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The feeding response of the larva of the egyptian cotton leafworm, Spodoptera littoralis boisd., to sugars and related compounds—I. Phagostimulatory and deterrent effects
Comp. Biochem. Physiol., 1972, Vol. 42A, pp. 899 to 914. Pergamon Press. Printed in Great Britain
THE FEEDING RESPONSE OF THE LARVA OF THE EGYPTIAN COTTON LEAFWORM, SPODOPTERA LITTORALIS BOISD., TO SUGARS AND RELATED COMPOUNDS-I. PHAGOSTIMULATORY AND DETERRENT EFFECTS* J. MEISNER,l
K. R. S. ASCHERl
and H. M. FLOWERS2
‘Department of Toxicology, The Volcani Institute of Agricultural Research, Bet Dagan; and rDepartment of Biophysics, The Weizmann Institute of Science, Rehovot, Israel (Received 11 November 1971)
Abstract-l. The effect of twenty-five sugars and related compounds on the feeding response of larvae of Spodoptera littoralis was investigated using the Styropor method. 2. Styropor lamellae were treated with 0.25 and 0.0625 M solutions. The weight of the lamellae consumed and the number and weight of fecal pellets, served as criteria of feeding stimulation. 3. Sucrose, raffinose, maltose, D-fnKtOSc, melibiose and D-glucose were found to be very active as phagostimulants. 4. The survival of larvae which had fed on sugar-treated Styropor for 3 days was the longest on sucrose, maltose, glucose, fructose, raffinose and melezitose. 5. Some of the sugars were assayed in pairs in different choice and nochoice experiments; no antagonistic or inhibitory effects were found.
INTRODUCTION THE ROLE of phagostimulants in the feeding behavior of phytophagous insects is the subject of intensive study. Various groups of compounds such as sugars, amino acids, vitamins, etc. were found to elicit feeding response in insects. Among the carbohydrates, a wide range of insects showed intensive response to sucrose (see, e.g., reviews by Lipke & Fraenkel, 1956; House, 1961; Davis, 1968; Schoonhoven, 1968). Other sugars, e.g., fructose, glucose, raffinose and maltose, were also effective as feeding stimulants (Davis, 1968). Among the polyhydric alcohols inositol exhibited feeding stimulation for some insect species (Ito, 1960; Yamamoto & Fraenkel, 1960). An earlier study (Meisner et al., 1970) was conducted on phagostimulants responsible for the attraction of the larvae of Spodoptera littoralis Boisd. [formerly called Prodeniu Zitura F. (Viette, 1963)] t o some of its host plants. From experiments with crude extracts of these plants, various fractions obtained therefrom,
* Contribution from The Volcani Israel. 1971 Series, No. 1988-E.
Institute
of Agricultural
Research,
Bet
Dagan,
900
J.
MEISNER,
K. R. S.
ASCHER
AND
H.M.
FLOWERS
and some pure nutrient chemicals, it was demonstrated preliminarily that sucrose, fructose and, to a lesser degree, glucose, were effective feeding stimulants for the larvae of this insect. The present study deals specifically with the stimulatory effect of carbohydrates and related compounds on the feeding response and survival of the larvae of S. littoralis. In addition, some of the sugars were tested in combination (of two sugars) to determine whether non-active sugars had an inhibitory effect on feeding. MATERIALS
AND
METHODS
Chemicals D-Arabinose, D-XylOW, D-glUCOSe, D-g&CtOSe, D-mannose, D-fructose, L-sorbose, L-rhamnose, L-fucose, N-acetyl-D-glucosamine, D-galacturonic acid, D-glucuronolactone; melibiose, sucrose, maltose, cellobiose, trehalose, raflinose, melezitose; D-mannitO1, Dsorbitol, galactitol (dulcitol), i-inositol, methyl o-D-glucopyranoside and calcium-Dgluconate were all from commercial sources. Bioassay technique Lamellae of a synthetic plastic material, Styropor (foamed polystyrene), were used as carriers for potential phagostimulatory substances. This material had been used with various methodological variations to assay feeding stimulation of larvae of the potato tuber moth, Gnorimoschema operculella Zell., codling moth, Cydia pommella L. and carob moth, Ectomyelois ceratoniae Zell. (Meisner & Ascher, 1968). For larvae of S. littoralis, thin, rectangular (6 x 3 cm) laminae of Styropor were cut from a small block with the help of a manually driven meat slicer (Meisner et al., 1970). The laminae were painted with solutions (ethanol-water, 1 : 1) of the test substances at known concentrations and left to dry for 24 hr. They were then placed singly or in pairs in closed Petri dishes (dia. 14 cm). Larvae, reared at 27°C on lucerne and weighing between 170 and 190 mg, were starved for 3 hr; then one weighed larva was introduced into each Petri dish. All experiments were conducted at 27°C. The feeding tests were of two types: (a) No-choice experiments, in which a single larva was confined to each Petri dish and exposed to one treated lamina. In earlier experiments we had also placed a control lamina treated with the solvent mixture only, into the same dish, but we soon noted that this lamina was left practically untouched; (b) Choice experiments, in which two or more laminae treated with different substances or with mixtures of them were offered simultaneously to the larva. No-choice experiments In the no-choice experiments, twenty-five sugars and related compounds were assayed in two concentrations, 0.25 and 0.0625 M (weight/volume). Each of the treatments was done in at least 20 x 1 larva replicates per compound, but many of them were repeated at least forty times. Every day the lamellae were taken out, weighed and returned. In our previous work with S. littoralis (Meisner et al., 1970), the rate of feeding was expressed as percentage of a standard-weight Styropor lamella consumed. Since then we have found it more convenient to use, instead, weight of Styropor consumed, in mg. Hence the weight of Styropor consumed after 24, 48 and 72 hr, and the total number and weight of fecal pellets, with the exclusion of the initial black ones which result from the prior feeding on lucerne, served us as the criteria of feeding stimulation in the present work. The fecal pellets were also counted. We had noted in the previous study (Meisner et al., 1970) that the size of the fecal pellets varied greatly with different plant extracts. A similar phenomenon could be seen also with different sugars and therefore the more objective criterion was the weight of the pellets. The number of dead (if any) larvae was recorded every day.
FEEDING RESPONSE OF EGYPTIAN
COTTON LEAFWORM LARVAE TO SUGARS
901
When a sugar-treated Styropor lamella was consumed at a rate of more than 10 mg/larva within 72 hr, we postulated that the sugar was highly phagostimulatory. Consumption of 5-10 mg/larva denoted moderate phagostimulatory activity. Sugars which elicited a feeding response of less than 5 mg Styropor/larva were considered to be weakly attractive or inactive. The lamellae were removed after the 3-day feeding, but the larvae were left in the Petri dishes. We continued to record daily mortality until all the larvae had died. In this type of experiment, larvae kept for 3 days on untreated lamellae (the lamellae were subsequently removed) served us as controls. The survival index was calculated according to the following formula : Survival index =
Total larva-days Initial number of larvae
Survival in excess of that of control larvae, as related to the amount of treated Styropor consumed during 3 days, was expressed by the formula A-B -3 c where A is the survival index on a treated Styropor lamella, B the survival index on untreated Styropor (control) and C the weight, in mg, of treated Styropor consumed per larva during 72 hr. This calculation was carried out only for survival indices reasonably greater than the control, i.e. above 3.0 (untreated control survival index was 2.5). Choice experiments In choice experiments we offered the larva simultaneously a lamella painted with a solution containing a mixture of 0.125 M of a weakly or non-attractive carbohydrate plus O-125 M sucrose, and a second lamella treated only with O-125 M sucrose. The two components separately at 0.125 M, and a mixture of the two sugars at a concentration of 0.125 M each, served as separate controls in these tests. These experiments were terminated after 48 hr. The weight of styropor consumed within this period, and the total weight of fecal pellets, again served as the criteria of phagostimulation.
RESULTS
No-choice experiments The feeding response of the larvae to various sugars and related compounds is summarized in Table 1. Sucrose was the most active sugar at both concentrations tested (0.25 and 0.0625 M). Raffinose, maltose, n-fructose, melibiose and n-glucose, in descending order of activity, elicited a strong feeding response at 0.25 M, whereas at 0.0625 M only sucrose and fructose were consumed at a rate of over 10 mg Styropor/larva during a period of 72 hr. Melezitose, trehalose, i-inositol and n-xylose produced a moderate feeding response (5-10 mg treated Styroporjarva within 72 hr) at 0.25 M. The rest of the carbohydrates assayed had a very slight or no phagostimulatory activity at all at both test concentrations. With some compounds of the last two groups, survival after 72 hr was poor. Since sucrose elicited the strongest feeding stimulus, it was of interest to assay the response to this sugar at a greater number of concentrations between 16 and 0.00001 per cent and also to determine the response threshold of the larvae to
RESPONSE
0.6
Control
;:: l-6 0.5 0.3 I.1 04 0.8 0.5 I.5 0.2
LARVAE
0.25 M
I.1
72 hr
z 73 106 62 52 35
70
0.4
24 hr
A:; 2.7 0.6 1.6 0.3 I.0 1.1 1.6 0.8
;:; 1.7 0.8
2:;
::;
33:;
8.9 5-3 4.5 7.3 3-5
48 hr 11.6 7.8
72 hr
COMPOUNDS
AT
7 ;z
::
:z
3;
;;
*z 55
179 112
Weight (mg)
96 16
59 9
33 22 Not taken
::
9
1:; 166
102 120 120
227 304
503 295 167 393 153
No.
