Pigmentation of Hyalophora cecropia larvae fed artificial diets containing carotenoid additives

J. Insect Pfrysiol., 1971, Vol. 17, pp. 1593 to 1598.

Pergnnton Press. Printed in Great Britain

PIGMENTATION OF HYALOPHORA CECROPIA LARVAE FED ARTIFICIAL DIETS CONTAINING CAROTENOID ADDITIVES RALPH

M. CLARK

Department of Biological Sciences, Faculty of Science and Mathematics, State University of New York College of Arts and Science, Plattsburgh, New York 12901 (Receitwd 5 October 1970) Abstract-Larvae of Hyalophora cecropia reared on artificial diets lacking carotenoids show abnormal coloration of bodies and dorsal tubercles. The addition of xanthophyll (lutein) to the diets of such larvae, if made prior to the fifth instar, allows the development of the colours normal to these larvae. If the xanthophyll-supplemented diet is not given until larvae are in the fifth instar, normal colours do not appear. INTRODUCTION

CECROPIAlarvae reared on Riddiford’s modification of the Adkinson-vanderzantLevengood diet for insects (RIDDIFORD,1968) d o not develop normal pigmentation. The general body colour is blue instead of the blue-green or green of leaf-reared larvae. Dorsal tubercules of fourth and fifth instar larvae are unpigmented, and the haemolymph and fat body are abnormally coloured (Riddiford, personal communication). The physiology of the insects does not appear to be upset in other respects. Growth rates of Cecropia reared on artificial diets are normal, and larvae reach normal size before pupation. Pupation proceeds readily, yielding normally coloured pupae. The emergence of adults is unhampered, and eggs from females reared on artificial diets are viable (RIDDIFORD,1968). The colour aberrations observed are common among insects reared on diets lacking carotenoids (DAHLMAN,1969), however, some insects, ScIz&ocerca gregaria for example, require &carotene for normal growth and reproduction as well as for normal pigmentation (HOWDENand HUNTER-JONES,1958; GILMORE, 1961). The absence of vitamin A or its precursors from diets alters the spectral response of houseflies (COHEN and BARKER, 1963) and causes histological aberrations of compound eyes of Manduca sexta, with concurrent loss of ability to orient to light (CARLSONet al., 1967). These observations raise the possibility that physiological malfunctions do exist but have not been observed in Cecropia and other insects raised on artificial diets lacking carotenoids. Several carotenoids are involved in insect coloration. One of the most abundant is p-carotene which may occur free, complexed with protein, or mixed with mesobiliverdin to form the green pigment called insectoverdin. P-Carotene forms the 1593

1594

RALPH

M.

CLARK

yellow component of insectoverdin and mesobiliverdin forms the blue component. VAN DER GEE~T(1968) stated that the blue colour of haemolymph of Pieris brassicae larvae reared on artificial diet was due to the absence of carotenoids from the diet. Carotenes are known to be responsible for many insect colours, including oranges and reds (GILMORE, 1965). The xanthophylls are another group of carotenoids commonly found in insects (CHROMARTIE,1959). These are found in the free state and as fatty esters (GILMORE,1961). DAHLMAN(1969) f ound that zanthophylls were the major carotenoid pigments of Manduca sexta. Since both xanthophyll and /?-carotene are abundant in leaves it is logical to test the effect of addition of these components to the artificial diets on which larvae are reared. This paper presents the results of an investigation into the effect on pigmentation of Cecropia larvae reared on diets containing these carotenoids. MATERIALS AND METHODS Six broods of larvae which hatched between April and August were utilized in this study. The first five broods were reared under continual fluorescent lighting at 26°C. The sixth brood was reared in a laboratory where temperatures varied between 26 and 3O”C, and where the light period ranged from 10 to 19 hr. Growth was normal under either set of conditions. The larvae were reared in covered plastic shoe boxes, the bottoms of which were covered with paper towels to facilitate frass removal. Boxes were cleaned daily at the time of feeding. Freshly cut cherry leaves (Prunus sp.) were sprayed with a solution containing the antibiotics kanamycin sulphate (Bristol Laboratories) and chlortetracycline hydrochloride (Sigma) at the concentrations used by RIDDIFORD(1967), immediately before feeding to the larvae. The population of larvae in the boxes was subdivided as necessary to alleviate crowded conditions. There were ultimately 5 fifth instar larvae per box. Larvae which commenced spinning prior to the time of feeding were removed to open boxes, as the high humidity in closed boxes interfered with the proper drying of cocoons. Larvae on diets were reared in 130 ml glass jars, covered with perforated aluminium foil, through the third instar. They were then transferred to plastic shoe boxes having raised galvanized wire screens which allowed frass to drop through but prevented larvae from eating the paper towels. Twigs were placed on the wire screens for larvae to crawl on, and for the attachment of their pads for ecdysis. Fresh slices of diet were placed in these containers as needed and aged, discoloured slices were removed. There were 5 to 7 larvae in each box by the onset of spinning. Diets were prepared according to the formulation of RIDDIFORD(1968) and modified by the addition of carotenoids as tabulated below (Table 1). These components were added when the antibiotic-vitamin solution (group 2) of Riddiford’s diet was mixed into the diet being prepared. In addition to the diets listed, a diet containing 1764 mg/l. lutein and 0.60 mg/l. /?-carotene was prepared.

