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J. stored Prod. Res. Vol. 34. No. I, pp. 59-74, 1998 0 1998 Elsevier Science Ltd. All rights reserved Pergamon
PII: S0022-474X(97)00019-2 Printed in Great Britain
0022-474X/98 519.00 + 0.00
The Assessment of Potential Attractants to Beetle Pests: Improvements to Laboratory
Pitfall Bioassay Methods COLIN MORGAN,‘* JOHN SHERINGTON,Z IRENE GUDRUPS
and NATALIE S. BOWDEN’
‘Central Science Laboratory, Sand Hutton, York Y04 ILZ, U.K. and ‘Natural Resources Institute, University of Greenwich, Central Avenue,
Chatham Maritime, Chatham, Kent ME4 4TB, U.K.
(Accepted 23 April 1997)
Abstract-A single pitfall bioassay has been used as a standard method for testing the response of small beetle pests to potential attractants. However, previous studies have suggested that there may be some potential for improvement and the need for a better understanding of the methodology. In this study, eight species of stored product insects were tested against known attractants to investigate certain aspects of the bioassay procedure likely to influence the sensitivity of the test. The insects tested were Ahusverus dew, Oryzuephilus surinumensis, Rhyzopertha dominica, Prostephanus truncatus, Sitophiius grnnarius, S. oryzae, S. zeamais and Stegobium puniceum. The factors investigated were the test duration, pitfall lip size and arena size.
For the majority of species tested, either the differentiation between control and attractant results was good, irrespective of the bioassay design, or changes to the bioassay design markedly improved the catch. However, this bioassay method may not be suitable for S. ream&. Of the assessment periods tested, it was generally found that 1 h was optimal, or at least as good as the longer alternatives. Although 100 mm arena sizes generally trapped larger numbers of insects, the differentiation between treatment and control samples was optimal with diifering arena sizes for the different insect species. Pitfall tip size appears to be the least important factor in increasing the differentiation between treatment and control samples.
Although these ihtdings go some way towards improving the methodology of the single pitfall bioassay, there remain some questions on the variability of the insect response under the test conditions. However, on the basis of previous experience aud the studies reported here, we are able to suggest the most effective design of the single pitfall bioassay for the species tested. Recommendations for particular aspects of the bioassay design have been chosen to achieve maximum differentiation between control and attractant. 0 1998 Elsevier Science Ltd. All rights reserved
Key words-attractant, pitfall bioassay, stored product insects, volatile
INTRODUCTION
The use of attractants in traps often significantly improves their performance, increasing the chances of detecting low-density insect infestations. However, the laboratory testing of potential attractants is often time consuming and expensive, ranging from the isolation and identification of suitable materials to the determination of release rates and quantities of attractant to use. Tests carried out under controlled laboratory conditions are, however, often critical in the development of a suitable lure.
*Corresponding author.
SPR 34/l< 59
60 Cohn Morgan et al.
Species
Table I. Attractants tested in pitfall bioassays
Attractant
Ahasverus advena
Oryzaephilus surinamensis Rhyzopertha dominica
Prostephanus truncatus Sitophilus granarius
Sitophilus oryzae
Sitophilus zeamais Stegobium paniceum
I-Octen-3-01; 200 pg ai per ml pentane; 5 ~1 treatments’ Attractant mixture based on natural pheromone2
Dominicalure 1 and 2 (1 : 1) in hexane 10 ng/pl; 20 pl per treatment’ Maize volatiles in dichloromethane; 20 pl per treatment’
2S,3R-Sitophilate; 2 ng per pl solvent; 5 ~1 per treatment4 Maize volatiles in dichloromethane; 20 pl per treatment’ Maize volatiles in dichloromethane; 20 pl per treatment’
Carob volatiles (CSL Ref. A35/36) 400 gh/pl; 5 pl treatments’
‘Pierce et al. (1991). ?Personal communication (Dr. John Chambers). “Pike et al. (1995). ‘Phillips et al. (1989). 5Personal observation (Colin Morgan). 6Williams et al. (1981). ‘Trematerra and Girgenti (1989).
At the Central Science Laboratory (CSL) and Natural Resources Institute (NRI), a single pitfall bioassay has been used for a number of years as a standard method for investigating potential attractants for a variety of stored product beetle pests. It was originally developed from the two-choice pitfall bioassay described by White and Birch (1987). However, results obtained from previous studies have often suggested that there was a potential for improvement and the need for a better understanding of the methodology. In particular, it was felt that refinement of the method might reduce some of the variation in bioassay results. This work was initiated at CSL (Morgan and Healey, 1993) and then expanded into a larger scale trial of some of the variables which may influence the response of insects to an attractant under bioassay conditions.
