Trehalose and other carbohydrates in Anthonomus grandis, Heliothis zea, and Heliothis virescens during growth and development

J. Insect Physiol., 1971, Vol. 17, pp. 657 to 675. Pergavwn Press. Printed in Great Britain

TREHALOSE AND OTHER CARBOHYDRATES IN ANTHONOMUS GRANDLY, HELIOTHIS ZEA, AND HELIOTHIS VIRESCENS DURING GROWTH AND DEVELOPMENT* WILLIAM CARMEN

C. NETTLES, JR., BRENDA PARRO, and CHARLES L. MANGUM

SHARBAUGH,

Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, 4115 Gourrier Avenue, Baton Rouge, Louisiana 70808 (Receiwed 19 August 1970) Ahtract-Trehalose was present in all stages of the boll weevil, Anthonmus grandis, and usually was the main free sugar found in the haemolymph and in extracts of whole insects. The quantity of trehalose per insect, which was maximal in late last&star larvae, gradually decreased during the pupal and pharate adult stages and declined more rapidly in young adults. In unfed adults, the most rapid decrease in trehalose levels occurred when glycogen was depleted. As would be expected from the higher sugar content of the boll, the titre of trehalose was higher in young and old weevils fed cotton bolls than in those fed squares (cotton flower buds); but when adults were 10 to 20 days old, the levels were similar regardless of whether weevils were fed either bolls or squares. Thus, at least part of their adult life, weevils were able to regulate trehalose levels in spite of sizeable differences in the sugar content of the adult diet. Trehalose levels also are regulated in weevils of other ages: A IO-fold increase in the sucrose content of the larval diet raised the carbohydrate and lipid content of late last&star larvae and pharate pupae; but the 60 to 69 per cent increase in trehalose was less than the increase in glucose, anthrone positive substances other than trehalose, glycogen, and total lipids, which shows that trehalose was more closely regulated than the other components. In weevils fed the sucrose-rich larval diet, trehalose levels dropped during metamorphosis so that trehalose levels of newly emerged adults were identical regardless of the carbohydrate content of the larval diet. As much as 400 pg per insect of sugars other than trehalose were present in late last&star larvae with a dry weight of about 5 mg each, but the quantity was dependent on carbohydrate content of the diet and on the nutritional state since a sucrose-rich diet increased the titre of these sugars and because these sugars were quickly metabolized when the larvae stopped feeding. Degradation of trehalose during extraction and storage was greatly reduced by extracting sugars with chloroform-methanol and ethanol and by deionizing extracts as quickly as possible. Assays were inaccurate when glucose ox&se was used for the determination of glucose in extracts of boll weevils and of Heliothis spp. because deionization removed most of a substance which very strongly inhibits the glucose oxidase assay in boll weevils and because the amount of glucose was considerably overestimated in deionized extracts of Heliothis zea and Heliothis virescens. * Mention of a proprietary product does not constitute an endorsement of this product by the U.S. Department of Agriculture. 657

658

W. C.

NETTLES, JR., B. PARRO, C.

SHARBAUCH, ANDC. L. MANCUM

INTRODUCTION TREHALOSE(Lu-D-glucopyranosyl-oL-D-glucopyranoside) usually is the main free sugar found in insects. Although trehalose is absent in certain stages of several species, it has been found in at least one stage of almost all insects studied. WYATT (1967) reviewed papers describing the distribution and metabolism of this sugar in insects. Quantitative trehalose determinations have been obtained from many species of insects ; but studies of changes in levels during growth and development have been limited to a comparatively small number of species and include only five orders (Homoptera, Orthoptera, Lepidoptera, Hymenoptera, and Diptera). In only a single species, Antherea pernri G&r., has trehalose been determined quantitatively in eggs, larvae, pupae, and adults (EGOROVAand SMOLIN, 1962). Research on changes in trehalose levels in adult insects has been especially neglected. Because of the diverse patterns which trehalose follows during growth and development in those few species which have been studied, additional research on other species is needed in order to better understand the role of this sugar in the metabolism of insects. The objectives of the present investigation were: to determine the changes in trehalose levels occurring during growth and development of the boll weevil, Anthonomus gnzndis Boheman, to discover whether trehalose is an important carbohydrate reserve, to determine the effects of aging, carbohydrate content of the diet, and starvation on trehalose levels, and to study trehalose levels in adults of two species of Lepidoptera, Heliothti zea (Boddie), and Heliothti virescens (F.). MATERIALS AND METHODS Boll wee&l rearing

The weevils were reared as described by BETZ (1966). Eggs were surface sterilized with cupric sulphate and mercuric chloride (NETTLES and BETZ, 1966). Larvae were fed a modification of the larval diet of EARLE et al. (1959). The modified diet contained 5 g of square powder, 3 g of soy protein, 1 g of yeast extract, and 25 mg of cysteine-HCl; 20 mg of inositol were added for each 100 ml of water. Adults were fed either small bolls or squares (flower buds) obtained from cotton plants. All insects were subjected to 14 hr of light and 10 hr of darkness each day in order to obtain reproducing adults. All stages of the weevils were reared at 27°C. Heliothis rearing Larvae of H. zea and H. virescens were fed the diet of BERGER(1963). Adults were held in glass jars with high humidity and were fed either 10% sucrose or 10% honey. Oviposition was on cheesecloth strips which subsequently were surface sterilized with cupric sulphate and mercuric chloride (NETTLES and BETZ, 1966) in a Biichner funnel. All insects were held at 14 hr of light and 10 hr of darkness per day and all stages of moths were reared at 27°C.