PIGMENTATION

OF HYALOPHORA

CECROPIA

LARVAB FED ARTIFICIAL DIETS

1595

Diets were refrigerated at 5°C until immediately before use. The vitamin A and /?-carotene were obtained in crystalline form from the Nutritional Biochemical Co. The xanthophyll (lutein) came in a solution at a concentration of 4 g/lb (8.82 mg/g). TABLE

~--SCHEDULE

OF CAROTENE ADDITIVES TO THE BASIC DIET

Diet

Carotene added (mg/l) None 1.20 060 757-o 17.64

Basic (Riddiford’s) Vitamin A &Carotene 1 j%Carotene 2 Lutein

A few fourth and fifth instar larvae raised on diets which did not promote normal pigmentation were transferred to leaves, or to other diets, to determine if they were still able to attain normal colouration. RESULTS

Table 2 shows the number of larvae started on the different diets, and reared through the fifth instar to spinning. Some larvae were switched from one diet to another, either while in an ecdysis or immediately following an ecdysis. These larvae are listed in Table 3. TABLE

2--TABULATION

OF THB NUMBER OF LARVAE REARRD THROUGH THE FIFTH INSTAR ON THE DIFFERENT DIETS

Brood

Diet

First instar

Fifth instar

2, 3, 4 1, 2, S, 6 1 5 6 6 6

Leaves Basic Vitamin A /?-Carotene 1 @Carotene 2 Lutein Lutein + p-carotene

478 155 23 20 20 20 20

330 109 18 12 16 15 14

TABLE 3--TABULATION

OF FOURTH AND FIFTH INSTAR LARVAE THAT WERE CHANGED FROM ONE DIET TO A SECOND DIET

Brood

From

To

Instar

No.

1 2 5 6 6

Vitamin A Basic Basic Basic Basic

Leaves Leaves Leaves Lutein Lutein

Fifth Fifth Fourth Fourth Fifth

2 4 4 3 5

1596

RALPH

M. CLARK

All larvae reared on leaves were normally pigmented. The body colour from the third instar until the larval-pupal ecdysis was blue-green or green. Dorsal abdominal tubercles (DAT) were bright yellow on fourth and fifth instar larvae. Dorsal thoracic tubercles (DTT) of segments two and three were red on the fourth instar and orange on the fifth instar larvae. Larvae reared on either basic diet, diet supplemented with vitamin A, or on diet containing 0.60 mg/l of p-carotene showed the same colour anomalies. Third instar larvae had normal, though weak, pigmentation, including pale red DTT. The body colour of fourth and fifth instar larvae was blue, and all dorsal tubercles were milk white. Lateral tubercles, which are blue with white tips on leaf-fed larvae, were normally coloured. The haemolymph from one of these larvae was blue. Because the j-carotene concentration of the first diet used was much lower than one would expect in leaves, a second was prepared which contained 757.0 mg/l of p-carotene. Larvae reared on this diet developed pale yellow DAT in the fourth instar and pink DTT. Fifth instar larvae showed the same colours, but the colours were much fainter than those seen in the earlier instar. The bodies of larvae fed this diet were blue, as was their haemolymph. No adverse effects due to the large amount of p-carotene in this diet were noted. Larvae reared on lutein-supplemented diet were normally pigmented throughout the first three instars. Fourth instar larvae had blue-green bodies and bright yellow DAT. Their DTT were red to red-orange. Larvae that were newly ecdysed into the fifth instar were normally pigmented, except that the DTT were a bright pink rather than the reddish-orange or orange of leaf-reared larvae. When these larvae matured, the coloration of the DTT was almost the normal orange. Larvae reared on diet supplemented with lutein plus 0.60 mg/l. p-carotene were identical in coloration to larvae reared on the lutein containing diet in all instars. The haemolymph of larvae fed diets containing lutein was a yellow-green. Fourth instar larvae which were switched from the basic diet to leaves developed normal coloration within 48 hr. These larvae were also normally pigmented during the fifth instar. Larvae changed from the basic diet to the lutein-supplemented diet developed the pigments of lutein-reared larvae if the change in diets was made before or during the fourth instar. Normal pigmentation of larvae was not attained if their diets were changed when they were ecdysing to, or were in, the fifth instar. When larvae were changed from the basic diet or from vitamin A-supplemented diet to leaves they developed pale yellow DAT and DTT. No orange appeared in the DTT. Larvae which had just ecdysed into the fifth instar developed little or no pigmentation of their dorsal tubercles if their diet was changed from the basic diet to the lutein-supplemented diet at the beginning of the fifth instar. DISCUSSION