MATERIALS AND METHODS
Experimental details
Unsexed adults, approximately l-3 weeks post-eclosion, were used in the study. The insect species studied were Ahasverus advena (Waltl), Oryzaephilus surinamensis (L.), Rhyzopertha dominica (F.j, Prosiephanus iruncaius (i-ioriij, Siiophiius granarius i;-.j, S. oryzae (Lj, S. zeamais Motschulsky and Stegobium paniceum (L.).
To assess the effectiveness of the bioassays, the insects were tested using an appropriate attractant as listed in Table 1.
Two sizes of arena were assessed: 100 mm and 200 mm in diameter (Figs 1 and 2). Arena bases were formed using either 150 mm or 270 mm diameter filter papers, each with a central hole of 25 mm in diameter. Glass tubes, 25 mm x 75 mm, were positioned over the holes and either stuck to the oaoer using wood adhesive (0 mm lipj or pushed through the oaner to form lins of 25 mm r--r ~~ \- ~~~~~~ r ~~--- ~~__ .~~_ r~~r-- ._ ~~~___ --=- or 5 mm. Tubes that were glued were left to dry for a minimum of 24 h prior to testing.
I IOmm diameter Lip: 0,2.5 or Smm
IlXhnm diameter
Glass tube (25 x 75mm) _ glued into central hole. treated or untreated with
attractant
Fig. I. Pitfall bioassay arena (100 mm) with components expanded for clarity.
The assessment of potential attractants to beetle pests 61
t3lass ate
“\
Lip: 0,2.5 or 5mm
/ L
2OOmtn diameter aluminium ring j
1
270mtn diameter
\ Melaminecoated Fluorl wood support
Glass tube (25 x 75mm) - glued into central hole.
coating
Filter paper disc: treated or untreated with
Fig. 2.
attractant
Pitfall bioassay arena (200 mm) with components expanded for clarity.
The filter papers were then positioned on pre-drilled staging, and aluminium rings, 100 mm or 200 mm in diameter, were placed centrally on the paper bases. All rings had previously been coated with a PTFE dispersion (Fluon), a minimum of 24 h before testing, to prevent insect escape.
Insects were introduced to the arenas, and the test was started immediately after placement of a filter paper disc, 20 mm in diameter, either untreated or attractant-treated, into the bottom of the pitfall tubes. To reduce air disturbance, the arenas were covered using either a 110 mm crystallizing dish for the 100 mm arena or a glass plate for the 200 mm arena. The numbers of insects found in individual pitfall tubes were recorded at 1 and 2 h, and at test completion at 3 h. Each of the 12 combinations (arena size x treatment x lip size) was replicated twice on each of four consecutive days with testing carried out between 14.00 and 18.00 h. Twenty insects were tested in each replicate arena. S. zeumais and P. truncatus were tested under conditions of 27 + 1°C and 70 f 5% r.h. The conditions for the other six species were set at 20°C and 50% r.h. All insect species were tested separately and in the dark, except during assessment when low-intensity red light was used.
Statistical analysis
A logistic model (McCullagh and Nelder, 1989) was used to examine the effect of arena size, lip size, treatment, day and their interactions on the probability of insects being trapped. Logistic models use the logit transformation for percentages (p)
hNP) = lo!& ( > loop_ p
The difference between treated and control samples is measured by the odds ratio
odds ratio = Pt ’ (loo - PC) (100 -Pt> x PC
where pt is the percentage trapped for the treated pitfall and pC is the percentage trapped for the control.
Each statistical analysis was carried out on three basic variables: the numbers caught in the pitfalls after 1, 2 and 3 h.
Tables 2-9 show the percentages of insects trapped and the odds ratios between treated and control data. The higher the odds ratio, the better the discrimination between treated and control samples. The effects were assessed by the “deviance” which has an approximate chi-squared distribution. The chi-squared tests of deviance refer to the logits and not to the percentage data. In statistical terms, the most relevant effects are the interaction of treatment with arena size, the interaction of treatment with lip size and the three-factor interaction of treatment x arena size x lip size.
62 Cohn Morgan et al.
RESULTS
In all cases, the traps with treated filter paper caught a higher proportion of insects than the untreated traps. Significant day effects were found for a number of species, indicating variability from day to day in the numbers of insects trapped.
A. advena (Table 2)
There was some indication of a treatment x arena size interaction for the 1 h results. Here, the odds ratios for the 200 mm arenas were larger than those for the corresponding lip size in the 100 mm arenas. This effect had disappeared by 2 h.
Lip size had no major effect on either the numbers trapped or the odds ratio between treated and control data.
The 100 mm arena trapped more insects than the 200 mm arena in both control and treated traps at each assessment period.
There were significant day effects at the 3 h assessments, indicating day to day variation in the numbers of insects trapped.
0. surinamensis (Table 3)
The significant interaction between arena and treatment for the 1 h values showed that, at this time, the treatment effect for the 200 mm arena was larger on the odds ratio scale than the effect for the 100 mm arena. This demonstrates that the 200 mm arena is better at differentiating treatments at this time. This interaction had disappeared by 2 h.