CARBOHYDRATES IN ANTHONOMUS

GRANDIS,

HELIOTHIS

ZEA, HELIOTHIS

VIREXENS

659

Se$araiion and determin4ztion of wars, giycogen, and total lipids Sugars, glycogen, and total lipids were separated by the procedure of Nettles and Betz (unpublished). At about 10 a.m., samples of insects (usually 10) were homogenized at top speed in a Virtis 45 homogenizer for 1 min in about 50 ml of chloroform-methanol (2 : 1). The homogenate was allowed to stand overnight and then was centrifuged for 10 min at a relative centrifugal force of 10,OOOg. The supematant was carefully decanted into a graduated separatory funnel and was partitioned by the addition of O-2 vol. of water. After the funnels were shaken and were allowed to stand overnight, the lower layer containing the lipids was drawn off, and the upper layer containing the sugars was quantitatively transferred to another container by washing with 80% ethanol. Solvent was removed from the lipids under reduced pressure at 30°C with a Buchler Rotary Evapo-mix. Total lipids were estimated gravimetrically. The precipitate remaining after treatment with chloroform-methanol was extracted twice with about 40 ml of 80% ethanol. (A wash bottle with a fine tip was used to add the ethanol.) After each addition, the centrifuge tubes were allowed to stand overnight before they were centrifuged at 10,OOOg for 10 min. The supematant from the two ethanol treatments was combined with the upper layer of the chloroform-methanol-water system, and the combined extracts were dried under reduced pressure at 30°C in test tubes with a Buchler Rotary Evapomix. The sugar extract was redissolved in 2 ml of water and was centrifuged for 10 min at 10,000 g. A measured portion was deionized by passing it through beds of ion exchange resins with water. Amberlite MB-3 was used in the moth studies and Dowex 1-Dowex 50 were used to deionize the weevil extracts. After drying the deionized extracts at 30°C under reduced pressure, the residue was redissolved in water (usually 1.5 to 1.7 ml) with the volume of water corresponding exactly to the volume of the portion of extract taken for deionization. Usually 100 ~1 portions of the deionized extract were used for sugar determinations. Trehalose was determined with anthrone reagent ( CARROLet al., 1956) following acid and alkali digestion by the method used by WYATT and KALF (1957). Trehalose, used as a standard, was also subjected to acid-alkali treatment. Glucose was estimated with glucose oxidase (Glucostat Special obtained from Worthington Biochemical Corporation) by the method described in the data sheet furnished by the manufacturer. No trehalose activity was ever detected in the glucose oxidase trehalase reagent. Total sugars were estimated with anthrone with glucose as a standard. All sugar analyses were performed in triplicate. Glycogen was extracted from the precipitate remaining after the second 80% ethanol extraction by homogenizing the precipitate in 10 ml of a 5% solution of trichloroacetic acid (TCA) with a Virtis 45 homogenizer operated at high speed for 1 min. (For complete extraction of glycogen, samples should be homogenized at top speed for 5 min (ROE et al., 1961).) After centrifuging, a portion containing less than 100 pg of glycogen was precipitated with 5 vol. of ethanol and glycogen was determined by the method of CARROLet al. (1956), which was scaled down to increase sensitivity. The glycogen obtained after centrifuging was redissolved in

660

W. C. NETTLES, JR., B. PARRO,C. SHARBAUGH, AND C. L. MANGUM

of water and reacted with 4-O ml of anthrone reagent for 15 min in a boiling water bath.

04 ml

For each extract, all sugar and glycogen analyses were replicated at least three times, and the mean of the replicates was used to compute the standard deviation. The number of samples of weevils analysed is shown in the Tables. Extraction of sugars with 80% ethanol at room temperature

A few samples were homogenized in about 40 ml of 80% ethanol with a Virtis homogenizer operated at top speed. After standing overnight, the homogenate was centrifuged. The supematant was taken to dryness under reduced pressure and the dried residue was stored at - 20°C for several weeks before being dissolved in 2 ml of water and deionized on layered beds of Dowex 1 and Dowex 50 ion exchange resins. The deionized extract, which was dried on a Buchler Rotary Evapo-mix and subsequently redissolved in water, was taken for wet chemical analyses. Thin-layer chromatography

Thin-layer chromatography was performed as described previously (NETTLES and BETZ, 1965) and the plates were sprayed with a mixture of aniline, diphenylamine, and phosphoric acid (HARRIS and MACWILLIAM, 1954). When the blood of boll weevils was chromatographed, the droplet of haemolymph obtained by puncturing the insect with a fine insect pin was applied directly to the thin-layer plated. RESULTS Evaluation of extraction procedures

When dried ethanolic extracts of weevils were stored at -20°C for several weeks before being deionized, the titre of glucose was about one-half as high as that of trehalose. However, when the samples were extracted first with chloroformmethanol, and then with SOT/, ethanol and the extracts were deionized without prior storage, glucose levels were only one-tenth to one-twentieth as high as were those of trehalose. In an experiment comparing the two extraction procedures, all samples were processed simultaneously and the extracts were deionized as quickly as possible after evaporation of solvent. Levels of trehalose were slightly lower and glucose levels were slightly higher (12 f 4 pg vs. 17 + 3 pg/insect) in the weevils extracted only with 80% ethanol than in those extracted with chloroform-methanol. In another experiment in which female weevils were fed 20% sucrose solutions for 10 days, trehalose levels were 21% lower and glucose levels were 335% higher in weevils stored in ethanol than in those processed without storage. We conclude that extraction of sugars with chloroform-methanol and ethanol is superior to extraction with ethanol alone and that inaccurate sugar determinations are obtained when weevils are stored in ethanol at - 20°C prior to extraction of sugars and when undeionized ethanol extracts are stored dry at -20°C. Therefore, weevils should be homogenized first in chloroform-methanol and then in 80% ethanol, and the extract should be deionized as soon as possible.

CARBOHYDRATES IN ANTHONOMUS

GRANDIS,

HELIOTHIS ZEA, HELIOTHIS

VIRESCENS

661

The data in Table 1 indicate that drying of weevil extracts probably causes losses of glucose. However, no change in technique was made to compensate for the loss because modifications would have affected the accuracy of the trehalose determinations. This was justified since the quantity of trehalose in the boll weevil is considerably greater than that of glucose and also because trehalose was the main object of this study. Additional losses of glucose probably were caused by retention of glucose on to the ion exchange resin (Table 1). Glucose levels reported for boll weevils in this paper should be considered as representing minimal values. TABLE

SUGAR BXTRACTS (1 mlequivalentto GLUCOSE CONTENT

10 weevils)

I-EFFECTS OF DRYING UNDBIONIZBD ON

pgg1ucosekS.D.