Fecal pellets produced during 72 hr in a a twenty-larva experiment
RELATED
O-0625 M
AND
Weight in mg of treated Styropor lamella consumed per larva, cumulative values after
TO VARIOUS SUGARS
$ One hundred per cent mortality.
31
1:: ;2: 41 I3 Not taken
145 185: 191 193 1;;
::
E 151 130 174 123 61
428 396 433 248 359 287 111 121 187
244
Weight (mg)
568
No.
twenty-larva experiment
Fecal pellets proz;;di$aring
RANGE,170-190mg) 0.25 AND 0.0625 M
(WEIGHT
t Ninety-five per cent died.
1.1
::;: 0.6 Not done
;:; 0.3
12:;
;:: 2.8 2.2
;:; 2.8
i:; 2.8
;:‘:
;:;
;:; 6.8
17.5 13.1 12.1
48 hr
;:; 2.1
4.4
;I;
24hr
;:; 0.3
* Ninety per cent died.
littoralis
Weight in mg of treated Styropor lamella consumed per larva, cumulative values after
OF S.
CeIIobiose D-Glucuronolactone L-Fucose CaIcium-D-gluconate
N-SCetyl-D-glUCOSSmine
Sucrose Raffinose Maltose D-Fructose Mehbiose D-Glucose Melezitose TrehaIose i-Inositol D-Xylose D-GaIactose D-Mannose L-Sorbose Methyl a-D-ghrcoside D-SorbitoI D-GaIacturonic acid L-Rhamnose D-i%nnitoI D-Arabinose GaIactitol
Sugars and related compounds
TABLE I-FEEDING
FEEDING
RESPONSE
OF EGYPTIAN
COTTON
LEAFWORM
LARVAE
TO
SUGARS
903
sucrose. The results are presented in Fig. 1, which shows the positive correlation between concentration of sucrose and larval response. The response threshold to sucrose was at O*OOlS per cent. I
I II
I1111
(>-
--of&hr
I
I
II I
20-
72 hr
16 -
u E ii4 c 12 8 b 8-8P6-
4-
l
_
8--/L 0
Controlb72 hr I
om
11l1111
0005 001
III
1111111
005
I
I
0.1 % Sucrose
Ill1111
05
I
1.0
1 l11llll
50
100 204
FIG. 1. Feeding response of larvae of S. littoralis (ranging in weight from 170 to 190 mg) to different concentrations of sucrose applied to Styropor lamellae. Five further concentrations from 0401 to 040001 per cent yielded values equal to or below those of control.
Values of larval survival which we consider as a certain indication of the nutritive value of sugars and related compounds, are presented in Table 2. The larval survival index was the greatest on sucros&.4 days. The sugars with a survival index ranging from 3 to 4 days were, in descending order: maltose, n-glucose, n-fructose, rafhnose and melezitose. The survival index of all the other compounds approached, or was even less than, that of the control (untreated Styropor). When we calculated the relation between the survival index in excess of control and Styropor consumption, the most nutritive sugar was D-&COSe, followed by sucrose (Table 2, last column).
904 TABLE
J. MEISNER,K.R.
S.
OF LARVAE OF S. littorah WITH VARIOUS SUGARS AND
~-SURVIVAL TREATED
Sugars and related compounds
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
ASCHERANDH.M.
FLOWERS
KEPT FOR 3 DAYS ON S~ROPOR RELATED COMPOUNDS AT 0.25 M
Survival index*
Sucrose Raffinose Maltose D-Fructose Melibiose D-Glucose Melezitose Trehalose i-Inositol D-Xylose D-Galactose D-Mannose L-Sorbose Methyl ol-D-glucoside D-Sorbitol D-Galacturonic acid L-Rhamnose D-Mannitol D-Arabinose Galactitol AJ-acetyl-D-glucosamine Cellobiose D-Glucuronolactone L-Fucose
4.4 3.2 3.8 3.3 2.8 3.6 3.2 2.4 2.2 1.8 2.8 2.8 2.1 2.7 2.3 2.5 2.2 2.7 2.1 2.6 2.4 1.8 2.0 2.0
Control
2.5
LAMELLAE
(A-WCt 8.9 4.0 7.7 6.5
x x x x 11.0 x 8.0 x
1O-2 10-s 10-2 1O-2 10-Z 1O-2
* Survival index = total larva-days/initial number of larvae. 7 A = Survival index on treated Styropor lamellae. B = Survival index on untreated Styropor lamellae (control). C = Weight in mg of treated Styropor consumed per larva during 72 hr (values taken from Table 1).
Choice experiments Experiments were carried out with the aim of elucidating whether slightly active or inactive sugars have merely no phagostimulatory effect, or if they inhibit feeding. To decide between these two possibilities, sugars of these two types were mixed with sucrose and offered to larvae. The results of these experiments are summarized in Table 3. The slightly active or inactive sugars tested, with the possible exception of fucose, did not reduce the activity of sucrose, when they were admixed with it at a concentration of 0.125 M each (Table 3, experimental layout c). In the choice tests (Table 3, experimental layout d), in which a lamella treated with a mixture of sucrose with a slightly active or non-active carbohydrate, each at 0.125 M, was pitted against O-125 M sucrose alone, there was an almost
EFFECTOF
(a) Sucrose 0.125 M (b) Sorbitol O-125 M (c) Sucrose 0.125 M + sorbitolO*125 M (d)
(a) Sucrose O-125 M (b) Arabinose 0.125 M (c) Sucrose 0.125 M + arabinose O-125 M (d)
(a) Sucrose O-125 M (b) Xylose 0.125 M (c) Sucrose 0.125 M + xylose 0.125 M (d)
(a) Sucrose 0.125 M (b) MannitolO~125 M (c) Sucrose 0.125 M+ mannito10.125 M (d)
2
3
4
ADDING
1
No choice
TABLE ~-THE
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
I
SLIGHTLY
II
INACTIVESUGARSTO
Sucrose 0.125 M + mannitol 0.125 M
Sucrose 0.125 M + xylose 0.125 M
Sucrose 0.125 M + arabinose 0.125 M
Sucrose 0.125 M + sorbito10.125 M
Choice
ACTIVEOR
ON PHAGOSTIMULATION
7.1 2.3 8.3
9.1 2.6 8.2
9.1 2.9 9.2
8.5 2.2 10.7
No choice
5.0
3.1
4.2
4.6
I
4.4
7.2
5.8
5.4
II
Choice
9.4
10.3
10.0
10.0
Total
Weight of treated Styropor comsumed in mg per larva during 48 hr
SUCROSE
OF S. littoralis
(continued overleaf)
12.3
11.1 5.0 12.4 5.8
7.5
13.8 3.8 11.0 14.0
6.3
7.6 0.8
12.2
13.8 2.4 12.7
12.8
14.0 1.5 13.8
Number of Weight of fecal pellets fecal pellets per larva in mg per during larva during 48 hr 48 hr
IN LARVAE
E
%
c
r?,
28 2 0 g P w z
(a) Sucrose 0.125 M (b) N-acetyl-Dglucosamine 0.125 M (c) Sucrose 0.125 M+ N-acetyl-Dglucosamine 0.125 M Sucrose (d) 0.125 M
7
Sucrose 0.125 M
(a) Sucrose 0.125 M (b) Gala&to1 0.125 M (c) Sucrose 0.125 M + galactitol 0.125 M (d)
6
Sucrose 0.125 M
(a) Sucrose 0.125 M (b) InositolO*l25 M (c) Sucrose 0.125 M + inositol O-125 M (d)
I
5
No choice II
Sucrose 0.125 M + N-acetyl-Dglucosamine 0.125 M
Sucrose O-125 M + galactitol 0.125 M
Sucrose 0.125 M + inositolO*l25 M
Choice
14.1
12.1 I.9 14.6
12.5
12.3
6.3
6.2
6.4 0.8
6.3
5.7 1.2 8.0
7.0
3.9 9.0
11.3
8.6
9.6
8.7
6.9 5.4 14.3
Weight of fecal pellets in mg per larva dur.ng 48 hr 12.1
7.3
5.1
5.6
5.1
Total
13.4 I.3
3.5
4.0
3.6
II
Number of fecal pellets per larva during 48 hr
7.1 0.8
7.6 I.6 11.5
7.4 3.3 6.7
I
Choice
Weight of treated Styropor consumed in mg per larva during 48 hr
No choice
TABLE 3 (cont.)
