Entomol. exp. appl. 43: 25-29, 1987
9 Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands
25
The role of a m m o n i a in the attraction of females of the Mediterranean fruit fly to
protein hydrolysate baits
M. Mazor 1, S. Gothilf 1 & R. Galun2
llnstitute of Plant Protection, ARO, The Volcani Center, Bet Dagan, Israel; and 2Department of
Parasitology, Hebrew University of Jerusalem, Hadassah Medical School, Jerusalem, Israel
Keywords: Mediterranean fruit fly, Ceratitis capitata, attractant, ammonia, protein hydrolysates
Abstract
Attraction of female Mediterranean fruitfly (medfly) to ammonia and to protein baits was studied with
an olfactometer. Ammonia bait (1 cc/trap) proved to be an effective lure. The maximum number of females
was caught in traps loaded with 0.01 M ammonia solution with a release rate of 5.28/zg/cc/h.
A positive correlation was found between female catch and the ammonia release rate of various protein
baits, especially from dry protein hydrolysate of which casein hydrolysate was the most effective. However,
casein hydrolysate was less attractive than certain ammonia solutions having a lower rate of ammonia release.
Elevation of the pH of the liquid commercial baits, Buminal and Naziman, increased the latter's efficacy
as medfly baits but the increased stimulation could not be strictly correlated with the increased rate of ammonia release. It is therefore suggested that medfly olfactory response to protein baits is affected not only by
ammonia but by other volatiles as well.
Introduction
Tephritid fruit flies require a source of protein to
complete egg maturation. This requirement is probably the main cause for the strong attraction of females towards decomposing proteinaceous substances, first observed by McPhail (1939). Based on
this behavioural reaction, hydrolysates of protein
have been included in baits to control fruit flies
(Steiner, 1952). Since ammonia is emitted from
decomposing proteins, it was assumed that attraction of fruit flies to protein baits is related to the
release of ammonia. Indeed ammonium salts have
long been used as a lure in fruit fly traps but with
conflicting conclusions concerning their effectiveness. Some workers suggested that ammonia is a
weak attractant (e.g. Ripley & Hepburn, 1929;
McPhail, 1939; Gow, 1954), while others considered it to be an effective lure (e.g. Hodson, 1948;
Frick, 1952; Prokopy, 1975; Reissig, 1975; Vita et
al., 1980). Recently Morton & Bateman (1981) and
Bateman & Morton (1981) studied the chemistry of
the components and volatiles of protein hydrolysates but did not identify any particular compound
as being responsible for the attraction of the
Queensland fruit fly-Dacus tryoni. However, they
did find that certain solutions of ammonium bicarbonate were much more effective attractants than
the standard protein baits, and that at low pH the
attraction of the fly to the protein baits is caused
by the small amounts of ammonia released from
the baits. At high pH, other attractive volatiles are
apparently released .from these baits. In the present
work, we have tested the attractancy of the female
Mediterranean fruit fly, Ceratitis capitata (Wied.)
(medfly), to ammonia solutions and several proteinaceous baits. We examined the relationship between the attractancy of these baits and their rate
of ammonia release.
Materials and methods
Medfly pupae originating from local fruit plan-
26
tations, reared for 5 to 10 generations in the laboratory, were supplied by the Israel Cohen Institute for
Biological Control, Rehovot, Israel. Pupae and the
emerging flies were kept in a room with windows,
under a natural day:night regime and a controlled
temperature of 25 + 1 ~
All experiments were
conducted in a separate room under the same conditions. Three- to 10-day-old females were used for
the experiments. Preliminary tests showed no
difference in the olfactory response of flies in this
age range. Flies were maintained on granulated sugar and water absorbed on cotton wool.
The following baits were tested: the enzymatic
hydrolysates of casein, lactalbumin, soy, and yeast
(ICN, Cleveland, OH, U.S.A.), all in dry powder
form; Buminal |
(Bayer, Leverkusen, F.D.R.),
Naziman (Nasid Ltd., Tel Aviv, Israel) and PIB 7
(Staley Manufacturing Co., Decatur, IL. U.S.A.), in
liquid form. The ammonia stock solution was a
concentrated volumetric solution (BDH) adjusted
to several concentrations between 0.0001 and
0.1 M.
The response of the medfly females to the baits
was examined in an olfactometer and in traps
described by Gothilf & Galun (1982). All tests were
of the two-choice type. During the test six traps
were suspended from the horizontally rotating
wheel of the olfactometer. Three traps were loaded
with one bait and three with a second bait or unbaited. One cc of the experimental bait, whether
liquid or dry powder, in a 3-cm-long • 1.5-cm i.d.