Lip size had a significant effect on the numbers trapped at both 1 h and 3 h. At these times, there were also significant interactions between lip size and treatment, showing a tendency for the smaller lip sizes to discriminate better between treated and control data.
The 100 mm arena trapped more than the 200 mm arena at all three assessment periods. There were significant day effects at the 1 and 3 h assessments, indicating a day to day variation
in the numbers of insects trapped.
R. dominica (Table 4)
Very few insects were trapped with the 2.5 or 5 mm lip. With no lip, the 100 mm arena discriminated between treated and control data better than the 200 mm arena (it had a higher odds ratio). In addition, the discrimination improved with time between 1 and 3 h.
P. truncatus (Table 5)
The significant three-factor interaction at 1 and 2 h complicates the interpretation. The lip size had a large effect on the numbers caught, with the 0 mm lip size catching more insects
than the other two lip sizes. There were significant interactions between lip size and treatment for both the 1 and 2 h results, so that, on the odds ratio scale, a lip size of 2.5 mm or 5 mm gave larger treatment effects than the bioassay with no lip. This effect was more pronounced for the 100 mm arena than for the 200 mm arena, but statistical comparisons may be distorted by the zero values for the 5 mm lip size, giving rise to infinite odds ratios.
S. granarius (Table 6)
Treatment differences were large for all bioassay variations. The arena size had little effect, with the 100 mm arena catching more than the 200 mm arena at 3 h only.
Lip size and its interaction with treatment were statistically significant at all three assessment periods. Overall, the best combination on the odds ratio scale was the 5 mm lip size for either the 200 or 100 mm arena. The 2.5 mm lip size seemed worse than both the 0 mm and 5 mm lip.
There were significant day effects at the 2 and 3 h assessments, indicating a day to day variation in the numbers of insects trapped.
S. oryzae (Table 7)
Treatment effects were generally large for all bioassay designs. Arena size had no significant effects or interactions.
Tab
le
2.
Perc
enta
ge
resp
onse
of
A
husu
erus
ad
uena
in
pi
tfal
l bi
oass
ays
Are
na
size
(m
m)
Lip
si
ze
(mm
) Ih
2h
3h
-I
07
C
ontr
ol
(%)
Tre
ated
(%
) O
dds
ratio
C
ontr
ol
(“IO
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
6
100
0 24
36
1.
8 34
46
1.
6 39
61
2.
5 I
2.5
16
34
2.6
28
50
2.6
39
61
2.5
3
5 IS
31
2.
5 30
49
2.
2 42
64
2.
5 3
200
0 13
25
2.
3 22
39
2.
2 33
51
2.
1 $
2.5
7 22
3.
1 19
35
2.
3 26
48
2.
6 u
5 7
28
5.2
13
37
4.0
21
45
3.1
8 S O
vera
ll 14
29
2.
6 24
43
2.
3 33
55
2.
5 5.
E
D
evia
nces
an
d si
gnif
ican
ce
prob
abili
ties
2 :: E
ffec
t d.
f. Ih
2h
3h
0 r!
Dev
ianc
e P
Dev
ianc
e P
Dev
ianc
e P
& i:
Tre
atm
ent
1 71
.5
0.00
0 72
.8
0.00
0 96
.8
0.00
0 A
rena
si
ze
I 23
.0
0.00
0 30
.9
0.00
0 36
.7
0.00
0 L
ip
size
2
5.0
0.08
2 1.
4 0.
491
1.2
0.54
9 i b
Day
3
2.1
0.44
0 4.
6 0.
204
8.6
0.03
5 u
Are
na
size
x
trea
tmen
t 1
3.1
0.07
8 1.
7 0.
192
0.01
0.
000
I
Lip
si
ze
x tr
eatm
ent
2 3.
8 0.
150
2.7
0.25
9 0.
5 0.
779
D
Are
na
size
x
lip
size
x
trea
tmen
t 2
0.6
0.72
3 2.
1 0.
346
0.8
0.68
6
8
Tab
le
3. P
erce
ntag
e re
spon
se
of O
ryza
ephi
lus
suri
nam
ensi
s in
pitf
all
bioa
ssay
s
Are
na
size
(m
m)
Lip
siz
e (m
m)
Ih
2h
3h
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
100
0 27
51
2.
9 46
64
2.
0 56
79
2.
9 2.
5 39
44
1.
2 54
72
2.
2 70
87
2.
9 5
27
31
1.2
41
59
2.1
54
74
2.4
200
0 5
20
4.6
15
35
3.0
24
67
6.3
2.5
3 13
5.
6 14
26
2.
1 20
43
3.