Sample

200 pg glucose standard 200 pg glucose in 1 *Oml of weevil extract, not dried before deionization 200 pg glucose in 1-O ml of weevil extract, dried before deionization 1-O ml of weevil extract, not dried before deionization 1-Oml of weevil extract, dried before deionization

20051 156f6 147f 11 21 + 1 6fO

The apparently poor recovery of glucose in Table 1 is partially explained by the data in Table 2. Inhibitor(s) of glucose oxidase (or some other part of the coupled glucose oxidase-peroxidase system) were present in the boil weevil. Most, but not all, of the inhibitor(s) were removed by deionization. CROMPTON and BIRT (1967) a 1so observed inhibition of glucose oxidase by insect extracts. TABLE

2--INHIBITION

OF GLUCOSE OXIDASEASSAYBYSUGAR

EXTRACTS OF BOLL WEEVILS

Sample

100 pg glucose standard 100 pg glucose + deionized boll weevil extract 1 weevil equivalent 4 weevil equivalents 100 pg glucose + undeionized boll weevil extract (1 weevil equivalent)

pg glucose found

100 98 87 4

Trehaiose in eggs, larvae, pupae, pharate adults, and newly emerged adult boll weevils Diet with normal concentration of sucrose. On a dry weight basis, 1 to 3-day-old boll weevil eggs contained 2*52&Q%% trehalose and 0.25% glucose. These samples were extracted with 80% ethanol, but little degradation of trehalose occurred since only traces of glucose were present. The data in Table 3 were obtained from weevils fed larval diet containing either 1*70/Osucrose (normal diet) or 16*30/osucrose (sucrose-rich diet). Trehalose

662

W. C. NETTLES,JR., B. PARRO, C. SHARBAUGH,AND C. L. MANCUM

levels in weevils from the normal diet decreased rather gradually during the pupal and pharate adult stage. From the late last-instar larva to the l-day postemergence adult, trehalose content decreased only about 20% and total lipids by about 37%. Trehalose levels of starved adult weevils (held on moistened cellucotton) continued to drop with the greatest decrease occurring during the second day of starvation. In 5 to 6 days, trehalose in unfed adults dropped 94% while total Iipids decreased 64%. In contrast, there was little difference in glucose content between late last-instar larvae, pharate pupae, and adults less than 24 hr after emergence; but in starved adults older than 24 hr, glucose dropped to barely detectable values. The quantity of total anthrone positive substances can only be taken as an approximation of the amount of total carbohydrates present in the sugar extract since some non-carbohydrates react with anthrone and also because all carbohydrates do not react equally, and some sugars do not react appreciably with anthrone. When the quantities of trehalose and total anthrone positive substances in weevils fed the normal diet were compared (Table 3), trehalose almost always accounted for most (89 to 102%) of the sugars present in pharate pupae, pupae, pharate adults, and unfed adults less than 48 hr after emergence. (The lower percentages of trehalose in starved adult weevils 72 hr and more after emergence probably was caused by analytical error because such small amounts of sugars were present in the older insects.) However, in late last-instar larvae, trehalose accounted for only 76% of the total anthrone positive substances present, and the quantity of sugar(s) other than trehalose and glucose (about 50 pg/weevil) was markedly higher in late last-instar larvae than in any other group of weevils fed the normal diet. It is significant that of all the insects analysed in this table, the late last-instar larvae were the only actively feeding insects and that the titre of sugar(s) other than trehalose and glucose dropped very quickly with the cessation of feeding in the pharate pupae. Sucrose and a small amount of fructose probably account for most of the sugars other than trehalose and glucose ; for convenience, these sugars will hereafter be called ‘sucrose’. Sucrose-rich diet. To determine whether trehalose levels are regulated in larvae, pupae, pharate adults, and newly emerged adults, weevils were fed a sucrose-rich larval diet (16.3% sucrose, Table 3). Normally weevils were fed a larval diet containing 1.7% sucrose. Compared to the normal diet, the sucrose-rich diet produced elevated levels of trehalose, glucose, ‘sucrose’, glycogen, and total lipids; but the titre of trehalose was affected least. Also, though trehalose levels were 60 to 69% higher in the late last instar larvae and in pharate pupae, the quantity decreased markedly (150 pg/insect) in the period between the formation of the pharate pupae and the emergence of the adults ; so newly emerged adults from either diet had trehalose levels that were practically identical. Trehalose in adult boll weevils. The results of sugar analyses of reproducing adult weevils fed either bolls or squares are shown in Table 4. Analyses were performed on both sexes ; but there usually was so little difference in trehalose levels between sexes that the values were averaged. During the first 6 days,

3-cA~oHYDRATE

AND

DIETS CONTAINING

(sucrose rich diet)

FED LARVAL

1.7%

SUCROSB

49f7 62f3 27f6 25+2 lOf3 6fl

4 5 3 4 7 5

155 +9 146Itll

124f4 133zk15

6 7

5 6

15lk 13 151+ 10

6 6

300*25 291 zk29

174+13 151f17

14 14

8 6

1%3+13 172+7

13fl 14f4

34+4 29f2

48+8 66f3

126+5 139 f 10

164f13 170 f 14

188 f 13 165 zk19

6+1 5+2

173 + 14 165 f 13

Sucrose rich diet 701 f 54 25+8 31+6 554k44

2fl If1

2+0 3+1

2+0 3+0

8f2 723

lOf3 9f5

-

Normal diet 248+18 10e3 188f6 7+0

Glucose

Total anthrone positive substances

16.3%

9+3 7+3 2+1 2&l 3f2 3f2

-

12 14

233 243

1188 1203

O-56 +O*ll o-53 f 0.12

-

3 10

376 232

-

-

1.65 * 0.09 1.61 + 0.06

1.78 f 0.13 1.89 + 0.12

0.23 + 0.04 0.16 f 0.03

0.35 z!I0.04 0.28 f 0.03

0.41 * 0.07 0.35 f 0.02

0.35 * 0.08 0.39 f 0.10

0.87 f 0.16 0.87 + 0.20

Total lipids

AND

-

Glycogen t

(normal diet)

50 9

‘Sucrose’ *

pg carbohydrate (as glucose) or mg lipid/insect k S.D.

SUCROSE

IN WmvILs

Trehalose

LIPID LEVELS

6 8

No. of samples

TOTAL

* Calculated by difference between total anthrone positive substances and the sum of trehalose and glucose. t Glvconen levels in larvae. nunae. and vouna adults fed normal diet reuorted bv NBRZBS and BBTZ (1965).