$ 1
b
7
g
F
.7;:
(a) Sucrose O-125 M (b) Cellobiose O-125 M (c) Sucrose 0.125 M + cellobiose O-125 M (d)
(a) Sucrose 0.125 M (b) Methyl a-n-glucoside 0.125 M (c) Sucrose 0.125 M + methyl a-~glucoside 0.125 M (d)
(a) Sucrose 0,125 M (b) Fucose O-125 M (c) Sucrose O-125 M + fucose O-125 M (d)
(a) Sucrose 0.125 M (b) Rhamnose 0.125 M (c) Sucrose 0.125 MS rhamnose 0.125 M (d)
8
9
10
11
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M + rhamnose 0.125 M
Sucrose 0.125 M + fucose 0.125 M
Sucrose 0.125 M + methyl or-n-ghrcoside 0.125 M
Sucrose 0.125 M + cellobiose 0.125 M 15.2
8.6 3.1 9.8
9.4 3.4 6.8
2.9
5.6
5.0
3.8
7.9
9.4
5.0
7.4 1.2 8.2
7.4
8.9 3.1 4.9
9.4
8.3
9.1 3.6
7.2
7.1 3.3 4.0
(continued ooerleuf)
11.3
14.1 2.4 13.9
14.7
15.4 4.5 10.0
15.4
13.3
10.0
10.7
8.6
4.0
4.7
14.1 44
6.0
6.0
13.1 2.5 8.7
8.5 3.5
8.2 2.2 7.4
2.9 8.LI E.9 6.LT
Z.&I
I.EI
s.91 0.s 6.LI
8.L .L*t 9.6 6.9 5.8 9.E 9.6
0.8 8-O
6.LI
9.0
2.8
I.S E*ZI 1.Z Z.ZI
LeEI
s.z Z.ZI
Z*ZI
+
m
m
‘j-ZI.0
asouuauI
sz1.0 asoJans
IN SZS~O asomnc;
asomnc;
IN SZI.0
Ill SZI.0 auomqouom3rqB IN SZI.0 + bu ~~1.0 =oJDns asom-ig
m sz1.0 aso-93
m gi.0 asouuem + m ~~1.0 asoJDng w szbo aso-m
m SZI-0 asovq~~ + m sz1.0 asoJ=q tu sz1.0 asoww ku SZI-0 a=JDns
INSZT.0
IN SZI-0 auo~xqouomxql + m sz1.0 as=w
tv sz1.0
=-ns
auo~x3~0uomxq~
FEDING
RESPONSE
OF EGYPTIAN
COTTON
LEAFWORM
LARVAE
TO SUGARS
909
equal distribution of consumption between the two lamellae, with the exception of: D-xylose, iV-acetyl-n-glucosamine, L-rharnnose and n-mannose-activity of mixture higher than sucrose; and methyl a-D-glucoside, L-fucose and n-glucuronolactone-activity of mixture lower than that of sucrose. The exceptionally high consumption rate of the xylose-sucrose mixture vs. sucrose alone, may indicate a synergistic effect. DISCUSSION
In the present study, the phagostimulatory effect of the sugars in which consumption was over 10 mg treated lamella/larva within 72 hr, decreased in the following order: At 0.125 M : sucrose > raffinose > maltose > fructose > melibiose > glucose. At 0.0625 M : sucrose > fructose > raffinose > glucose > maltose > melibiose. As judged by human response, sugars are usually listed in decreasing order of sweetness as fructose > sucrose > glucose > galactose > mannose > lactose > raffi1964). Evidently, the order of sweetness established for nose (Shallenberger, human beings and the feeding response of larvae of S. littoralis to various sugars are different. It should be noted that the sweet-taste threshold of sucrose for the human tongue lies between 3 x 1O-3 and 3 x 10ea M (Richter & Campbell, 1940), whereas sensitivity to sucrose within the range of 1 x lo-’ to 1 x 10d6 M is characteristic of many insect species (Dethier, 1955). In most insects investigated, sucrose, raffinose, maltose, n-fructose and n-glucose were found highly active, though their exact relative order as regards phagostimulation differed from species to species. Sucrose was found to be the most acceptable sugar in a great number of insects, though several exceptions are known. One example is the confused flour beetle, Tribolium con&sum, for which maltose had a higher phagostimulatory rate than sucrose at O-1 M (Loschiavo, 1965). n-Fructose, which elicited a strong feeding response in our study, was found to be highly attractive to many other insects. Thus, the adults of the yellow-fever mosquito, Aedes aegypti (Galun & Fraenkel, 1957), the fruit fly, Drosophila melanogaster (Hasset, 1948), the Mexican bean beetle, Epilachna vurivestis (Augustine et al., 1964) and the larvae of the spruce budworm, Choristoneura fumiferana (Heron, 1965), responded to fructose in a degree similar to or even more than to sucrose. A relatively low activity of fructose was found in the two varieties of the imported fire ant, Solenopsis saevissima saevissima and Solenopsis saevissima richteri (Ricks & Vinson, 1970). The least phagostimulatory hexose in the active group was glucose. This holds true also for some other insect species. Thus, the Mexican bean beetle, E. varivestis (Augustine et al., 1964), the cabbage looper, Trichoplusia ni (Gothilf & Beck, 1967) and the blowfly, Phormia regina (Dethier, 1955) showed a higher feeding rate on fructose than on glucose, whereas in the potato beetle, Leptinotarsa decemlineata, glucose at O-1 M gave a much higher feeding response than fructose (Hsiao & Fraenkel, 1968). In Chrysops vittatus, glucose was also superior to fructose, which was explained by the feeding habits of the fly (Lall, 1970). Only
910
J. MEISNER,K. R. S. ASCHERAND H. M. FLOWERS
a moderate feeding rate is elicited by glucose in the sweet-clover weevil, Sitoniu (Akeson et al., 1970), and it is only slightly active in the prairie grain wireworm, Crenicera aeripennis destructor (Davis, 1961). According to Shallenberger (1964), a freshly prepared solution of a-n-glucose is sweeter for human beings than a mutarotated one. When we compared the activity of a freshly prepared solution of 0.25 M n-glucose, which is principally in the 01form, with that of one aged for a few days, which contains a mixture of the 01and the ,!I form, no difference was found in the consumption rate of the treated Styropor lamellae. Dethier (1955) also recognized that the threshold values for fresh and for aged, mutarotated solutions of glucose are not significantly different. Evans (1963) confirmed these findings: he demonstrated convincingly that there is no difference between the response of blow-flies to cx-n-glucose and @-glucose prepared from it by recrystallization from hot acetic acid. It cannot be excluded, however, that with some other sugars, mutarotation in solution may have some influence on phagostimulation. This question will still have to be investigated more thoroughly, although it sounds far-fetched to compare the activity of a carbohydrate in solution with that of its dried residue on a Styropor lamella. Among the compound sugars in this group we found that melibiose, which is usually considered as inactive, has a strongly phagostimulatory effect on the larvae of S. ZittoraZis. Solely, Sutherland (1971) very recently demonstrated that melibiose stimulated considerable feeding in the grass grub, Costelytra zealandica. To the best of our knowledge, these are, therefore, the first reports on the high phagostimulatory activity of this sugar. The inactivity of melibiose in the honeybee was explained by von Frisch (1934) as being due to the presence of a galactose group and to the lack of an 01linkage, an opinion shared by Dethier (1955) to interpret results in the blowfly. A merely slightly phagostimulatory effect of this sugar could be detected in the Colorado beetle (Hsiao & Fraenkel, 1968) and in spruce budworm larvae (Heron, 1965). The hexoses n-mannose, n-galactose and L-sorbose were found to be slightly active in this study. D-Mannose showed a medium effectiveness in the sweetclover weevil (Akeson et al., 1970) and a slight effectiveness in the potato beetle (Hsiao & Fraenkel, 1968); it was inactive in the blowfly (Dethier, 1955) and spruce budworm larvae (Heron, 1965), and toxic in the honeybee (Staudenmayer, 1939). n-Calactose showed only a medium activity in the potato beetle (Hsiao & Fraenkel, 1968) and a slight one in the spruce budworm (Heron, 1965). A direct relationship between the quantity of sugar-coated Styropor consumed by the larvae and the weight of fecal pellets excreted is not always apparent (see Table 1). This may be due to the fact that since our chief goal in the present paper was the phagostimulatory aspect, we did not choose a physiologically defined period, nor did we collect residual food from the gut (cf. Waldbauer, 1968). We have certain cases in Table 1, in which excretion seemed to be greater than ingestion (methyl-ol-n-glucoside at both 0.25 and O-0625 M, arabinose at 0.25 M and n-galactose at O-0625 M). An examination of the influence of feeding different cylindricollis
FEEDING RESPONSE OF EGYPTIAN
COTTON
LEAFWORM
LARVAE TO SUGARS
911