0 s
5 4
IO
2.9
18
27
1.7
27
38
1.7
5
Ove
rall
17
28
1.9
31
47
2.0
42
65
2.5
Z
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s D
3 E
ffec
t d.
f. Ih
2h
3h
Z
Dev
ianc
e D
evia
nce
Dev
ianc
e F
P
P
P
Tre
atm
ent
1 35
.8
0.00
0 56
.4
0.00
0 11
3.8
0.00
0 A
rena
si
ze
1 21
8.9
0.00
0 23
2.9
0.00
0 22
2.3
0.00
0 L
ip s
ize
2 13
.6
0.00
1 4.
1 0.
129
10.2
0.
006
Day
3
28.1
0.
000
6.4
0.09
4 13
.2
0.00
4 A
rena
si
ze x
tre
atm
ent
1 9.
6 0.
002
0 1.
000
0.4
0.52
7 L
ip s
ize
x tr
eatm
ent
2 9.
3 0.
010
0.6
0.74
1 9.
6 0.
008
Are
na
size
x l
ip s
ize
x tr
eatm
ent
2 2.
0 0.
369
1,44
2 0.
486
5.72
2 0.
057
Tab
le
4. P
erce
ntag
e re
spon
se
of R
hyzo
pert
ha
dom
inica
m
pIt
tal
bioa
ssay
s
Are
na
size
(m
m)
Lip
siz
e (m
m)
Ih
2h
3h
Con
trol
(%
) T
reat
ed
(“IO
) O
dds
ratio
C
ontr
ol
(%)
Tre
ated
(%
) O
dds
ratio
C
ontr
ol
(%)
Tre
ated
(%
) O
dds
ratio
100
0 19
51
5.
8 29
73
6.
7 35
84
9.
5 2.
5 0
3 in
fini
te
0 6
Infi
nite
2
9 5.
1 5
1 1
1.0
1 I.
0 2
1 0.
7 20
0 0
13
19
1.6
1;
41
3.0
28
50
2.5
2.5
1 0
0.0
1 I
0.5
1 1
I.0
5 0
0 In
fini
te
0 I
Infi
nite
1
2 1.
5 O
vera
ll 6
13
2.6
8 20
2.
8 12
24
2s
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s
Eff
ect
d.f.
Ih
2h
3h
Dev
ianc
e P
Dev
ianc
e P
Dev
ianc
e P
Tre
atm
ent
1 47
.8
0.00
0 89
.0
0.00
0 87
.2
0.00
0 A
rena
si
ze
I 41
.5
0.00
0 32
.5
0.00
0 32
.4
0.00
0 L
ip s
ize
2 35
1.8
0.00
0 53
4.9
0.00
0 62
9.4
0.00
0 D
ay
3 40
.0
0.00
0 11
.9
0.00
0 15
.0
0.00
1 A
rena
si
ze x
tre
atm
ent
1 15
.4
0.00
0 6.
7 0.
035
13.7
0.
001
Lip
siz
e x
trea
tmen
t 2
1.1
0.58
8 0.
2 0.
896
5.6
0.06
2 A
rena
si
ze x
lip
siz
e x
trea
tmen
t 2
4.9
0.08
7 7.
9 0.
019
2.8
0.24
3
Tab
le
5.
Perc
enta
ge
resp
onse
of
P
rosr
epha
nus
trun
catu
s in
pi
tfal
l bi
oass
ays
Are
na
size
(m
m)
Lip
si
ze
(mm
) Ih
2h
3h
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(X)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
100
0 28
46
2.
2 33
60
3.
1 38
71
4.
1 2.
5 3
I4
6.6
3 I9
9.
0 8
27
4.5
5 0
II
Infi
nite
0
I3
Infi
nite
3
I6
5.8
200
0 23
46
2.
9 29
59
3.
5 33
69
4.
7 s
2.5
3 23
9.
1 5
26
6.8
II
29
3.4
5 5
g I9
4.
4 8
24
3.9
13
26
2.4
Ove
rall
IO
21
3.2
I3
33
3.4
17
40
3.1
@
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s I 5
Eff
ect
d.f.
Ih
2h
3h
z e D
evia
nce
P
Dev
ianc
e P
D
evia
nce
P
Tre
atm
ent
I 98
0.
000
140.
5 0.
000
141.
4 0.
000
Are
na
size
I
2.2
0.13
8 4.
7 0.
030
2.7
0.03
0 L
ip
size
2
182.
6 0.
000
249.
7 0.
000
268.
5 0.
000
Day
3
IO.1
0.
018
5.6
0.13
3 3.
2 0.
362
Are
na
size
x
trea
tmen
t I
0 1.
000
0.6
0.43
9 0.
6 0.
439
Lip
si
ze
x tr
eatm
ent
2 12
.9
0.00
2 6.
8 0.
033
1.4
0.49
7 A
rena
si
ze
x lio
si
ze
x tr
eatm
ent
2 7.