Late last-instar larvae Pharate pupae Emerged adults (O-6 hr) Male Female

Late last-instar larvae Pharate pupae Pupae and pharate adults 0-24hr 24-48 hr Adults (emerged) 0-2hr Male Female 16-24hr Male Female 24-48 hr Male Female 72-96 hr Male Female 120-144 hr Male Female

Age-stage

TABLE

6 6

6 6

12 6

-6

6

1 2

4 6

10 15

17 20

40 6

127

12 6

6 12

6 6

Square-fed weevils

52+4

37+4 -

4Of6 49+66

53+3 47f9

71+5 64fl4

33 f 2

44+4** 41 f7

49*11 36+8

30+3 24+6

33f4 24+4

Square-fed weevils

Trehalose

0 (6)

-

2(12) 2 (6)

O(6)

3 (2)

Boll-fed weevils

O(6)

-

3 (6) 1 (6)

0 (12)

l(6)

Square-fed weevils

Glucose *

pg sugar (as glucose)/weevil -t_S.D.

BOLL WEEVILS FED EITHER BOLLS OR SQUARES FORK

Boll-fed weevils

OF ADULT

Numbers in parentheses show number of samples analyzed in triplicate. t Males only.

l

Boll-fed weevils

Days fed bolls or squares

No. of samples

TABLED-SUGARCONTENT

56 f 5

43*4 -

49*4 52k6

62+5 59+10

90516 79f16

Boll-fed weevils

41+2

47&S? 45f8

53 + 14 40+7

40_+5 30+4

47f4 33+3

Square-fed weevils

Total anthrone positive substances

TO~ODAYS

F

r

P

2

! ?

F

0

F 3 ,o

F

1 L y

2

.?

a

CA~O~RATBS

IN ANTHONOMUS

GRANDIS,

HELIOTXIS

ZEh, HELIOTHIS

VIRESCENS

665

content of square-fed weevils did not change appreciably. Trehalose levels in boll-fed weevils declined the first 6 days but still remained markedly higher than in square-fed adults. Between 10 and 20 days, however, there was little difference in trehalose levels between boll- and square-fed weevils. At 40 days, the trehalose content of boll-fed insects again appeared to be higher than that of square-fed individuals. If some of the values in Table 4 seem to be somewhat lower than those in Table 3, it is because the weevils studied in Table 4 already were 1 to 2 days after emergence when they were placed in contact with bolls and squares. Thus, they were actually 1 to 2 days older than the number of days indicated. Many of the glucose analyses were discarded because of a malfunctioning spectrophotometer, but the remaining determinations (Table 4) show that the quantity of glucose in these weevils was the lowest encountered in this, study. Glucose, present at trace levels, averaged from 0 to 3 pg/insect in the boll- and square-fed adult weevils. A comparison of the levels of trehalose and total anthrone positive substances shows that both the percentages and quantities of sugars other than trehalose and glucose tended to decrease with age in both boll- and square-fed adults. Chromatography of sugar extracts. Thin-layer chromatography confirmed the results of the chemical analyses: trehalose was the main sugar present in whole boll weevils and that it was present in all stages. Trehalose has been identified in extracts of boll weevil eggs, larvae, pupae, and adults by GLC with an SE-52 column (Nettles and Burks, unpublished data, method of SWEELEY et al., 1963). Trehalose also accounted for most of the sugars detected in the haemolymph by TLC. Trehalose in adults of H. virescens and H. zea. The results of sugar analyses of adults of 2 species of noctuids, H. oirescens and H. zea are shown in Table 5. Variation in trehalose levels among individual insects was much greater in the moths than in the weevils, but the difference probably is due to the comparatively small number of moths analysed. In wirescens, trehalose tended to be slightly higher in females than in males. The titre of trehalose was lowest in unfed virescens adults less than 24 hr old. In fed moths, trehalose levels increased the first 2 to 3 days and then remained essentially constant. Glucose determinations with glucose oxidase on virescens extracts obviously were high because the sum of glucose and trehalose was greater than that of the total anthrone positive substances. Also, the colour produced was atypical. With deionized extracts of H. zea pupae, and pharate adults, the unusual colour produced after incubation with glucose oxidase faded 40% in 22 hr and 41% after 30 hr at 25°C. Therefore, since the glucose standards faded only slightly during the same period, we increased the accuracy of the glucose determinations in H. zea somewhat by allowing the samples to stand for 24 hr between termination of the incubation with glucose oxidase and the spectrophotometric reading. H. zea samples were allowed to stand, but the oirescens samples were read soon after the termination of the reaction. The glucose analyses of virescens samples consequently read high trehdose

807 (803-809) 495 (-)

901 (-) 563 (437-688) 867 (-)

5 8 2

5

6

7

(97;%06)

725 (655-796)

650 (548-726)

6

4

637 (557-718)

4

3

669 (623-714)

6

(1) 558 (-) 710 (612-809)

H. virescens * 293 (-) 468 (-) 578 (538-618)

Males

Females

Trehalose

2

-

No. of females *

394 (-)

140 (-)

196 (128-292)

170 (127-213)

148 (-) 137 (114-160)

1256 (-)

994 (-) 585 (431-739)

707 (590-788)

177 (124-229) 122 (114-149)

691 (642-739)

123 (-) 131 (120-141)

Males

538 (-)

942 (852-1120)

F

$ P r

1067 (936-l 198)

“8

1 %

P rn

5 ,o

3

1 .P

.P z

9

741 (682-799)

664 (594-734)

584 (-) 730 (631-828)

-

Females

Total anthrone positive substances

325 (-) 491 (-) 580 (547-612)

Females

150 (123-176)

118 (108-127)

120 (-) 107 (-) 127 (113-141)

Males

Glucose

Mean pg sugar (as glucose)/moth + range

spp. ADULTS FED 10% SUCROSB SOLUTION

3

No. of males *

LEVELS OF Heliothis

1


Age (days)

TABLE S-SUGAR

751 (567-990) 750 (-) 943 (-) 538 (407-669)

436 (308-708) 623 (469-l 102) 355 (250-769) 497 (350-1135) 497 (167-565)

9

10


1

2

4

8

-

383 (-) 312 (157-701)

1191 (562-1996)

482 (358-813)

H. zeut:

619 (589-650)

1152 (1086-1218)

758 (601-915)

825 (624-962)

655 (462-847)

(124-876)

(2358)

(31%)

(535-555)

110 (78-123)

110 (-) 712 (-) 151 (103-198)

(lOl?i8)

168 (151-184)

* Usually 2 to 3 moths/sample. t Usually 1 moth/sample. $ Glucose allowed to stand for 24 hr before being read in the spectrophotometer.