carbohydrates on the composition of the fecal pellets in S. littoralis may throw some light on these findings. It was evident from the number : weight ratio of fecal pellets in Table 1 that the size of the pellets varied greatly for different carbohydrates; a similar phenomenon was previously described by us for various plant extracts on Styropor (Meisner et aE., 1970). As to the survival index of the larvae of S. littoralis, which was in the order sucrose > maltose > glucose > fructose > raffinose > melezitose, our results are more or less in agreement with those of Galun & Fraenkel (1957) for A. aegypti but not for Sarcophaga bullata and Mwa domestica, for both of which survival was high on mannose and galactose, contrary to our results. The data of Hasset (1948) on the survival rate of D. melanogaster adults on sugars at M/10 are in agreement with the low survival index found by us for mannose, galactose and melibiose. When we calculated the relation between the survival index (minus control values) and the amount of Styropor consumed, n-glucose and sucrose were found to be the two sugars nutritionally most efficient for survival. The survival indices of the larvae on different sugars are thus not only a function of the total weight consumed, but may be also influenced by differences in the metabolization and utilization of these compounds. Experiments in progress to examine the fate of a number of the ingested sugars considered in the present work will be described in a later publication. In experiments with the housefly (Galun & Fraenkel, 1957), the blowfly (Dethier, 1955), the mealworm, Tenebrio molitor (Fraenkel, 1955) and the sweetclover weevil (Akeson et al., 1970), it was found that many slightly active or inactive sugars, when admixed with sucrose, glucose or starch, had a strong feeding deterrent effect. This phenomenon was found in the housefly in choice experiments with solutions of sucrose mixed with xylose, sorbose or ribose, in the ratio 1 : 1, and to a lesser extent with rhamnose, arabinose, a-methyl mannoside or inositol. Dulcitol was the only inactive sugar that did not deter the flies when admixed with sucrose (Galun & Fraenkel, 1957). Dethier (1955) showed that mannose, which is poorly phagostimulatory in the blowfly, caused a tenfold elevation of the threshold of fructose. No such change could be found on adding mannose to other attractive sugars. Dethier concluded for the blowfly that “it had been impossible for the most part to demonstrate any competitive inhibition between sugars”. Further light was thrown on this problem by Fraenkel (1955), who kept larvae of T. molitor on an artificial diet containing glucose as the main carbohydrate constituent. The total carbohydrate content was kept constant at 80 percent. In no-choice experiments on this diet, an inhibitory effect was still obtained at the following concentrations, when the concentration of glucose was slightly reduced and the following sugars were introduced: 10% galactose; 5% arabinose, rhamnose, mannose or sorbose; 2% xylose, ribose, or-methyl mannoside or dulcitol (galactitol) ; and 1y. a-methyl glucoside. Akeson et al. (1970), in nochoice experiments, demonstrated that residues of sucrose in a 1 : 1 ratio with
912
J. MEISNER, K. R. S. ASZHERANDH. M. FLOWERS
arabinose, glucuronic acid or, to a lesser extent, mannitol, on sweet-clover root discs, are strongly deterrent to S. cylindricollis. In our no-choice experiments with mixtures of sugars, there was no reduction in feeding with the possible exception of with fucose-sucrose. In the choice experiments, we found that n-xylose, N-acetyl-n-glucosamine, L-rhamnose and n-mannose in a 1 : 1 ratio with sucrose, enhanced feeding. This was clearly not in agreement with similar work by other authors, albeit with insect species different from ours. In this type of experiment methyl or-D-glucopyranoside, L-fucose and n-glucuronolactone reduced the phagostimulatory effect of sucrose for 5’. littoralis. It cannot be excluded that the differences between our results and those of similar work by other authors are due to the fact that the latter offered the sugars in aqueous solution (Dethier, 1955 ; Galun & Fraenkel, 1957), in a semisynthetic, wet diet (Fraenkel, 1955) or applied on wet root discs of plant origin (Akeson et al., 1970), whereas in our work the sugars were offered to the insects on an inert carrier in mixtures considered as dry. However, the concept of “dry” sugar is open to discussion. As pointed out by Dethier (1955), crystals of sugar dried in a desiccator for a month elicit a positive response in the blowfly due to a monolayer of water invariably forming on their surface, resulting in a solution. The existence of a viscous secretion at the tip of insect labellar taste hairs was demonstrated, e.g. in Vanessa, Leptinotarsa and Calliphora (see review by Stiirckow, 1967), confirming an earlier postulation of Tiensuu (1952) on the presence of such a secretion. Tiensuu had suggested, however, that this secretion was responsible for the stimulation of some insects by dry sugars, but Stiirckow (1967) found that, on the contrary, the secretion inhibited the response to sucrose. Acknowledgement-Our technical help.
thanks are expressed to Mrs. Shulamit Cohen for her excellent
REFERENCES AKESONW. R., HASKINSF. A., GORZ H. J. & MANCLITZG. R. (1970) Feeding response of the sweetclover weevil to various sugars and related compounds. J. econ. Ent. 63, 1079-1080. AUGUSTINEM. G., FISK F. W., DAVIDSONR. H., LAPIDUS J. B. & CLEARY R. W. (1964) Host-plant selection by the Mexican bean beetle, Epilachna varivestis. Ann. ent. Sot. Am. 57, 127-134. DAVISG. R. F. (1961) The biting response of larvae of the prairie grain wireworm, Crenicera aeripennis destructor (Brown) (Coleoptera: Elateridae) to various extracts of germinating rye seed. Can. J. 2001. 39, 299-303. DAVIS G. R. F. (1968) Phagostimulation and consideration of its role in artificial diets. Bull. ent. Sot. Am. 14, 27-30. DETHIERV. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Q. Rev. Biol. 30, 348-371. EVANSD. R. (1963) Chemical structure and stimulation by carbohydrates. In O&action and Taste I (Edited by ZOTI‘ERMAN Y.), pp. 165-176. Proc. 1st Int. Symp., Stockholm, September 1962, Pergamon Press, Oxford. FRAENKELG, (1955) Inhibitory effects of sugars on the growth of the mealworm, Tenebrio molitor L. J. cell. camp. Physiol. 45, 393-408.
FEEDINGRESPONSEOF EGYPTIANCOTTONLEAFWORMLARVAETO SUGARS VON FRISCH K. (1934) Uber den Geschmacksinn der Biene.
913
Ein Betrag zur vergleichenden Physiologie des Geschmacks. 2. vergl. Physiol. 21, 1-156. GALUN R. & FRAENKELG. (1957) Physiological effects of carbohydrates in the nutrition of a mosquito, Aedes aegypti and two flies, Sarcophaga bullata and Musca domestica. J. cell. camp. Physiol. 50, l-23. GOTHILF S. & BECK S. D. (1967) Larval feeding behaviour of the cabbage looper, Trichoplusia ni. J. Insect Physiol. 13, 1039-1053. HASSETC. H. (1948) The utilization of sugars and other substances by Drosophila. Biof. Bull. 95, 114-123. HERON R. J. (1965) The role of chemotactic stimuli in the feeding behaviour of spruce budworm larvae on white spruce. Can. J. Zool. 43, 247-269. HOUSEH. L. (1961) Insect nutrition. A. Rev. Ent. 6, 13-26. HSIAO T. H. & FRAENKELG. (1968) The influence of nutrient chemicals on the feeding behavior of the Colorado potato beetle, Leptinotarsa decemlineata. Ann. ent. Sot. Am. 61, 44-54. IT0 T. (1960) Effect of sugars on feeding of larvae of the silkworm, Bombyx mori. J. Insect Physiol. 5, 95-107. LALL S. B. (1970) Nectar sugars as chemostimulants for the tarsal taste sensilla of haematophagous tabanids (Diptera). J. med. Ent. 7, 103-106. LIPKE H. & FRAENKELG. (1956) Insect nutrition. A. Rev. Ent. 1, 17-44. LOSCHIAVO S. R. (1965) The chemosensory influence of some extracts of brewer’s yeast and cereal products on the feeding behaviour of the confused flour beetle, Tribolium confusum (Coleoptera: Tenebrionidae). Ann. ent. Sot. Am. 58, 578-588. MEISNER J. & ASCHER K. R. S. (1968) A method to assay the phagostimulatory effect towards insects of plant extracts applied to styroporR disks. Riv. Parassit. 29, 74-77. MEISNERJ., ASCHERK. R. S. & KAMHI J. (1970) Feeding stimulants for the larva of the Egyptian cotton leafworm, Spodoptera littoralis Boisd.-I. Assaying the larval response to extracts of several host plants and to some pure substances with the styroporR method. Wld Rev. Pest Control 9, 104-l 18. RICHTERC. P. & CAMPBELLK. H. (1940) Sucrose taste threshold of rats and humans. Am. J. Physiol. 128, 291-297. RICKS B. L. & VINSON S. B. (1970) Feeding acceptability of certain insects and various water-soluble compounds to two varieties of the imported fire ant. J. econ. Ent. 63, 145-148. SCHOONHOVEN L. M. (1968) Chemosensory bases of host plant selection. A. Rev. Ent. 13, 115-136. SHALLENBERGER R. S. (1964) Sweetening agents in foods. Agric. Sci. Rev. 2, l-10. STAUDENMAYER T. (1939) Die Giftigkeit der Mannose fi.ir Bienen und andere Insekten. Z. vergl. Physiol. 26, 664-668. STURCKOWB. (1967) Occurrence of a viscous substance at the tip of the labellar hair of the blowfly. In 02faction and Taste II (Edited by HAYASHIT.), pp. 707-720. Proc. 2nd Int. Symp., Tokyo, Sept. 1965. Pergamon Press, Oxford. SUTHERLAND0. R. W. (1971) Feeding behaviour of the grass grub Costelytra zealundica (White) (Coleoptera: Melolonthinae)-1. The influence of carbohydrates. N.Z. J. Sci. 14, 18-24. TIENSUUL. (1952) On the tarsal chemical sense, and its significance and distribution in the class Insecta. Trans. IXth Int. Congr. Ent., Amsterdam, Vol. I, pp. 253-254. VIETTE P. (1963) Le complexe de “Prodenia litura (Fabricius)” dans la rkgion malgache (Lep. Noctuidae). Bull. mens. Sot. linn. Lyon 32, 145-148. [Abstract seen in Rev. appl. Ent. A 53, 421 (1965).] WALDBAUERG. P. (1968) The consumption and utilization of food by insects. Adv. Insect Physiol. 5, 229-288. 32
914
J. MEISNER, K. R. S. ASCHERANDH. M. FLOWERS
YAMAMOTO R. T. & F RAEZNKEL G. (1960) Assay of the principal gustatory stimulant for the tobacco hornworm, Protoparce sexta, from solanaceous plants. Ann. ent. Sot. Am. 53, 499-503. Key Word Index-Phagostimulation; sucrose; melibiose.
