedes aegypti, but also to strains from
Penang, Key West and Trinidad. It was also attrac-
tive to field-caught Aedes stimul,ans, and to Gulex
pipiens at evening. A solution of L-lysine exposed in
the cage stimulates the resting mosquitoes to take
flight, and they react to it as to a guinea pig or human
hand. The concentration ofL-lysine in human blood 7
and sweat• is about 27 µgm. per ml. Since it may be
distilled from aqueous solution and is attractive at
l p.p.m., L-lysine could thus be the main attractive
factor of mammalian blood and body emanations.
Full details of the work will be published elsewhere.
We are indebted to the Defence Research Board of
Canada for financial support of these investigations.
A. w. A. BROWN
A. G. CARMICHAEL
Department of Zoology,
University of W astern Ontario,
London, Canada.
1
Burgess, L., and Brown, A. W. A., Bull, Ent. Bes., 48, 783 (1957).
• Laannan, J'. J'., .Acad, Proefschr. Leukn, 144 (1955),
' Schaerffenberg, B., and Kupka, E., Oesterr. Zool. Z., 3, 410 (1951).
• Schaerffenberg, B., and Kupka, E., Naturwi,a,, 46, 457 (1959).
• Rudolfs, W., Bull. New Jersey Agne, Ec,;p. Sta., 327, 23 (1922).
• Moore, S., and Stein, W. H., J, Biol. Ol~m., 211, 893 (1954),
'Stein, W. H., and Moore, 8., J. Biol. Chem., 211, 915 (1954).
'Hier, S. W., Cornbleet, T., and Bergeim, 0., J, Biol. Chem., 166,327
(1946),
A Method of dissolving the Protective
Spittle Masses of Frog Hopper Nymphs
DURING the course of biochemical investigations
into the nature and composition of the spittle masses
produced by cercopid nymphs (Phil,aenus spumarius
L. and Neophilaenus sp.) it was observed that these
masses decomposed within a few minutes of being
placed in a 2/3 N solution of sulphuric acid.
Since Cercopidae are of considerable economic
inportance, especially Aeneol,amia varia saccharina
in the cane sugar industry, and are protected from
the action of insecticides by the spittle which they
produce, it was realized that the above observation
might be utilized in the measures taken for their
control.
It waa shown in the laboratory that the rate of
solution of spittle masses (Fig. 1) was dependent on
the normality but largely independent of the nature
of the acid, hydrochloric and nitric being as effective
as sulphuric acid. Since the rate of solution is not
only dependent on the normality of the acid, but
also on size of spittle mass and amount of agitation,
the values given in Fig. 1 therefore relate only to
spittle masses of the size produced by late fourth
and early fifth instar nymphs, and subjected to gentle
agitation. Field trials were then carried out using
sulphuric acid at various concentrations. This acid
was chosen because of its low vapour pressure. Diel-
drin (0 ·06 per cent), added as Shell 'Dieldrex', was
added to each solution to kill exposed nymphs and
thus prevent the production of new spittle masses.
The results are given in Table 1, which also shows
the effect of weather on both the rate and total
destruction of spittle masses.