3 0.
027
8.4
0.01
5 2.
5 0.
291
Tab
le
6.
Perc
enta
ge
resp
onse
of
Si
toph
ilus
gra
nari
us
in
pitf
all
bioa
ssay
s
Are
na
size
(m
m)
Lip
si
ze
(mm
) Ih
2h
3h
-I
B
100
200
Ove
rall
Eff
ect
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
k?
g 0
11
55
10.2
23
82
15
.3
37
92
20.1
D
2.
5 17
36
2.
8 32
64
3.
9 58
94
11
.1
w
5 3
36
17.8
14
75
17
.7
34
94
28.4
e
0 8
45
10.1
21
78
13
.3
32
86
13.4
4
2.5
11
42
5.8
28
76
8.2
44
92
14.2
:
5 4
52
28.0
9
68
22.1
14
81
25
.6
2 9
44
8.2
21
74
10.5
37
90
15
.3
p
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s S G
d.
f. Ih
2h
3h
ry
S
Dev
ianc
e P
D
evia
nce
P
Dev
ianc
e P
6 t
Tre
atm
ent
1 33
2.1
0.00
0 57
0.4
0.00
0 66
6.3
0.00
0 A
rena
si
ze
I 0.
1 0.
752
0.8
0.33
1 19
.8
0.00
0
Lip
si
ze
2 5.
0 0.
082
;
13.7
0.
001
36.8
0.
000
F D
ay
3 5.
7 0.
127
9.7
0.02
1 17
.2
0.00
1 m
A
rena
si
ze
x tr
eatm
ent
1 2.
1 0.
147
2.1
0.14
7 0.
2 0.
655
D
Lip
si
ze
x tr
eatm
ent
2 27
.0
0.00
0 24
.5
0.00
0 5.
4 0.
067
D
Are
na
size
x
lip
size
x
trea
tmen
t 2
1.3
0.51
7 3.
0 0.
221
1.1
0.58
4
Tab
le
7. P
erce
ntag
e re
spon
se
of S
itop
hilu
s or
yzue
in
pitf
all
bioa
ssay
s
Are
na
size
(m
m)
Lip
siz
e (m
m)
Ih
2h
3h
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
100
0 24
75
9.
5 36
87
12
.1
41
94
21.6
2.
5 34
65
3.
6 57
79
2.
7 68
89
3.
7 5
I4
63
10.8
28
79
10
.0
33
86
12.5
20
0 0
24
72
8.1
47
85
6.4
58
93
9.7
c1
2.5
24
69
7.0
40
84
8.0
45
&9
10.4
S.
5
9 65
18
.8
21
82
16.8
32
85
12
.0
Ove
rall
21
68
7.8
38
83
7.7
46
89
9.8
f Z
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s 6
E&
t d.
f. Ih
2h
3h
2 ?
Dev
ianc
e P
D
evia
nce
P
Dev
ianc
e P
Tre
atm
ent
I 46
3.3
0.00
0 43
3.0
0.00
0 45
9.3
o.oo
o A
rena
si
ze
I 0.
8 0.
371
0.2
0.65
5 0.
3 0.
584
Lip
siz
e 2
18.7
0.
000
25.3
0.
000
31.8
0.
000
Day
3
68.9
0.
000
52.5
0.
000
54.8
0.
000
Are
na
size
x t
reat
men
t I
2.8
0.09
4 2.
8 0.
094
0.4
0.52
7 L
ip s
ize
x tr
eatm
ent
2 15
.7
0.00
0 15
.8
0.00
0 7.
8 0.
020
Are
na
size
x I
iD si
ze x
tre
atm
ent
2 3.
2 0.
203
II.0
0.
004
9.0
0.01
I
Are
na
size
(m
m)
Tab
le
8.
Perc
enta
ge
resp
onse
of
Si
6oph
ilus
zea
mai
s in
pi
tfal
l bi
oass
ays
Lip
si
ze
(mm
) lh
2h
3h
Con
trol
(X
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(X
) T
reat
ed
(X)
Odd
s ra
tio
100
200
Ove
rall
Eff
ect
0 2.5
5 0 2.5
5 d.f.
52
59
1.4
66
76
1.7
73
85
2.1
45
JO
2.9
62
74
1.8
73
78
1.3
61
54
0.7
JJ
64
0.5
81
74
0.6
0 I3
In
fini
te
4 18
5.
8 I
19
3.2
8 11
1.
6 11
24
2.
5 I3
29
2.
9 2
13
7.4
8 21
3.
2 9
30
4.1
28
37
1.5
38
46
1.4
43
53
1.5
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s
Ih
2h
3h
Dev
ianc
e D
D
evia
nce
D
Dev
ianc
e D
Tre
atm
ent
1 23
.5
0.00
0 21
.2
0.00
0 30
.0
0.00
0 A
rena
si
ze
1 58
1.2
0.00
0 65
2.2
0.00
0 72
1.3
0.00
0 L
ip
size
2
0.8
0.67
0 0.