glass tube (exposure surface of 1.8 cm2), was put
in each baited trap. Traps of different treatments
were placed alternately in the olfactometer. 250 or
500 female flies were placed in the olfactometer before the start of testing and the same flies were used
for several tests, usually one test per day, but sometimes two. Dead flies were replaced by live females
of the same age before every test. Each test lasted
one hour. At the end of that time the entrance holes
of the traps were plugged with a piece of cotton
wool and the traps were transferred to a refrigerator
for a few minutes. With this procedure the flies became less active and could be counted more easily.
The trapped flies were collected with an aspirator,
counted, and released back into the olfactometer.
For a given bait in a test, the number of flies captured in the three traps was recorded and the test
was repeated 5 - 1 2 times. Four olfactometers were
used so that four tests could be run simultaneously.
The release rate of ammonia from protein
hydrolysate baits and from ammonia solutions was
determined as follows: 1 cc of an experimental bait
was put in a 3-cm-long x 1.5-cm-i.d. glass tube of
the type used in the trap, two or three such tubes
were prepared when materials of low ammonia release rate were tested. The tubes were placed in a
100-ml flask. Air was pulled with a pump over the
sample at a rate of 150 ml/min and then into two
consecutive tubes, through cinter glass filters, each
containing 10 ml distilled water. The emitted ammonia was trapped mainly in the first water trap;
the second trap contained less than 10% of the
released ammonia. The duration of this procedure
ranged from 1 to 4 h, depending on the expected release rate of ammonia from the experimental material. The amount of ammonia trapped in the distilled water was determined by the phenolchlorite
method (Solorzano, 1969) and was calculated as #g
ammonia released from 1 cc experimental material
per hour. The release rate was checked twice for
each bait and only small differences were found.
Except for Fig. 2, the average of the two checks is
given. All the chemical work was carried out at an
ambient temp. of 25 _+ 1 ~
Results and discussion
Before questioning the role of ammonia in medfly attraction to baits, it was important to assess the
relationship between rate of ammonia release from
a bait and the olfactory reaction of the flies. Using
a bait of pure ammonia solution, which released
only ammonia (ignoring water molecules which
had no stimulatory effect in the present experimental environment), a clear correlation was found between the concentration of the solution and its
stimulation (Fig. 1) and between the concentration
and rate of ammonia release (Fig. 2). The relationship between concentration of ammonia solution
and the number of trapped females is linear,
characteristic of log stimulus - response relation
up to a concentration of 0.01 M (Fig. 1). The relative ammonia release rate at maximum stimulation
was 5.28/~g/cc/h. Beyond 0.01 M the correlation
was negative, i.e., at higher concentrations with
higher release rates of ammonia, the solutions became less and less attractive.
The olfactory response of medfly females to vari-
27
80
0
1
I
I
I
I
70
Bait
Mean* 9
catch _+ s . e .
Ammonia release
tzg/cc/h
60
Casein hydrolysate
Lactalbumin hydrolysate
Buminal
Soy hydrolysate
Naziman
PIB 7
Yeast hydrolysate
Control
192.0_+ 5.8 a
141.0+_ 7.4 b
135.6_+ 6.0 b
109.2+ 10.6 c
92.4_+ 9.7 c
83.6+ 9.5 c
29.4+_ 6.1 d
4.5
3.2
2.0
0.4
1.4
0.1
0.1
0.1
0
50
r~
t,U
.-I
4O
3O
* Average of five tests. Average of control was calculated from
catches in unbaited traps in all tests. No. followed by same
letter not significantly different at 5~ level.
h
20
iO
0
I
1
0.0001
0001
0005
AMMONIA
I
I
0.01
I
0.05 O I
CONCENTRATION
(M)
Fig. 1. Attraction of 9 medflies to traps baited with ammonia
solutions of different concentrations. Dots: means of ten tests.
Bars: +_S.E.
A
2O
0
o
'
I
'
I
I
I
'
I
'
_- V -" 1 5 8 . 8 3 X + 0.79
l/
iq
16
IM
t'r
I.iJ
t~O
,,~
Table 1. Attraction of medfly females to protein hydrolysate
baits and their corresponding ammonia releases.