12

11

837 (727-947)

8

(4OY45) -

484 (-)

154 (42-l 89)

(49:;38)

114 (103-125)

524 (136-911)

195 (136-254)

(1:5:84)

126 (126-126)

573 (190-883)

653 (295-1442)

514 (267-844)

799 (666-1259)

635 (401-861)

915 (-) 2589 (-) 745 (513-977)

956 (569-1097)

1019 (987-1050)

(201?090) -

$

8

x p

(-)

e i

x 2

i

%

8 8

5 2

g

P

$

8

2577

(69:286)

709 (490-1012)

663 (604-721)

2296 (1347-3244)

849 (656-1042)

(678-1122)

697 (484-909) 909

668

W. C. NETTLES, JR., B. PARRO, C. SHARBAUGH, AND C. L. MANGUM

and demonstrated that erroneous data may be obtained even from deionized moth extracts assayed with glucose oxidase. From the difference between the titre of trehalose and of total anthrone positive substances, trehalose was apparently the only sugar present in appreciable quantities the first 6 days in the virescens males. Sugar(s) other than trehalose probably appeared in older males because the percentage of trehalose (but not the quantity per moth) fell noticeably when they were 7 days and more after emergence. With only a single exception (11 days), the percentage of sugars other than trehalose varied but little with age in the females. In the moths 11 days after emergence trehalose was about 25 to 40% higher than expected, and sugars other than trehalose were about 17-fold higher and accounted for an average of about 1400 pg/moth with as much as 2160 pg/moth present. The moths 11 days after emergence apparently had recently fed on the sucrose solution. However, it may be significant that trehalose levels increased only slightly despite the large amount of sugar available for trehalose synthesis. There was a sharp increase in trehalose levels when H. sea adults fed on the 10% sucrose solution for 1 day and the change was greater in females than in males. By the second day, however, the titre of trehalose in both sexes had dropped to levels similar to those in unfed moths less than 24 hr after emergence and thereafter, it changed only slightly with age. The unusually large amounts of total anthrone positive substances (about 2200 pg) and glucose (about 500 pg) in a single female 2 days after emergence are interesting because of the rather low level of trehalose (383 pg as glucose equivalents); thus, trehalose levels did not rise even though there were sizeable quantities of precursors present in the body of the insect. On the basis of quantity/insect, trehalose levels began lower in unfed virescens but increased with age until the titre of trehalose was higher in virescens than in zea. This result was somewhat surprising because zea is larger and heavier than virescens. However, some of the zeu, showed symptoms (deformed wings) of a dietary fatty acid deficiency which may have influenced the results.

DISCUSSION

Trehalose levels during growth and development In the boll weevil, trehalose is the main sugar present in the haemolyrnph and in extracts of whole insects and is found in eggs, larvae, pupae, pharate adults, and adults. The quantity, which is maximal in late last&star larvae, gradually decreases during the pupal and pharate adult stage and declines more rapidly in young adults. Decreases in trehalose levels in the interval between the formation of mature larvae and newly emerged adults also have been observed in whole Antheruea per@ (EGOROVA and SMOLIN, 1962), Pieri> brussicue (L.) (MOREAU, 1969), and in Chrysti shanghuiensis Smith (TAKEDAand HUKUSIMA,1963). However, the levels did not change markedly during the larval-pupal ecdysis in whole boll weevils and in whole P. brassicae (MOREAU, 1969) though a pronounced decrease

CARBOHYDRATES IN ANTHONOMUS

GRANDLY, HELIOTHIS ZEA, HELIOTHIS

VIRES33NS

669

in blood or in whole insects does occur at this time in A. pernyi (EGOROVAand SMOLIN, 1962), Bombyx mori (DUCH&XAU-BOSONet al., 1963), Galleria mellonella (LENARTOWICZet al., 1967), Pseduletia ( = Leucania) sepanztu Walker (LIU and FENG, 1965), Samia Cynthia ricini (Donovan) (CHANGet al., 1964), and Hyalophwa cecropk (WYATT and KALF, 1957). Levels in the blood of Schistocerca gregaria (HOWDEN and KILBY, 1960) and Locusta migratoria migratorioides (HILL and GOLDSWORTHY,1968) also drop during ecdysis. The data on trehalose in whole adult boll weevils are significant because this is the most complete study of trehalose in adult insects and also because of the effects of age and carbohydrate content of the diet. It is not surprising that young and old adults fed bolls have trehalose levels decidedly higher than those found in square-fed weevils of the same age since the sugar content of bolls is five to eight times higher than that of squares (NETTLES and BETZ, 1965). Glycogen and lipid levels also are higher in boll-fed than in square-fed weevils (BRAZZELet al., 1957 ; LAMBREMONTet aZ., 1964; NETTLES and BETZ, 1965). But the similar titres of trehalose in weevils fed either bolls or squares for 10 to 20 days were unexpected and show that, during part of their adult life, weevils are able to regulate or maintain trehalose at a fairly constant level despite sizeable differences in the sugar content of the adult diet. Older weevils may lose the ability to regulate trehalose because levels of this sugar in insects 40 days after emergence fed bolls were higher than in those of the same age fed squares, and age also affected glycogen since the glycogen content of 40-day post-emergence weevils fed either bolls or squares was markedly lower than that of 10 to 20 days after emergence fed the same diet (NETTLES and BETZ, 1965). On a dry weight basis, the percentage composition of trehalose, glycogen, and lipids in the boll weevil in several developmental stages is shown in Table 6. Degradation of carbohydrates during drying prevented carbohydrate analyses from being performed on dried samples and the values are approximations since carbohydrate, lipid, and dry weight determinations were performed on different samples. The calculations are based on dry weights (Nettles, unpublished data) obtained over a period of several years. Trehalose levels in boll weevil larvae and pupae are comparable to the levels found in several other species, but trehalose content of eggs is higher and that of adults is lower than that of other species (CROMPTONand BIRT, 1967; LENARTOWICZ et al., 1967; WYATT, 1967; QUICKENDEN and ROFZMHILD, 1969; QUICKENDEN,1970). Trehalose as an energy reserve Since glycogen and lipids are known to be important energy reserves of insects, we compared the percentage composition (dry weight basis) of trehalose, glycogen, and total lipids in weevils of different ages to determine the importance of trehalose as a reserve of the boll weevil (Table 6). Total lipid levels were always higher than those of glycogen. Also, except with young adults and pupae and pharate adults older than 24 hr, the titre of glycogen was higher than that of trehalose. Thus, in comparison with the amounts of glycogen and lipids, the