6 0.
741
1.0
0.60
7 D
ay
3 0.
8 0.
850
2.5
0.47
5 2.
2 0.
532
Are
na
size
x
trea
tmen
t 1
13.5
0.
000
18.7
0.
000
19.9
0.
000
Lip
si
ze
x tr
eatm
ent
2 8.
4 0.
015
10.1
0.
006
3.2
0.20
2 A
rena
si
ze
x lip
si
ze
x tr
eatm
ent
2 26
.2
0.00
0 6.
0 0.
049
6.0
0.05
0
Tab
le
9. P
erce
ntag
e re
spon
se
of S
tego
bium
pa
nice
um
in p
itfal
l bi
oass
ays
Are
na
size
(m
m)
100
Lip
siz
e (m
m)
Ih
2h
3h
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
Con
trol
(%
) T
reat
ed
(%)
Odd
s ra
tio
0 21
40
2.
5 31
56
2.
8 40
14
4.
2
200
Ove
rall
Eff
ect
2.5
5 0 2.5
5 d.f.
17
43
3.7
24
61
5.0
35
18
6.8
22
46
3.0
28
61
4.1
36
82
7.8
11
28
3.0
22
45
3.0
30
54
2.8
3 16
7.
4 9
23
2.9
16
39
3.4
2 6
21
4.3
13
26
2.5
18
41
3.0
13
32
3.1
21
45
3.1
27
61
4.2
;
Dev
ianc
es
and
sign
ific
ance
pr
obab
ilitie
s (I
;: &
Ih
2h
3h
E
F D
evia
nce
D
Dev
ianc
e D
D
evia
nce
4
Tre
atm
ent
1 10
6.2
0.00
0 13
8.2
0.00
0 21
1.3
0.00
0 A
rena
si
ze
1 85
.0
0.00
0 92
.6
0.00
0 11
7.8
0.00
0 L
ip s
ize
2 5.
6 0.
061
13.4
0.
001
8.1
0.01
7 D
ay
3 17
.0
0.00
1 16
.8
0.00
1 13
.8
0.00
3 A
rena
si
ze x
tre
atm
ent
1 1.
5 0.
221
1.7
0.19
2 10
.1
0.00
1 L
ip s
ize
x tr
eatm
ent
2 2.
3 0.
317
1.8
0.40
7 3.
2 0.
202
Are
na
size
x l
ip s
ize
x tr
eatm
ent
2 0.
1 0.
974
1.9
0.39
4 1.
1 0.
569
The assessment of potential attractants to beetle pests 71
The 5 mm lip size traps generally caught fewer insects than the traps with smaller lips. The interaction between lip size and treatment was not straightforward. It seems that the treatment effect for the 2.5 mm lip was generally less than the effect for the other two lip sizes, with no consistent difference in treatment effect between the 0 mm and 5 mm lip sizes.
There was little difference between the three assessment periods, with the lip size by treatment deviance being larger for the 1 and 2 h results.
There were significant day effects at all three assessment periods, indicating a day to day variation in the numbers of insects trapped.
S. zeamais (Table 8)
Overall, there was little difference between treated and control traps, especially for the 100 mm arenas, suggesting that the bioassay does not effectively differentiate between treatment and controls. Additionally, the significant three-factor interaction makes the interpretation of the results difficult.
The arena size had a very large effect on the numbers caught, with the 200 mm arena catching fewer insects than the 100 mm arena. The treatment effects, however, were larger for the 200 mm arena than for the 100 mm arena at all three times (on the odds ratio scale).
There was a significant interaction between lip size and treatment for the 1 and 2 h results, in addition to a three-factor interaction. For the 200 mm arena, the treatment effect for the 0 mm lip was larger than the effect for the other lip sizes at 1 and 2 h. This effect was not apparent for the 100 mm arena.
Stegobium paniceum (Table 9)
The arena size had a large effect on the numbers of insects caught, with the 100 mm arena catching more than the 200 mm arena. At 3 h, there was a significant interaction between arena size and treatment, with the 100 mm arenas giving a larger treatment effect (on the odds ratio scale) at all lip sizes.
The lip size had a small effect on the numbers caught, but there were no significant interactions with treatment.
There were significant day effects at all three assessment periods, indicating a day to day variation in the numbers of insects trapped.
DISCUSSION
For almost all of the species tested, there appeared to be evidence of a significant day to day variation in the results. Previous experience of insect behavioural bioassays has also shown similar day to day variations. These variations do not exhibit any obvious pattern. Bearing in mind the controlled conditions under which these bioassays have been conducted and the probability that insect behaviour will be more variable under field conditions, our preferred practice is to carry out the bioassays over a number of days. However, as this implies some acceptance of a variation in results, other workers may opt for a different approach.