8
IM
_J
IM
r,r
4
0
0
i
0
I
0.02
AMMONIA
i
I
0.04
,
I
0.06
CONCENTRATION
i
I
,
0.08
l
O. I
(M)
Fig. 2. Ammonia release rate from ammonia solutions of
different concentrations.
ous baits and their corresponding release rates o f
a m m o n i a are summarized in Table 1. In this table
the various baits were c o m p a r e d to an unbaited
control. Since the control catch was practically nill,
statistical analysis was unwarrented. However,
within the bait series, significant differences were
detected as indicated in the table. The most attractive a m o n g the tested baits was casein hydrolysate;
next was lactalbumin hydrolysate and Buminal;
and less stimulatory were soy hydrolysate, Naziman, PIB 7 and yeast hydrolysate. A positive correlation was f o u n d between the stimulatory effect o f
the baits and the a m o u n t s o f a m m o n i a released
from them (r =0.77). If the dry baits are considered
as a separate group, an even stronger relationship
(r=0.96) exists between a m m o n i a release and
stimulation (Fig. 3).
In spite o f the p r o n o u n c e d relationship between
medfly attraction and a m m o n i a release from the
dry baits shown in Fig. 3, it seems that attraction
to the dry baits is also affected by other volatiles.
In Table 2 various concentrations o f a m m o n i a solution were c o m p a r e d to casein hydrolysate in pairs.
The flies preferred a m m o n i a solutions at concentrations o f 0.01 M and 0.0075 M although their
a m m o n i a release, especially o f the latter, is less
than that o f the casein. There was no difference between the reaction o f flies to casein hydrolysate and
to a m m o n i a solutions o f 0.005 M and 0.0025 M
whose a m m o n i a release rates are m u c h smaller
than that o f the casein. It seems, therefore, that alt h o u g h a m m o n i a release can be used as a criterion
for stimulation o f dry protein hydrolysates, it is not
the only c o m p o u n d which affects the behavioral
28
I
200
I
I
I
Y = 52.17X § 3 0 . 8 0
150
1-
IJJ
_1
I00
:E
IJJ
M.
50
YH
o
0
AMMONIA
I
I
I
1
0.8
1.6
2.4
3.2
RELEASE
RATE
(IJg/ec/hour)
Fig. 3. Relationship between rate of a m m o n i a release and 9
catch in traps baited with dry protein hydrolysates. (CH) casein
hydrolysate; (LH) laetaibumin hydrolysate; (SH) soy hydrolysate; (YH) yeast hydrolysate. Dots: See Fig. 1, but 5 tests. Bars:
see Fig. 1.
Table 2. Medfly attraction to a m m o n i a solutions and to casein
hydrolysate.
Test
1
2
3
4
5
Bait
Ammonia0.001M
Casein hydrolysate
Ammonia0.0025 M
Casein hydrolysate
A m m o n i a 0.005 M
Casein hydrolysate
A m m o n i a 0.0075 M
Casein hydrolysate
A m m o n i a 0.01 M
Casein hydrolysate
Mean* 9
catch_+ s.c.
A m m o n i a release**
tzg/cc/h
23.7+_2.0b
40.4+ 3.3 a
21.2+1.4a
24.0_+ 2.3 a
27.7+2.5 a
24.1 • 4.0 a
46.4 +- 3.1 a
20.0_+ 1.8 b
56.8•
a
11.5 +_ 1.7 b
0.1
3.1
1.2
3.1
1.6
3.1
2.0
3.1
2.4
3.1
* Average of ten tests. Significancy, see Table 1.
** Values of release from a m m o n i a solutions calculated from
regression line of Fig. 2.
reaction of the flies to these baits, and that other
volatiles, apparently repellents, are involved in the
fly-bait relationship.
As for the three liquid baits, it is improbable that
their attractiveness resulted only from their ammonia release which was low compared with the release rate of dry baits of similar attractiveness (Table 1). That such low rates of ammonia release are
not enough for the recorded females catches, can
also be deduced from the tests with pure ammonia
which were mentioned before (Figs. 1 and 2).