carbohydrates;
Spodoptera
littoralis;
Styropor;
THE FEEDING RESPONSE OF THE LARVA OF THE EGYPTIAN COTTON LEAFWORM, SPODOPTERA LITTORALIS BOISD., TO SUGARS AND RELATED COMPOUNDS-I. PHAGOSTIMULATORY AND DETERRENT EFFECTS* J. MEISNER,l
K. R. S. ASCHERl
and H. M. FLOWERS2
‘Department of Toxicology, The Volcani Institute of Agricultural Research, Bet Dagan; and rDepartment of Biophysics, The Weizmann Institute of Science, Rehovot, Israel (Received 11 November 1971)
Abstract-l. The effect of twenty-five sugars and related compounds on the feeding response of larvae of Spodoptera littoralis was investigated using the Styropor method. 2. Styropor lamellae were treated with 0.25 and 0.0625 M solutions. The weight of the lamellae consumed and the number and weight of fecal pellets, served as criteria of feeding stimulation. 3. Sucrose, raffinose, maltose, D-fnKtOSc, melibiose and D-glucose were found to be very active as phagostimulants. 4. The survival of larvae which had fed on sugar-treated Styropor for 3 days was the longest on sucrose, maltose, glucose, fructose, raffinose and melezitose. 5. Some of the sugars were assayed in pairs in different choice and nochoice experiments; no antagonistic or inhibitory effects were found.
INTRODUCTION THE ROLE of phagostimulants in the feeding behavior of phytophagous insects is the subject of intensive study. Various groups of compounds such as sugars, amino acids, vitamins, etc. were found to elicit feeding response in insects. Among the carbohydrates, a wide range of insects showed intensive response to sucrose (see, e.g., reviews by Lipke & Fraenkel, 1956; House, 1961; Davis, 1968; Schoonhoven, 1968). Other sugars, e.g., fructose, glucose, raffinose and maltose, were also effective as feeding stimulants (Davis, 1968). Among the polyhydric alcohols inositol exhibited feeding stimulation for some insect species (Ito, 1960; Yamamoto & Fraenkel, 1960). An earlier study (Meisner et al., 1970) was conducted on phagostimulants responsible for the attraction of the larvae of Spodoptera littoralis Boisd. [formerly called Prodeniu Zitura F. (Viette, 1963)] t o some of its host plants. From experiments with crude extracts of these plants, various fractions obtained therefrom,
* Contribution from The Volcani Israel. 1971 Series, No. 1988-E.
Institute
of Agricultural
Research,
Bet
Dagan,
900
J.
MEISNER,
K. R. S.
ASCHER
AND
H.M.
FLOWERS
and some pure nutrient chemicals, it was demonstrated preliminarily that sucrose, fructose and, to a lesser degree, glucose, were effective feeding stimulants for the larvae of this insect. The present study deals specifically with the stimulatory effect of carbohydrates and related compounds on the feeding response and survival of the larvae of S. littoralis. In addition, some of the sugars were tested in combination (of two sugars) to determine whether non-active sugars had an inhibitory effect on feeding. MATERIALS
AND
METHODS
Chemicals D-Arabinose, D-XylOW, D-glUCOSe, D-g&CtOSe, D-mannose, D-fructose, L-sorbose, L-rhamnose, L-fucose, N-acetyl-D-glucosamine, D-galacturonic acid, D-glucuronolactone; melibiose, sucrose, maltose, cellobiose, trehalose, raflinose, melezitose; D-mannitO1, Dsorbitol, galactitol (dulcitol), i-inositol, methyl o-D-glucopyranoside and calcium-Dgluconate were all from commercial sources. Bioassay technique Lamellae of a synthetic plastic material, Styropor (foamed polystyrene), were used as carriers for potential phagostimulatory substances. This material had been used with various methodological variations to assay feeding stimulation of larvae of the potato tuber moth, Gnorimoschema operculella Zell., codling moth, Cydia pommella L. and carob moth, Ectomyelois ceratoniae Zell. (Meisner & Ascher, 1968). For larvae of S. littoralis, thin, rectangular (6 x 3 cm) laminae of Styropor were cut from a small block with the help of a manually driven meat slicer (Meisner et al., 1970). The laminae were painted with solutions (ethanol-water, 1 : 1) of the test substances at known concentrations and left to dry for 24 hr. They were then placed singly or in pairs in closed Petri dishes (dia. 14 cm). Larvae, reared at 27°C on lucerne and weighing between 170 and 190 mg, were starved for 3 hr; then one weighed larva was introduced into each Petri dish. All experiments were conducted at 27°C. The feeding tests were of two types: (a) No-choice experiments, in which a single larva was confined to each Petri dish and exposed to one treated lamina. In earlier experiments we had also placed a control lamina treated with the solvent mixture only, into the same dish, but we soon noted that this lamina was left practically untouched; (b) Choice experiments, in which two or more laminae treated with different substances or with mixtures of them were offered simultaneously to the larva. No-choice experiments In the no-choice experiments, twenty-five sugars and related compounds were assayed in two concentrations, 0.25 and 0.0625 M (weight/volume). Each of the treatments was done in at least 20 x 1 larva replicates per compound, but many of them were repeated at least forty times. Every day the lamellae were taken out, weighed and returned. In our previous work with S. littoralis (Meisner et al., 1970), the rate of feeding was expressed as percentage of a standard-weight Styropor lamella consumed. Since then we have found it more convenient to use, instead, weight of Styropor consumed, in mg. Hence the weight of Styropor consumed after 24, 48 and 72 hr, and the total number and weight of fecal pellets, with the exclusion of the initial black ones which result from the prior feeding on lucerne, served us as the criteria of feeding stimulation in the present work. The fecal pellets were also counted. We had noted in the previous study (Meisner et al., 1970) that the size of the fecal pellets varied greatly with different plant extracts. A similar phenomenon could be seen also with different sugars and therefore the more objective criterion was the weight of the pellets. The number of dead (if any) larvae was recorded every day.
FEEDING RESPONSE OF EGYPTIAN
COTTON LEAFWORM LARVAE TO SUGARS
901
When a sugar-treated Styropor lamella was consumed at a rate of more than 10 mg/larva within 72 hr, we postulated that the sugar was highly phagostimulatory. Consumption of 5-10 mg/larva denoted moderate phagostimulatory activity. Sugars which elicited a feeding response of less than 5 mg Styropor/larva were considered to be weakly attractive or inactive. The lamellae were removed after the 3-day feeding, but the larvae were left in the Petri dishes. We continued to record daily mortality until all the larvae had died. In this type of experiment, larvae kept for 3 days on untreated lamellae (the lamellae were subsequently removed) served us as controls. The survival index was calculated according to the following formula : Survival index =
Total larva-days Initial number of larvae
Survival in excess of that of control larvae, as related to the amount of treated Styropor consumed during 3 days, was expressed by the formula A-B -3 c where A is the survival index on a treated Styropor lamella, B the survival index on untreated Styropor (control) and C the weight, in mg, of treated Styropor consumed per larva during 72 hr. This calculation was carried out only for survival indices reasonably greater than the control, i.e. above 3.0 (untreated control survival index was 2.5). Choice experiments In choice experiments we offered the larva simultaneously a lamella painted with a solution containing a mixture of 0.125 M of a weakly or non-attractive carbohydrate plus O-125 M sucrose, and a second lamella treated only with O-125 M sucrose. The two components separately at 0.125 M, and a mixture of the two sugars at a concentration of 0.125 M each, served as separate controls in these tests. These experiments were terminated after 48 hr. The weight of styropor consumed within this period, and the total weight of fecal pellets, again served as the criteria of phagostimulation.
RESULTS
No-choice experiments The feeding response of the larvae to various sugars and related compounds is summarized in Table 1. Sucrose was the most active sugar at both concentrations tested (0.25 and 0.0625 M). Raffinose, maltose, n-fructose, melibiose and n-glucose, in descending order of activity, elicited a strong feeding response at 0.25 M, whereas at 0.0625 M only sucrose and fructose were consumed at a rate of over 10 mg Styropor/larva during a period of 72 hr. Melezitose, trehalose, i-inositol and n-xylose produced a moderate feeding response (5-10 mg treated Styroporjarva within 72 hr) at 0.25 M. The rest of the carbohydrates assayed had a very slight or no phagostimulatory activity at all at both test concentrations. With some compounds of the last two groups, survival after 72 hr was poor. Since sucrose elicited the strongest feeding stimulus, it was of interest to assay the response to this sugar at a greater number of concentrations between 16 and 0.00001 per cent and also to determine the response threshold of the larvae to
RESPONSE
0.6
Control
;:: l-6 0.5 0.3 I.1 04 0.8 0.5 I.5 0.2
LARVAE
0.25 M
I.1
72 hr
z 73 106 62 52 35
70
0.4
24 hr
A:; 2.7 0.6 1.6 0.3 I.0 1.1 1.6 0.8
;:; 1.7 0.8
2:;
::;
33:;
8.9 5-3 4.5 7.3 3-5
48 hr 11.6 7.8
72 hr
COMPOUNDS
AT
7 ;z
::
:z
3;
;;
*z 55
179 112
Weight (mg)
96 16
59 9
33 22 Not taken
::
9
1:; 166
102 120 120
227 304
503 295 167 393 153
No.