670

W. C. NETTLES, JR., B. PARRO,C. SHARBAUGH, ANDC. L. MANGUM

quantity of trehalose is sometimes rather small. But trehalose is a sizeable reserve of the boll weevil: it accounts for from 2.8 to 4.0% of the dry weight of eggs, late last-instar larvae, pupae, and pharate adults, and newly emerged adults. Also, it probably is significant that the largest amounts are present in those forms that are unable to feed for several days (eggs, late last-instar larvae, pupae, pharate adults, TABLE ~-APPROXIMATE PERCHNTAGES OF TREHALOSE, GLYCOGEN,ANDTOTALLIPIDSPRESWT IN ‘II-IIIBOLL WEBVIL Approximate percentage of dry weight Stage-age

Eggs Late last instar larvae Pupae and pharate adults Emerged adults Reproducing o-2 hr 10-20 days (square-fed) Diapausing About 15 days (boll-fed) * t $ 8

Trehalose

Glycogen *

Total lipids

2.5 4.0 3.8

11.2 5.9 -

26.1 t 17.4 -

3.9 0.5

0.2 0.4

13.8 19.9:

0.5 5

2.3

37.7:

Calculated from NETTLESand BETZ (1965). Calculated from BUMGARNER and LAMBREMONT (1966). Calculated from LAMBIIIIMONT et al. (1964). Unpublished data of Nettles et al.

and to a limited extent in newly emerged adults). Surprisingly low levels of trehalose (0.5%) and total soluble carbohydrates (glycogen plus trehalose: 0.9% in square-fed and 2.7 per cent in boll-fed weevils) are present in mature adults. But the mobility gained from flight makes food readily accessible to the adult so high levels of carbohydrates in the adult may not be as important as in other stages.

Regulation of trehalose Trehalose levels of insects are regulated to a certain extent and the supporting data are drawn from experiments involving starvation, injection of sugars or saline solution, injury, circadian rhythms, and analyses of insects during growth and development (WYATT, 1967). Since most reports were limited to blood trehalose, it is interesting that ROCKSTEIN and SRIVASTAVA(1967) found trehalose levels in flight muscle of houseflies, Musca domestica, tended to level off after decreasing for 24 hr after emergence. From our analyses of whole insects, several reasons exist for concluding that trehalose levels are regulated in the boll weevil. Although glycogen content decreased 99% in the interval between the late last-instar larvae and newly emerged adults, trehalose levels dropped only 31%. (Glycogen undoubtedly is a precursor of trehalose, and the decrease in trehalose probably would have been

CARBOHYDRATES IN ANTHONOMUS

GRANDIS, HELlOTHIS 2.24, HELIOTHIS

VIIWSCENS

671

smaller if appreciable quantities of glycogen were present in newly emerged adults.) However, when sucrose in the larval diet was increased lo-fold, the trehalose content of late last-instar larvae and pharate pupae increased only 60 to 69%. Usually trehalose accounted for almost all sugars present in the boll weevil, but 57% of the free sugars found in late last-instar larvae fed the carbohydrate-rich diet were sugars other than trehalose. Therefore, when large amounts of sugars other than trehalose were present, trehalose levels in the boll weevil were not greatly affected. More signiiicantly, even though the sucrose-rich diet produced elevated titres of trehalose in the late last-instar larvae and pharate pupae, trehalose content later dropped to normal levels in newly emerged adults. Also, though sucrose-rich larval diet produced higher levels of carbohydrates and lipids in the late last-instar larvae and pharate pupae of the boll weevil, it had less effect on trehalose than it did on the titre of glucose, sugars other than trehalose and glucose, glycogen, and total lipids. In addition, as noted, despite differences in the sugar content of bolls and squares, there was little if any difference in trehalose levels in adults fed either bolls or squares for 10 to 20 days. Additional evidence that regulation of trehalose occurs in boll weevils was obtained in a study of diapausing and reproducing insects (Nettles et aZ., unpublished data). Although glycogen content was obviously affected by sex, physiological state (whether the insect was reproducing or in diapause), and short periods of fasting, these three factors had slight to no effect on trehalose levels in adult weevils 13 to 17 days after emergence. Therefore, we conclude that trehalose levels are regulated in the boll weevil and it is of interest that this is the first time that regulation of trehalose has been demonstrated in whole insects. Despite a large amount of variation, which probably was a result of small sample size, the titre of trehalose also appeared to be regulated in adults of H. zea and H. virescens. In both species of moths, trehalose content rose following the ingestion of sucrose; but the increase was much more pronounced in zea than in virescens. By the second day, trehalose reached levels which remained fairly constant for the remainder of adult life in both species. In zeu, especially, an equilibration period seemed necessary before the insects adjusted to the diet of 10% sucrose; this is similar to the young boll-fed adult weevils: they required several days to adjust to the high sugar content of bolls. As additional evidence of regulation, it probably is significant that trehalose levels were not greatly elevated in those moths in which large quantities of sugars other than trehalose were present. But in the only other study of adult Lepidoptera, trehalose levels in P. Zmsskze were not as well-regulated because MOREAU (1969) found that the titre of the sugar dropped &I% as the adult aged.