Following the results of Bateman & Morton
(1981) who succeeded in improving the attractancy
of yeast hydrolyate to Dacus tryoni by elevating its
pH, we examined this process with Buminal and
Naziman which are commonly used in bait sprays
of fruit trees. The pH of the commercial preparations is 5.8 for Buminal and 4.5 for Naziman. At
these pHs their ammonia release rate is relatively
low (Table 1). Using 5N NaOH, the pH of both
baits was adjusted also to 7.5, 8, 8.5 and 9. The
catch corresponding to a given pH was compared
to that of an unbaited control. Again the control
catch was virtually nill and results were analyzed as
in Table 1. Table 3 illustrates the differences detected among the various formulations. Buminal at pH
of 7.5, 8 and 8.5 was more attractive to the females
than at pH of 5.8 and 9, with no significant difference in number of females trapped between pHs of
7.5, 8 and 8.5. Naziman showed a slightly different
pattern of relationship between attractancy and
pH: at pH 8 it was more attractive than at lower or
higher pHs. (Table 3). It is obvious from these
results that the attractiveness of these liquid baits
Table 3. Female catch and a m m o n i a release of Buminal and
Naziman at different pHs.
pH
Mean* 9
catch _+ s.e.
A m m o n i a release
#g/cc/h
Naziman
4.5
7.5
8.0
8.5
9.0
Control
22.2+_2.9
42.1+_2.9
75.3 +_5.3
53.4+_5.6
25.2 • 5.4
0.3
c
b
a
b
c
0.1
2.5
6.4
15.8
26.8
Buminal
5.8
7.5
8.0
8.5
9.0
Control
29.8+2.5
45.7+2.1
49.4+6.1
49.9 _+4.4
36.9+4.2
0.2
b
a
a
a
ab
0.4
4.6
7.4
11.5
23.6
*
Average of twelve tests. Significancy, see Table 1.
29
to medfly females can be increased by elevating the
pH. Although increase in pH was followed by
higher ammonia release, the reason for the increase
in attractiveness cannot be attributed solely to
elevated ammonia release, because the relation between pH, ammonia release and female catch was
different and specific for each of the two liquid
baits (Table 2). It can therefore be assumed that
volatiles other than ammonia also play a role in female medfly attraction to these baits.
We show here in agreement with the findings of
Bateman & Morton (1981) with the Queensland
fruit fly, that ammonia is a very effective attractant
for female medfly. It may be an effective lure also
for many other fruit flies. As for the protein baits,
their stimulation of medfly females could not be
attributed solely to their ammonia volatiles. It is
suggested that other unidentified volatiles have an
important effect on the olfactory reaction of the fly
to protein baits.
Acknowledgement
Contribution from the Agricultural Research Organization, (ARO), Bet Dagan, Israel. No. 1653-E,
1986 series.
R~sum~
Le role de l'ammoniaque dans l'attraction des
femelles de la mouche mdditerandenne aux attractifs ?l base d'hydrolysats de proteines
Uattraction de la femelle de la mouche mOditeranOenne h l'ammoniaque et aux attractifs protOiniques a 6t6 6tudiOe au moyen de l'olfactomOtre
dOcrit par Gothilf & Galun (1982). Uammoniaque
(1 cc/piege) s'avOra ~tre un attractif efficace. Le
nombre maximum de femelles fut attrapp6 dans les
piOges chargOs avec une solution d'ammoniaque
0.01 M ayant une vitesse de diffusion de
5.28/tzg/cc/h.
Une corrOlation positive a &6 trouvde entre les
captures de femelles et les vitesses de diffusion de
divers attractifs protOiniques, particulierement ceux
base d'hydrolysats de protOines secs parmi
lesquels le plus effectif a 6t6 l'hydrolysat de casOine.
Toutefois, l'hydrolysat de casOine a 6t6 moins attractif que certaines solutions d'ammoniaque ayant
une vitesse de diffusion plus basse. Une hausse du
pH des attractifs liquides commerciaux, Buminal et
Naziman, a augment6 leur efficacit6 en tant qu'attractifs pour la mouche mOditeranOenne; toutefois
la stimulation plus intense n'a pu 6tre mise en correlation stricte avec une vitesse accrue de la diffusion
de l'ammoniac. C'est pourquoi il est suggOr6 que la
rOaction olfactive de la mouche mOditerranOenne
est rOgie, non seulement par l'ammoniaque mais
aussi par d'autres substances volatiles.
References
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799- 801.
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Accepted: July 5, 1986.