Fecal pellets produced during 72 hr in a a twenty-larva experiment
RELATED
O-0625 M
AND
Weight in mg of treated Styropor lamella consumed per larva, cumulative values after
TO VARIOUS SUGARS
$ One hundred per cent mortality.
31
1:: ;2: 41 I3 Not taken
145 185: 191 193 1;;
::
E 151 130 174 123 61
428 396 433 248 359 287 111 121 187
244
Weight (mg)
568
No.
twenty-larva experiment
Fecal pellets proz;;di$aring
RANGE,170-190mg) 0.25 AND 0.0625 M
(WEIGHT
t Ninety-five per cent died.
1.1
::;: 0.6 Not done
;:; 0.3
12:;
;:: 2.8 2.2
;:; 2.8
i:; 2.8
;:‘:
;:;
;:; 6.8
17.5 13.1 12.1
48 hr
;:; 2.1
4.4
;I;
24hr
;:; 0.3
* Ninety per cent died.
littoralis
Weight in mg of treated Styropor lamella consumed per larva, cumulative values after
OF S.
CeIIobiose D-Glucuronolactone L-Fucose CaIcium-D-gluconate
N-SCetyl-D-glUCOSSmine
Sucrose Raffinose Maltose D-Fructose Mehbiose D-Glucose Melezitose TrehaIose i-Inositol D-Xylose D-GaIactose D-Mannose L-Sorbose Methyl a-D-ghrcoside D-SorbitoI D-GaIacturonic acid L-Rhamnose D-i%nnitoI D-Arabinose GaIactitol
Sugars and related compounds
TABLE I-FEEDING
FEEDING
RESPONSE
OF EGYPTIAN
COTTON
LEAFWORM
LARVAE
TO
SUGARS
903
sucrose. The results are presented in Fig. 1, which shows the positive correlation between concentration of sucrose and larval response. The response threshold to sucrose was at O*OOlS per cent. I
I II
I1111
(>-
--of&hr
I
I
II I
20-
72 hr
16 -
u E ii4 c 12 8 b 8-8P6-
4-
l
_
8--/L 0
Controlb72 hr I
om
11l1111
0005 001
III
1111111
005
I
I
0.1 % Sucrose
Ill1111
05
I
1.0
1 l11llll
50
100 204
FIG. 1. Feeding response of larvae of S. littoralis (ranging in weight from 170 to 190 mg) to different concentrations of sucrose applied to Styropor lamellae. Five further concentrations from 0401 to 040001 per cent yielded values equal to or below those of control.
Values of larval survival which we consider as a certain indication of the nutritive value of sugars and related compounds, are presented in Table 2. The larval survival index was the greatest on sucros&.4 days. The sugars with a survival index ranging from 3 to 4 days were, in descending order: maltose, n-glucose, n-fructose, rafhnose and melezitose. The survival index of all the other compounds approached, or was even less than, that of the control (untreated Styropor). When we calculated the relation between the survival index in excess of control and Styropor consumption, the most nutritive sugar was D-&COSe, followed by sucrose (Table 2, last column).
904 TABLE
J. MEISNER,K.R.
S.
OF LARVAE OF S. littorah WITH VARIOUS SUGARS AND
~-SURVIVAL TREATED
Sugars and related compounds
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
ASCHERANDH.M.
FLOWERS
KEPT FOR 3 DAYS ON S~ROPOR RELATED COMPOUNDS AT 0.25 M
Survival index*
Sucrose Raffinose Maltose D-Fructose Melibiose D-Glucose Melezitose Trehalose i-Inositol D-Xylose D-Galactose D-Mannose L-Sorbose Methyl ol-D-glucoside D-Sorbitol D-Galacturonic acid L-Rhamnose D-Mannitol D-Arabinose Galactitol AJ-acetyl-D-glucosamine Cellobiose D-Glucuronolactone L-Fucose
4.4 3.2 3.8 3.3 2.8 3.6 3.2 2.4 2.2 1.8 2.8 2.8 2.1 2.7 2.3 2.5 2.2 2.7 2.1 2.6 2.4 1.8 2.0 2.0
Control
2.5
LAMELLAE
(A-WCt 8.9 4.0 7.7 6.5
x x x x 11.0 x 8.0 x
1O-2 10-s 10-2 1O-2 10-Z 1O-2
* Survival index = total larva-days/initial number of larvae. 7 A = Survival index on treated Styropor lamellae. B = Survival index on untreated Styropor lamellae (control). C = Weight in mg of treated Styropor consumed per larva during 72 hr (values taken from Table 1).
Choice experiments Experiments were carried out with the aim of elucidating whether slightly active or inactive sugars have merely no phagostimulatory effect, or if they inhibit feeding. To decide between these two possibilities, sugars of these two types were mixed with sucrose and offered to larvae. The results of these experiments are summarized in Table 3. The slightly active or inactive sugars tested, with the possible exception of fucose, did not reduce the activity of sucrose, when they were admixed with it at a concentration of 0.125 M each (Table 3, experimental layout c). In the choice tests (Table 3, experimental layout d), in which a lamella treated with a mixture of sucrose with a slightly active or non-active carbohydrate, each at 0.125 M, was pitted against O-125 M sucrose alone, there was an almost
EFFECTOF
(a) Sucrose 0.125 M (b) Sorbitol O-125 M (c) Sucrose 0.125 M + sorbitolO*125 M (d)
(a) Sucrose O-125 M (b) Arabinose 0.125 M (c) Sucrose 0.125 M + arabinose O-125 M (d)
(a) Sucrose O-125 M (b) Xylose 0.125 M (c) Sucrose 0.125 M + xylose 0.125 M (d)
(a) Sucrose 0.125 M (b) MannitolO~125 M (c) Sucrose 0.125 M+ mannito10.125 M (d)
2
3
4
ADDING
1
No choice
TABLE ~-THE
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
I
SLIGHTLY
II
INACTIVESUGARSTO
Sucrose 0.125 M + mannitol 0.125 M
Sucrose 0.125 M + xylose 0.125 M
Sucrose 0.125 M + arabinose 0.125 M
Sucrose 0.125 M + sorbito10.125 M
Choice
ACTIVEOR
ON PHAGOSTIMULATION
7.1 2.3 8.3
9.1 2.6 8.2
9.1 2.9 9.2
8.5 2.2 10.7
No choice
5.0
3.1
4.2
4.6
I
4.4
7.2
5.8
5.4
II
Choice
9.4
10.3
10.0
10.0
Total
Weight of treated Styropor comsumed in mg per larva during 48 hr
SUCROSE
OF S. littoralis
(continued overleaf)
12.3
11.1 5.0 12.4 5.8
7.5
13.8 3.8 11.0 14.0
6.3
7.6 0.8
12.2
13.8 2.4 12.7
12.8
14.0 1.5 13.8
Number of Weight of fecal pellets fecal pellets per larva in mg per during larva during 48 hr 48 hr
IN LARVAE
E
%
c
r?,
28 2 0 g P w z
(a) Sucrose 0.125 M (b) N-acetyl-Dglucosamine 0.125 M (c) Sucrose 0.125 M+ N-acetyl-Dglucosamine 0.125 M Sucrose (d) 0.125 M
7
Sucrose 0.125 M
(a) Sucrose 0.125 M (b) Gala&to1 0.125 M (c) Sucrose 0.125 M + galactitol 0.125 M (d)
6
Sucrose 0.125 M
(a) Sucrose 0.125 M (b) InositolO*l25 M (c) Sucrose 0.125 M + inositol O-125 M (d)
I
5
No choice II
Sucrose 0.125 M + N-acetyl-Dglucosamine 0.125 M
Sucrose O-125 M + galactitol 0.125 M
Sucrose 0.125 M + inositolO*l25 M
Choice
14.1
12.1 I.9 14.6
12.5
12.3
6.3
6.2
6.4 0.8
6.3
5.7 1.2 8.0
7.0
3.9 9.0
11.3
8.6
9.6
8.7
6.9 5.4 14.3
Weight of fecal pellets in mg per larva dur.ng 48 hr 12.1
7.3
5.1
5.6
5.1
Total
13.4 I.3
3.5
4.0
3.6
II
Number of fecal pellets per larva during 48 hr
7.1 0.8
7.6 I.6 11.5
7.4 3.3 6.7
I
Choice
Weight of treated Styropor consumed in mg per larva during 48 hr
No choice
TABLE 3 (cont.)