Sugars other than trehalose and glucose Usually trehalose is the principal free sugar present in the boll weevil. However, we sometimes detected sizeable quantities of substances that reacted with anthrone and decomposed during acid-alkali treatment. These unidentified

672

W. C. NETTLES,JR., B. PARRO,C. SHARBAUGH, AND C. L. MANGIJM

compounds probably consisted mainly of sucrose and a small amount of fructose and, in fact, we have verified the presence of both in whole insects by recent gas chromatographic analyses (Nettles and Burks, unpublished data). When whole late last-instar larvae from the sucrose rich diet were washed three times before being homogenized, sucrose and fructose were found in the extracts of the whole insect. Sucrose, unlike fructose, was not detected in the blood. Thus, sucrose was present inside the insect, probably in the alimentary tract or fat body. Under certain conditions, however, sucrose and other disaccharides are present in the haemolymph of adult insects (Nettles, unpublished data). VAN HANDEL (1968) found that non-intestinal oligosaccharidases with activity towards sucrose and maltose were present in several species of insects. He was unable to explain the significance of these enzymes, but the oligosaccharidases may be responsible for the hydrolysis of sucrose that appears in blood when starving insects feed on sucrose-rich diets such as nectar. The presence of sucrose in the boll weevil is not unusual since this sugar was found in other species of insects by WYATT et al. (1956), WYSS-HUBER et al. (1961), EHRHARDT(1962), EGOROVAand SMOLIN (1962) MAURIZIO (1965, 1967), CROMPTONand BIRT (1967), and ALUMOT et al. (1969). The quantity of ‘sucrose’ (anthrone positive sugars other than trehalose and glucose) in the boll weevil was dependent on the sucrose concentration of the diet and the nutritional state of the insect. In late last-instar larvae feeding on the normal diet, ‘sucrose’ accounted for 50 pg or 20% of the free sugars. In larvae of the same age fed diet containing ten times the usual concentration of sucrose, about 376 pg or 54% of the free sugars were present as ‘sucrose’. This is by far the highest percentage of ‘sucrose’ and the lowest percentage of trehalose found in boll weevils in this study. Continuous feeding is necessary to maintain the high ‘sucrose’ levels because the titre of ‘sucrose’ dropped quickly in pharate pupae from both diets when feeding ceased; however, the decrease was more gradual in weevils obtained from the sucrose rich diet. The rapid disappearance of ‘sucrose’ when feediig stopped is very similar to the data reported by MAURIZIO (1967) who found that the large quantities of glucose and fructose, present in the blood of feeding larvae of the honey bee Apis meZZz@raL., later disappeared when mature larvae stopped feeding. Sugars injected into adult mosquitoes also are metabolized rather quickly (VAN HANDEL, 1968, 1969a, b). Degradation of trehalose during extraction and storage In two earlier studies of trehalose in insects (GEIGY et al., 1959; WYSS-HUBER et al., 1961) trehalose was degraded when insects were extracted with or stored in ethanol at room temperature. However, these data seem to have been overlooked by many workers including ourselves. Only when we switched from extraction with 8OO/oethanol to extraction with chloroform-methanol and ethanol and began deionizing extracts as soon as possible did we realize that trehalose was converted to glucose when whole insects were stored at - 20°C and also when dried ethanolic prior to deionization. Degradation of trehalose extracts were stored at -20°C during extraction and storage was minimized, and perhaps prevented, by extracting

CARBOHYDRATRS IN ANTHONOMUS

GRANDIS, HELIOTIilS ZEA, HELIOTHIS

VIRESCENS

673

the weevils with chloroform-methanol and ethanol and by deionizing the extracts as quickly as possible. We were unable to determine whether extraction with chloroform-methanol, or immediate deionization, or a combination of the two was responsible for minimizing the conversion of trehalose to glucose. But chloroform-methanol may have at least some effect since the titre of glucose is somewhat lower in weevils extracted with chloroform-methanol than in those extracted with 80% ethanol. The failure of ethanol to inactivate boll weevil trehalase is to be expected since ethanol has been used in the purification of trehalase from insects (PETRYSZYNand SZARKOWSKA,1959; FRIEDMAN, 1960). Although small amounts of trehalose and large amounts of glucose and fructose are lost during deionization (Table 1 and unpublished data of McDonald, Nettles, and Burks), the losses of trehalose were small compared with those that occurred during storage of undeionized ethanolic extracts. GEIGY et al. (1959), WYSS-HUBERet al. (1961), EGOROVAand SMOLIN (1962), and VAN HANDEL(1969a) used hot ethanol to minimize degradation of trehalose, but even with this technique, GEIGY et al. (1959) had to cut adult Sarcophqa bulkzta into pieces to increase the rate of penetration of alcohol into the tissues. Also, LENARTOWICZ et al. (1967) concluded that extraction with 10% TCA at 0°C was a preferable procedure and that even then, a minute quantity of trehaIose was converted to glucose. Extraction with TCA is undesirable for certain purposes, however, since 5% TCA hydrolyses sucrose (Nettles and Betz, unpublished data). Acknowledgements-We thank EVBRARDOVOGEL, Louisiana State University, for translating Maurizio’s papers; MITSUO YOSHIMEKI,Louisiana State University, for translating tables of several of the Japanese papers, and G. T. CHEN, Louisiana State University, for translating tables of several of the Chinese papers.

REFERENCES ALUMOT E., LENSKY Y., and HOLSTEIN P. (1969) Sugars and trehalase in the reproductive

organs and hemolymph of the queen and drone honey bees (Apis mellifca L. var. Ligwtica Spin.). Comp. Biochem. Physiol. 28,1419-1425. BERGER R. S. (1963) Laboratory techniques for rearing Heliothis species on artificial medium. U.S. Dept. Agric. ARS-33-84 4pp. BETZN. L. (1966) Improved laboratory methods for rearing the boll weevil. y. econ. ht. 59,374-376. BRAZZELJ. R., NJWSOM L. D., ROUSSELJ. S., GAINES R. C., and C&CIO T. (1957) The effect of food on fat accumulation of resistant and susceptible boll weevils. g. econ. Ent. 50, 459-462. BUMGARNERJ. E. and LAMBRJIMONTE. N. (1966) The lipid-class spectrum and fatty acid content of the boll weevil egg. Con@. Biochem. Physiol. 18,975-981. CARROLN. V., LONGLEY R. W., and ROE J. H. (1956) The determination of glycogen in liver and muscle by use of the anthrone reagent. J. biol. Chem. 220,583-593. CHANG C., LIV F., and FP.NG H. (1964) Studies on metabolism of Erisilkworm during metamorphosis-II. The properties of trehalase and its effect on metabolism. Acta ent. sin. 13,494-502. CROMPTONM. and Bmr L. M. (1967) Changee in the amounts of carbohydrates, phosphagen, and related compounds during the metamorphosis of the blowfly, Lucilia ngrina. J.