The factors tested in this study, i.e. the test duration, pitfall lip size and arena size, were considered to be likely to influence the response of the insects to the pitfall bioassay. Trap catch is a function of a variety of interactive factors, such as the insect activity, behavioural characteristics and population density, which themselves may be influenced by changes in the physical presentation and environment of the trap.
The results from this study demonstrate that a single standard bioassay for a wide range of species may be inappropriate as the optimal design differs widely for the species tested here. However, the most consistent optimal factor was the test duration. Generally, the shortest (1 h) test was either preferable or as good as any other test duration in maximizing the response differential between the treated and control samples. This suggests that, if a test material is attractive, the insects will be successfully trapped rather rapidly. This is presumably partly due to the relatively short distances over which the insects have to travel to the source. After 1 h, insects in both the treated and control arenas will continue to be trapped, and this may have the effect of reducing the differential between an attractive lure and the control. In addition, it may be more convenient, where the test duration can be of any length, to adopt the shortest available.
12 Cohn Morgan et al.
Although larger numbers of insects were trapped in the 100 mm arenas, the difference between the treated and control samples, on a logit scale, varied according to the insect species being tested. In a 100 mm arena, the likelihood of an insect falling into the pitfall by chance is higher than for the larger arena due to the arena to pitfall size ratio. How much this may influence the results will depend on other factors, such as the insect species and insect activity levels. In this study, it was noted that the more active species, such as 0. surinamensis and A. advena, gave a greater response differentiation in the 200 mm arenas. Conversely, less active species, such as P. truncatus and S. paniceum, seemed best suited to 100 mm arena bioassays.
Obeng-Ofori (1993) found that, although faster moving insect species made more frequent encounters with pitfall traps, the likelihood of falling into the trap was dependent on the behaviour of the species at the trap rim. His findings showed that, in general, the larger species, such as P. truncatus and Sitophilus spp., although slower and less likely to encounter the pitfall trap, were more likely to fall in once an encounter was made. In our tests, P. truncatus showed the lowest capture rate after R. dominica, although we do not have data on the frequency of encounters to show whether this was due to a low encounter rate or success at avoiding falling into the trap itself. Obeng-Ofori (1993) also found that 0. surinamensis, although having the highest frequency of encounters with the trap rim, was the least prone to capture. He put this down to its successful “skirting” behaviour, in contrast with P. truncatus which was unsuccessful in this behaviour, perhaps due to its greater weight. Munro (1966) states that 0. surinamensis has adhesive setae on its tarsi, which may help to prevent falling. In the experiments reported here, 0. surinamensis successfully evaded capture in the control traps, although trap catch was substantially increased with the addition of the lure, confirming that it is able, at least to some degree, to prevent itself from falling.
Apart from the tests using P. truncatus and S. zeamais, bioassays were performed at 20°C. Previous tests with 0. surinamensis (Morgan, unpublished observation) had shown much higher activity levels at higher temperatures, often resulting in unacceptably high control “responses” and problems in differentiating the response between attractants and controls. Tests using P. truncatus and S. zeamais were performed at 27”C, reflecting the warmer conditions under which these insects are of importance. The trap catch of S. zeamais in the smaller arenas was high in both the control and test pitfalls, resulting in poor differentiation. This may be improved by reducing the test ambient temperature.
Although differing lip sizes often showed statistically significant differences in catch, treatment x lip size interactions did not always accompany this. Thus, commonly, no particular lip size was preferred over another, although one particular lip size was sometimes preferred. The effect of lip size appears to be the least important factor in maximizing the differential between treated and control assays. Some of the lip size effects seem to be inconsistent, e.g. with the 2.5 mm lip size being apparently less suitable for the detection of treatment differences. This anomaly is difficult to interpret, but again may be explained as a consequence of a variety of factors, such as insect species, morphological dimensions, inclination to climb, levels of activity, etc. One of the most dramatic differences seen in trap catch by altering the lip size was shown by R. dominica, where any lip size above the arena surface severely restricted catch, but reasonable odds ratios were obtained by a combination of no lip with the smaller arena. This may be a reflection of the sluggish nature of this species, which is disinclined to attempt climbing and prone to losing its balance while investigating the pitfall rim.
A summary of the conditions which may be employed to best differentiate between treatment and control pitfall tests for the different insect species is presented in Table 10. Where there is some
Species and temperature
Table 10. Suggested test variables for single pitfall bioassays
Arena size (mm) Lip size (mm) Time (h)
Ahawerus advena, 2O’C Oryzaephilus surinamensis, 20°C Rhyzopertha dominica, 20°C Prostephanus truncatus, 21°C Sitophilus granarius, 20°C Sitophilus oryzae, 20°C Sitophilus zeamais, 27°C Stegobium paniceum, 20°C
200 5 1 200 0 3
10&M 2.:,5 lb 100/200 of5 11213 100/200 11213
200 015 100 2.515 :
The assessment of potential attractants to beetle pests 73
doubt about the conditions under which the best differentiation may be made, alternatives are given. This table represents our suggestions based on the findings of this study, but it must be pointed out that these may need further validation.