$ 1
b
7
g
F
.7;:
(a) Sucrose O-125 M (b) Cellobiose O-125 M (c) Sucrose 0.125 M + cellobiose O-125 M (d)
(a) Sucrose 0.125 M (b) Methyl a-n-glucoside 0.125 M (c) Sucrose 0.125 M + methyl a-~glucoside 0.125 M (d)
(a) Sucrose 0,125 M (b) Fucose O-125 M (c) Sucrose O-125 M + fucose O-125 M (d)
(a) Sucrose 0.125 M (b) Rhamnose 0.125 M (c) Sucrose 0.125 MS rhamnose 0.125 M (d)
8
9
10
11
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M
Sucrose 0.125 M + rhamnose 0.125 M
Sucrose 0.125 M + fucose 0.125 M
Sucrose 0.125 M + methyl or-n-ghrcoside 0.125 M
Sucrose 0.125 M + cellobiose 0.125 M 15.2
8.6 3.1 9.8
9.4 3.4 6.8
2.9
5.6
5.0
3.8
7.9
9.4
5.0
7.4 1.2 8.2
7.4
8.9 3.1 4.9
9.4
8.3
9.1 3.6
7.2
7.1 3.3 4.0
(continued ooerleuf)
11.3
14.1 2.4 13.9
14.7
15.4 4.5 10.0
15.4
13.3
10.0
10.7
8.6
4.0
4.7
14.1 44
6.0
6.0
13.1 2.5 8.7
8.5 3.5
8.2 2.2 7.4
2.9 8.LI E.9 6.LT
Z.&I
I.EI
s.91 0.s 6.LI
8.L .L*t 9.6 6.9 5.8 9.E 9.6
0.8 8-O
6.LI
9.0
2.8
I.S E*ZI 1.Z Z.ZI
LeEI
s.z Z.ZI
Z*ZI
+
m
m
‘j-ZI.0
asouuauI
sz1.0 asoJans
IN SZS~O asomnc;
asomnc;
IN SZI.0
Ill SZI.0 auomqouom3rqB IN SZI.0 + bu ~~1.0 =oJDns asom-ig
m sz1.0 aso-93
m gi.0 asouuem + m ~~1.0 asoJDng w szbo aso-m
m SZI-0 asovq~~ + m sz1.0 asoJ=q tu sz1.0 asoww ku SZI-0 a=JDns
INSZT.0
IN SZI-0 auo~xqouomxql + m sz1.0 as=w
tv sz1.0
=-ns
auo~x3~0uomxq~
FEDING
RESPONSE
OF EGYPTIAN
COTTON
LEAFWORM
LARVAE
TO SUGARS
909
equal distribution of consumption between the two lamellae, with the exception of: D-xylose, iV-acetyl-n-glucosamine, L-rharnnose and n-mannose-activity of mixture higher than sucrose; and methyl a-D-glucoside, L-fucose and n-glucuronolactone-activity of mixture lower than that of sucrose. The exceptionally high consumption rate of the xylose-sucrose mixture vs. sucrose alone, may indicate a synergistic effect. DISCUSSION
In the present study, the phagostimulatory effect of the sugars in which consumption was over 10 mg treated lamella/larva within 72 hr, decreased in the following order: At 0.125 M : sucrose > raffinose > maltose > fructose > melibiose > glucose. At 0.0625 M : sucrose > fructose > raffinose > glucose > maltose > melibiose. As judged by human response, sugars are usually listed in decreasing order of sweetness as fructose > sucrose > glucose > galactose > mannose > lactose > raffi1964). Evidently, the order of sweetness established for nose (Shallenberger, human beings and the feeding response of larvae of S. littoralis to various sugars are different. It should be noted that the sweet-taste threshold of sucrose for the human tongue lies between 3 x 1O-3 and 3 x 10ea M (Richter & Campbell, 1940), whereas sensitivity to sucrose within the range of 1 x lo-’ to 1 x 10d6 M is characteristic of many insect species (Dethier, 1955). In most insects investigated, sucrose, raffinose, maltose, n-fructose and n-glucose were found highly active, though their exact relative order as regards phagostimulation differed from species to species. Sucrose was found to be the most acceptable sugar in a great number of insects, though several exceptions are known. One example is the confused flour beetle, Tribolium con&sum, for which maltose had a higher phagostimulatory rate than sucrose at O-1 M (Loschiavo, 1965). n-Fructose, which elicited a strong feeding response in our study, was found to be highly attractive to many other insects. Thus, the adults of the yellow-fever mosquito, Aedes aegypti (Galun & Fraenkel, 1957), the fruit fly, Drosophila melanogaster (Hasset, 1948), the Mexican bean beetle, Epilachna vurivestis (Augustine et al., 1964) and the larvae of the spruce budworm, Choristoneura fumiferana (Heron, 1965), responded to fructose in a degree similar to or even more than to sucrose. A relatively low activity of fructose was found in the two varieties of the imported fire ant, Solenopsis saevissima saevissima and Solenopsis saevissima richteri (Ricks & Vinson, 1970). The least phagostimulatory hexose in the active group was glucose. This holds true also for some other insect species. Thus, the Mexican bean beetle, E. varivestis (Augustine et al., 1964), the cabbage looper, Trichoplusia ni (Gothilf & Beck, 1967) and the blowfly, Phormia regina (Dethier, 1955) showed a higher feeding rate on fructose than on glucose, whereas in the potato beetle, Leptinotarsa decemlineata, glucose at O-1 M gave a much higher feeding response than fructose (Hsiao & Fraenkel, 1968). In Chrysops vittatus, glucose was also superior to fructose, which was explained by the feeding habits of the fly (Lall, 1970). Only
910
J. MEISNER,K. R. S. ASCHERAND H. M. FLOWERS
a moderate feeding rate is elicited by glucose in the sweet-clover weevil, Sitoniu (Akeson et al., 1970), and it is only slightly active in the prairie grain wireworm, Crenicera aeripennis destructor (Davis, 1961). According to Shallenberger (1964), a freshly prepared solution of a-n-glucose is sweeter for human beings than a mutarotated one. When we compared the activity of a freshly prepared solution of 0.25 M n-glucose, which is principally in the 01form, with that of one aged for a few days, which contains a mixture of the 01and the ,!I form, no difference was found in the consumption rate of the treated Styropor lamellae. Dethier (1955) also recognized that the threshold values for fresh and for aged, mutarotated solutions of glucose are not significantly different. Evans (1963) confirmed these findings: he demonstrated convincingly that there is no difference between the response of blow-flies to cx-n-glucose and @-glucose prepared from it by recrystallization from hot acetic acid. It cannot be excluded, however, that with some other sugars, mutarotation in solution may have some influence on phagostimulation. This question will still have to be investigated more thoroughly, although it sounds far-fetched to compare the activity of a carbohydrate in solution with that of its dried residue on a Styropor lamella. Among the compound sugars in this group we found that melibiose, which is usually considered as inactive, has a strongly phagostimulatory effect on the larvae of S. ZittoraZis. Solely, Sutherland (1971) very recently demonstrated that melibiose stimulated considerable feeding in the grass grub, Costelytra zealandica. To the best of our knowledge, these are, therefore, the first reports on the high phagostimulatory activity of this sugar. The inactivity of melibiose in the honeybee was explained by von Frisch (1934) as being due to the presence of a galactose group and to the lack of an 01linkage, an opinion shared by Dethier (1955) to interpret results in the blowfly. A merely slightly phagostimulatory effect of this sugar could be detected in the Colorado beetle (Hsiao & Fraenkel, 1968) and in spruce budworm larvae (Heron, 1965). The hexoses n-mannose, n-galactose and L-sorbose were found to be slightly active in this study. D-Mannose showed a medium effectiveness in the sweetclover weevil (Akeson et al., 1970) and a slight effectiveness in the potato beetle (Hsiao & Fraenkel, 1968); it was inactive in the blowfly (Dethier, 1955) and spruce budworm larvae (Heron, 1965), and toxic in the honeybee (Staudenmayer, 1939). n-Calactose showed only a medium activity in the potato beetle (Hsiao & Fraenkel, 1968) and a slight one in the spruce budworm (Heron, 1965). A direct relationship between the quantity of sugar-coated Styropor consumed by the larvae and the weight of fecal pellets excreted is not always apparent (see Table 1). This may be due to the fact that since our chief goal in the present paper was the phagostimulatory aspect, we did not choose a physiologically defined period, nor did we collect residual food from the gut (cf. Waldbauer, 1968). We have certain cases in Table 1, in which excretion seemed to be greater than ingestion (methyl-ol-n-glucoside at both 0.25 and O-0625 M, arabinose at 0.25 M and n-galactose at O-0625 M). An examination of the influence of feeding different cylindricollis
FEEDING RESPONSE OF EGYPTIAN
COTTON
LEAFWORM
LARVAE TO SUGARS
911