Insect Physiol. 13,1575-1592. 28

674

W. C. NETTLJZS,JR., B. PARRO, C. SHARBAUGH, ANDC. L. MANGUM

DUCH~TEAU-BOSSON G., JEUNIAUXC., and FLORKINM. (1963) Contributions a la biochimie du ver g soie-XXVII. TrChalose, tr&alase et mue. Arch. int. Physiol. Biochim. 71, 566-576. EARLE N. W., GAINES R. C., and ROUSSELJ. S. (1959) A larval diet for the boll weevil containing an acetone powder of cotton squares. J. econ. Ent. 52, 710-712. EGOROVA T. A. and SMOLINA. N. (1962) Trehalose in the organism of the oak silkworm in different stages of its development. Biokhimiya 27, 407-410 (English Translation). EHRHARDTP. (1962) Untersuchungen zur Stofiechselphysiologie von Megoura oiciae Buckt., einer phloemsaugenden Aphide. 2. vergl. Physiol. 46, 169-211. FRIEDMANS. (1960) The purification and properties of trehalase isolated from Phormia regina Meig. Archs Biochem. Biophys. 87, 252-258. GEIGY R., HUBERM., WEINMAND., and WYATT G. R. (1959) Demonstration of trehalose in the vector of trypanosomiasis: The tsetse fly. Acta trap. 16,255-262. HARRISG. and MACWILLIAMI. C. (1954) A dipping technique for revealing sugars on paper chromatograms. Chemy Ind. Land. 73, 249. HILL L. and GOLDSWORTHY G. J. (1969) Growth, feeding activity, and the utilization of reserves in larvae of Locustu.J. Insect Physiol. 14,1085-1098. HOWDEN G. F. and KILBY B. A. (1960) Biochemical studies on insect haemolymph-I. Variations in reducing power with age and the effect of diet. J. Insect Physiol. 4,258-269. LAMBREMONT E. N., BLUM M. S., and SCHRADER R. M. (1964) Storage and fatty acid composition of triglycerides during adult diapause of the boll weevil. Ann. ent. Sot. Am. 57, 526-532. LENARTOWICZ E., ZALUSKAH., and NIEMIERKOS. (1967) Carbohydrates in the wax moth during development. Acta biochim. pol. 14,267-275. Lru F. and FENG H. (1965) The metabolism of blood sugars of the armyworm. Acta ent. sin. 14,432-440. MAURIZIOA. (1965) Untersuchungen fiber das Zuckerbild der HBmolymphe der Honigbeine (Apis mellificu L.)-I. Das Zucherbild des Blutes erwachsener Bienen. J. Insect PhysioZ. 11,745-763. MAURIZIOA. (1967) Untersuchungen fiber das Zuckerbild der HBmolymphe der Honigbeine (Apis mellificu L.)-II. Das Zuckerbild des Blutes der Bienenbrut. 2. Bienenforsch. 9,74-87. MOREAUR. (1969) Observations sur les variations du taux de trhhalose au tours du dt%eloppement direct et de la diapause, chez Pieris brassicae L. C. R. Acad. Sci., Paris 268,14411444. NETTLESW. C., JR. and BETZ N. L. (1965) Glycogen in the boll weevil with respect to diapause, age, and diet. Ann. ent. Sot. Am. 58,721-726. NETTLES W. C., JR. and BETZ N. L. (1966) Surface sterilization of eggs of the boll weevil with cupric sulfate. r. econ. Ent. 59,239. PETRYSZYNC. and SZARKOWSKA L. (1959) Trehalase activity during metamorphosis of the hawk moth, Celerio euphorbiae L. Bull. Acad. pal. Sci. Cl. II. Ser. Sci. biol. 7,491+93. QUICKENDEN K. L. (1970) Carbohydrates in eggs of the grasshopper Aulocura elliotti, during development. J. Insect Physiol. 16, 171-183. QUICKENDEN K. L. and ROEMHILDG. R. (1969) Maternal age and density effects on carbohydrate partitioned to eggs of the grasshopper, Aulocara elliotti. J. Insect Physiol. 15, 1215-1223. ROE J. H., BAILEY J. M., GRAY R. R., and ROBINSONJ. N. (1961) Complete removal of glycogen from tissues by extraction with cold trichloroacetic acid solution. J. biol. Chem. 236,12&l 246. ROCKSTEINM. and SRIVASTAVA P. N. (1967) Trehalose in the flight muscle of the house fly, Musca domestica L., in relation to age. Experientia 23,636-637. SAITO S. (1963) Trehalose in the body fluid of the silkworm, B&X mori. L. J. Insect Physiol. 9, 509-519.

CARBOHYDRATES IN ANTHONOMUS

GRANDLY, HELIOTHIS ZEA, HELlOTHIS

VIRESCENS

675

SWEELEY C. C., BENTLEY R., MAKITAM., and WELLS W. W. (1963) Gas-liquid chromatography of trimethylsilyl derivates of sugars and related substances. J. Am. Chem. Sot. 85,2497-2507. TAKEDA S. and HUKUSIMAS. (1963) Behaviors of the trehalose and glycerol in Mone-ma f&avescens Walker (Lepidoptera: Cochlidiidae) and its parasite, Chrysis shunghaiensis Smith, during various stages of development. Res. Bull. Fat. Agric. G&-ken prefect. Univ. l&88-94. VAN HANDEL (1968) Utilization injected maltose sucrose by evidence for oligosaccharidases. Camp. Physiol. 24, VAN HANDEL (1969a) Metabolism hexoses in intact mosquito: of glucose trehalose as Comp. Biochem. 29, 413-421. HANDELE. The equilibrium sorbitol 2 in the mosquito. Camp. Physiol. 29, WYATT G. (1967) The of sugars polysaccharides in Adv. Insect 4, 287-360. G. R. G. F. (1957) The of insect Trehalose and carbohydrates. J. Physiol. 40,833-847. G. R., T. C., WYATT S. (1956) The of insect lymph. Organic of the of the Bombyx mori, two other J. gen. 39,853-868. WY.+HUBER STREIBELH., E., and R. (1961) Papierchromatographischer Nachweis verschiedener Zucker und spektrophotometrische Bestimmung des Glykogens in Extrakten von Glossinen, Reduviiden und Ornithodorus moubata. Acta trctp. 18, 46-57.