Tests conducted earlier at CSL (Morgan and Healey, 1993) showed that, for 0. surinamensis, differences in response between treated and control replicates in 200 mm arenas were more likely to be statistically significant (p < 0.05) at the 3 h assessment than at the 1 and 2 h assessments. In this investigation, the 3 h assessment period also gave the best level of differentiation, as measured by the odds ratio, when a 0 mm lip was used with a 200 mm arena. The next best test period was 1 h, using a lip size of either 0 or 2.5 mm.
The suitability of the single pitfall bioassay for the different insect species under test appears to fall into one of three categories. Firstly, there are those insect species, e.g. S. granarius and S. oryzae, for which treatment differentiation is good almost irrespective of the bioassay design. Secondly, for some species, such as A. advena, P. truncatus, R. dominica, S. paniceum and 0. surinamensis, there are distinct recommendations for bioassay design which enhance treatment differentiation. Lastly, there appear to be some insect species, e.g. S. zeamais, which show a low response to the treatment irrespective of the bioassay design. It is possible, however, the use of a more powerful attractant.
that the response may be improved by
CONCLUSIONS
This study shows that the test generally gives better, or as good, results at I h as at 2 or 3 h. Although 100 mm arenas tend to catch greater numbers of insects, the differentiation between test and control samples may be better with the larger arena, depending on the insect species under study. Lip size appears to be the least important variable studied in influencing the differentiation between test and control samples.
It has been established that, for a number of insect species, either the treatment effects are generally largely irrespective of the bioassay design, or a specific recommendation for bioassay design markedly improves the treatment differentiation. For a few species, however, the response to the treatment remains low despite alterations in bioassay design. It is unclear whether this is due to an unsuitable methodology or to some other reason relating to the attractiveness of the volatiles.
Optimization of the methods used for laboratory testing of potential attractants may enable improvements to be made in both the sensitivity and dependability of the results. Numerous factors influence the success of lures in field applications which have not been incorporated into the laboratory bioassays detailed in this study. However, laboratory bioassays, when combined with pilot studies in the field, can prove to be both cost effective and reliable for the early screening of attractants and, as such, represent a particularly useful tool for the development of a successful lure.
REFERENCES
McCullagh P. and Nelder J. A. (1989) Genera/&d Linear Models, 2nd Edn. Chapman and Hall, London. Morgan C. P. and Healey V. S. (1993) The assessment of potential attractants against beetle pests: improvements to the
laboratory pitfall bioassay. In Proceedings of a Working Group Meeting on Use of Pheromones and Other Semiochemicals in Infegrated Control (Edited by McVeigh L. J. and Hall D. R.), Bulletin-OILB-SROP 16 (IO), pp. 255-259. Chatham, Kent.
Munro J. W. (1966) Pests of Stored Products. Rentokil Library, Hutchinson, London. Obeng-Ofori D. (1993) The behaviour of 9 stored product beetles at pitfall trap arenas and their capture in millet.
Entomologia Experimentalis et Applicata 66, I6 l-l 69. Phillips J. K., Chong J. M., Andersen J. F. and Burkholder W. E. (1989) Determination of the enantiomeric composition
of (R*,S*)-I-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius. Entomologia Experimenralis et Applicata 51, 149-l 53.
Pierce A. M., Pierce H. D. Jr., Borden J. H. and Oehlschlager A. C. (1991) Fungal volatiles: semiochemicals for stored-product beetles (Coleoptera: Cucujidae). Journal of Chemical Ecology 17, 581-597.
Pike V., Smith J. L., White R. D. and Hall D. R. (1995) Studies of responses of stored-product pests, Prostephanus truncatus (Horn) and Sirophilus zeamais Motsch., to food volatiles. In Proceedings of rhe 6th Internarional Working Conference on Stored-Product Profection (Edited by Highley E., Wright E. J., Banks H. J. and Champ B.), pp. 566-569. Canberra, Australia.
74 Colin Morgan ef al.
Trematerra P. and Girgenti P. (1989) Influence of pheromone and food attractants on trapping of S. oryrue: a new trap. Journal of Applied Entomology 108, 12-20.
White P. R. and Birch M. C. (1987) Female sex pheromone of A. punctatum. Extraction, identification and bioassays. Journal of Chemical Ecology 13, 1695-1706.
Williams H. J., Silverstein R. M., Burkholder W. E. and Khorramshahi A. (1981) Dominicalure 1 and 2: components of aggregation pheromone from male lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). Journal of Chemical Ecology 7, 159-180.