carbohydrates on the composition of the fecal pellets in S. littoralis may throw some light on these findings. It was evident from the number : weight ratio of fecal pellets in Table 1 that the size of the pellets varied greatly for different carbohydrates; a similar phenomenon was previously described by us for various plant extracts on Styropor (Meisner et aE., 1970). As to the survival index of the larvae of S. littoralis, which was in the order sucrose > maltose > glucose > fructose > raffinose > melezitose, our results are more or less in agreement with those of Galun & Fraenkel (1957) for A. aegypti but not for Sarcophaga bullata and Mwa domestica, for both of which survival was high on mannose and galactose, contrary to our results. The data of Hasset (1948) on the survival rate of D. melanogaster adults on sugars at M/10 are in agreement with the low survival index found by us for mannose, galactose and melibiose. When we calculated the relation between the survival index (minus control values) and the amount of Styropor consumed, n-glucose and sucrose were found to be the two sugars nutritionally most efficient for survival. The survival indices of the larvae on different sugars are thus not only a function of the total weight consumed, but may be also influenced by differences in the metabolization and utilization of these compounds. Experiments in progress to examine the fate of a number of the ingested sugars considered in the present work will be described in a later publication. In experiments with the housefly (Galun & Fraenkel, 1957), the blowfly (Dethier, 1955), the mealworm, Tenebrio molitor (Fraenkel, 1955) and the sweetclover weevil (Akeson et al., 1970), it was found that many slightly active or inactive sugars, when admixed with sucrose, glucose or starch, had a strong feeding deterrent effect. This phenomenon was found in the housefly in choice experiments with solutions of sucrose mixed with xylose, sorbose or ribose, in the ratio 1 : 1, and to a lesser extent with rhamnose, arabinose, a-methyl mannoside or inositol. Dulcitol was the only inactive sugar that did not deter the flies when admixed with sucrose (Galun & Fraenkel, 1957). Dethier (1955) showed that mannose, which is poorly phagostimulatory in the blowfly, caused a tenfold elevation of the threshold of fructose. No such change could be found on adding mannose to other attractive sugars. Dethier concluded for the blowfly that “it had been impossible for the most part to demonstrate any competitive inhibition between sugars”. Further light was thrown on this problem by Fraenkel (1955), who kept larvae of T. molitor on an artificial diet containing glucose as the main carbohydrate constituent. The total carbohydrate content was kept constant at 80 percent. In no-choice experiments on this diet, an inhibitory effect was still obtained at the following concentrations, when the concentration of glucose was slightly reduced and the following sugars were introduced: 10% galactose; 5% arabinose, rhamnose, mannose or sorbose; 2% xylose, ribose, or-methyl mannoside or dulcitol (galactitol) ; and 1y. a-methyl glucoside. Akeson et al. (1970), in nochoice experiments, demonstrated that residues of sucrose in a 1 : 1 ratio with
912
J. MEISNER, K. R. S. ASZHERANDH. M. FLOWERS
arabinose, glucuronic acid or, to a lesser extent, mannitol, on sweet-clover root discs, are strongly deterrent to S. cylindricollis. In our no-choice experiments with mixtures of sugars, there was no reduction in feeding with the possible exception of with fucose-sucrose. In the choice experiments, we found that n-xylose, N-acetyl-n-glucosamine, L-rhamnose and n-mannose in a 1 : 1 ratio with sucrose, enhanced feeding. This was clearly not in agreement with similar work by other authors, albeit with insect species different from ours. In this type of experiment methyl or-D-glucopyranoside, L-fucose and n-glucuronolactone reduced the phagostimulatory effect of sucrose for 5’. littoralis. It cannot be excluded that the differences between our results and those of similar work by other authors are due to the fact that the latter offered the sugars in aqueous solution (Dethier, 1955 ; Galun & Fraenkel, 1957), in a semisynthetic, wet diet (Fraenkel, 1955) or applied on wet root discs of plant origin (Akeson et al., 1970), whereas in our work the sugars were offered to the insects on an inert carrier in mixtures considered as dry. However, the concept of “dry” sugar is open to discussion. As pointed out by Dethier (1955), crystals of sugar dried in a desiccator for a month elicit a positive response in the blowfly due to a monolayer of water invariably forming on their surface, resulting in a solution. The existence of a viscous secretion at the tip of insect labellar taste hairs was demonstrated, e.g. in Vanessa, Leptinotarsa and Calliphora (see review by Stiirckow, 1967), confirming an earlier postulation of Tiensuu (1952) on the presence of such a secretion. Tiensuu had suggested, however, that this secretion was responsible for the stimulation of some insects by dry sugars, but Stiirckow (1967) found that, on the contrary, the secretion inhibited the response to sucrose. Acknowledgement-Our technical help.
thanks are expressed to Mrs. Shulamit Cohen for her excellent
REFERENCES AKESONW. R., HASKINSF. A., GORZ H. J. & MANCLITZG. R. (1970) Feeding response of the sweetclover weevil to various sugars and related compounds. J. econ. Ent. 63, 1079-1080. AUGUSTINEM. G., FISK F. W., DAVIDSONR. H., LAPIDUS J. B. & CLEARY R. W. (1964) Host-plant selection by the Mexican bean beetle, Epilachna varivestis. Ann. ent. Sot. Am. 57, 127-134. DAVISG. R. F. (1961) The biting response of larvae of the prairie grain wireworm, Crenicera aeripennis destructor (Brown) (Coleoptera: Elateridae) to various extracts of germinating rye seed. Can. J. 2001. 39, 299-303. DAVIS G. R. F. (1968) Phagostimulation and consideration of its role in artificial diets. Bull. ent. Sot. Am. 14, 27-30. DETHIERV. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Q. Rev. Biol. 30, 348-371. EVANSD. R. (1963) Chemical structure and stimulation by carbohydrates. In O&action and Taste I (Edited by ZOTI‘ERMAN Y.), pp. 165-176. Proc. 1st Int. Symp., Stockholm, September 1962, Pergamon Press, Oxford. FRAENKELG, (1955) Inhibitory effects of sugars on the growth of the mealworm, Tenebrio molitor L. J. cell. camp. Physiol. 45, 393-408.
FEEDINGRESPONSEOF EGYPTIANCOTTONLEAFWORMLARVAETO SUGARS VON FRISCH K. (1934) Uber den Geschmacksinn der Biene.
913
Ein Betrag zur vergleichenden Physiologie des Geschmacks. 2. vergl. Physiol. 21, 1-156. GALUN R. & FRAENKELG. (1957) Physiological effects of carbohydrates in the nutrition of a mosquito, Aedes aegypti and two flies, Sarcophaga bullata and Musca domestica. J. cell. camp. Physiol. 50, l-23. GOTHILF S. & BECK S. D. (1967) Larval feeding behaviour of the cabbage looper, Trichoplusia ni. J. Insect Physiol. 13, 1039-1053. HASSETC. H. (1948) The utilization of sugars and other substances by Drosophila. Biof. Bull. 95, 114-123. HERON R. J. (1965) The role of chemotactic stimuli in the feeding behaviour of spruce budworm larvae on white spruce. Can. J. Zool. 43, 247-269. HOUSEH. L. (1961) Insect nutrition. A. Rev. Ent. 6, 13-26. HSIAO T. H. & FRAENKELG. (1968) The influence of nutrient chemicals on the feeding behavior of the Colorado potato beetle, Leptinotarsa decemlineata. Ann. ent. Sot. Am. 61, 44-54. IT0 T. (1960) Effect of sugars on feeding of larvae of the silkworm, Bombyx mori. J. Insect Physiol. 5, 95-107. LALL S. B. (1970) Nectar sugars as chemostimulants for the tarsal taste sensilla of haematophagous tabanids (Diptera). J. med. Ent. 7, 103-106. LIPKE H. & FRAENKELG. (1956) Insect nutrition. A. Rev. Ent. 1, 17-44. LOSCHIAVO S. R. (1965) The chemosensory influence of some extracts of brewer’s yeast and cereal products on the feeding behaviour of the confused flour beetle, Tribolium confusum (Coleoptera: Tenebrionidae). Ann. ent. Sot. Am. 58, 578-588. MEISNER J. & ASCHER K. R. S. (1968) A method to assay the phagostimulatory effect towards insects of plant extracts applied to styroporR disks. Riv. Parassit. 29, 74-77. MEISNERJ., ASCHERK. R. S. & KAMHI J. (1970) Feeding stimulants for the larva of the Egyptian cotton leafworm, Spodoptera littoralis Boisd.-I. Assaying the larval response to extracts of several host plants and to some pure substances with the styroporR method. Wld Rev. Pest Control 9, 104-l 18. RICHTERC. P. & CAMPBELLK. H. (1940) Sucrose taste threshold of rats and humans. Am. J. Physiol. 128, 291-297. RICKS B. L. & VINSON S. B. (1970) Feeding acceptability of certain insects and various water-soluble compounds to two varieties of the imported fire ant. J. econ. Ent. 63, 145-148. SCHOONHOVEN L. M. (1968) Chemosensory bases of host plant selection. A. Rev. Ent. 13, 115-136. SHALLENBERGER R. S. (1964) Sweetening agents in foods. Agric. Sci. Rev. 2, l-10. STAUDENMAYER T. (1939) Die Giftigkeit der Mannose fi.ir Bienen und andere Insekten. Z. vergl. Physiol. 26, 664-668. STURCKOWB. (1967) Occurrence of a viscous substance at the tip of the labellar hair of the blowfly. In 02faction and Taste II (Edited by HAYASHIT.), pp. 707-720. Proc. 2nd Int. Symp., Tokyo, Sept. 1965. Pergamon Press, Oxford. SUTHERLAND0. R. W. (1971) Feeding behaviour of the grass grub Costelytra zealundica (White) (Coleoptera: Melolonthinae)-1. The influence of carbohydrates. N.Z. J. Sci. 14, 18-24. TIENSUUL. (1952) On the tarsal chemical sense, and its significance and distribution in the class Insecta. Trans. IXth Int. Congr. Ent., Amsterdam, Vol. I, pp. 253-254. VIETTE P. (1963) Le complexe de “Prodenia litura (Fabricius)” dans la rkgion malgache (Lep. Noctuidae). Bull. mens. Sot. linn. Lyon 32, 145-148. [Abstract seen in Rev. appl. Ent. A 53, 421 (1965).] WALDBAUERG. P. (1968) The consumption and utilization of food by insects. Adv. Insect Physiol. 5, 229-288. 32
914
J. MEISNER, K. R. S. ASCHERANDH. M. FLOWERS
YAMAMOTO R. T. & F RAEZNKEL G. (1960) Assay of the principal gustatory stimulant for the tobacco hornworm, Protoparce sexta, from solanaceous plants. Ann. ent. Sot. Am. 53, 499-503. Key Word Index-Phagostimulation; sucrose; melibiose.
carbohydrates;
Spodoptera
littoralis;
Styropor;