These results indicate that the colour aberrations of Cecropia larvae fed artificial diets are mainly, if not entirely, due to the absence of xanthophylls from

PIGMENTATION

OF HYALOPHORA

CECROPIA

LARVAE FED ARTIFICIAL

DIETS

1597

their diets. That these colour anomalies are not due to genetic factors is shown by the fact that all larvae reared on leaves have normal colouration, and by the finding that fourth instar larvae transferred from the basic diet to leaves, or to luteinsupplemented diet, are able to develop normal coloration. This dependence on a xanthophyll is not surprising, since xanthophylls contribute to the colouration of many insects, including the swallowtails P. xuthus and P. protenor demeiruis (OHNISHI, 1959), and the tobacco hornworm (DAHLMAN,1969). Xanthophylls and other carotenoids are also found in the yellow-green or green haemolymph of insects (GOODWINand SRISUKH,1951; HACKMAN,1952; VAN DER GEEST, 1968). Carotenoids form the yellow component of these haemolymph pigments, and in their absence the haemolymph is blue because of the presence of the blue component of hemolymph pigment, mesobiliverdin. The Cecropia larvae with blue haemolymph observed in this study were reared on diets lacking lutein. Larvae reared on lutein-containing diets possessed yellow-green haemolymph, thus showing that the lutein contributed to haemolymph colour in Cecropia. p-carotene is found in P. brasicae haemolymph and in the haemolymph of other insects (HACKMAN,1952). It is the only carotenoid in locust haemolymph (GOODWINand SRISUKH,1951). H owever, in the studies reported here p-carotene did not seem to contribute to haemolymph coloration, even when present in a high enough concentration to contribute to tubercle pigmentation. The demonstration that p-carotene could pigment dorsal tubercles of Cecropia larvae suggests that the observations of others that p-carotene was not utilized by certain insects on artificial diets may be because of a low concentration of this carotenoid in the diets used. The effect of the higher concentration of B-carotene in lutein-supplemented diets was not examined, and the possibility exists that pcarotene or other carotenoids may be necessary for completely normal tubercle pigmentation. The ability of larvae to utilize p-carotene also suggests that its importance to normal functioning of Cecropia should be examined more carefully. HOWDENand HUNTER-JONES(1958) and COHEN and BARKER(1963) showed that its derivative, vitamin A, was important to locusts and to houseflies. CARLSONet al. (1967) found extensive lesions in the compound eyes of Manduca sexta reared on vitamin-Adeficient diets. However, this p-carotene derivative does not seem to be necessary to the functioning of most insects. Vitamin A may actually be injurious to Cecropia larvae. Preliminary experiments in which the concentration of vitamin A added to the diet was 240 mg/l indicated that the larvae reared on such diets had difficulty in ecdysing. Cuticles were discoloured and survival rates were low. These experiments were not extensive enough to be conclusive and were not continued. The presence of the weak red pigmentation of thoracic tubercles of third instar larvae reared on diets lacking carotenoids indicates that there are enough of these carotenoids in most of the eggs of Cecropia to produce near normal coloration through the third instar. In a few cases a faint tubercle pigmentation was also observed on fourth instar larvae reared on these diets. DADD (1961) found that

1598

RALPH M. CLARK

locust eggs contained enough carotenoids to support normal larval growth on carotene-free diets. These observations also indicate that the larvae is able to maintain a reserve supply of these carotenoids for a considerable period of time. One of the more interesting results was the failure of fifth instar larvae to develop normal colouration of thoracic tubercles when transferred from diets lacking carotenoids to leaves or to lutein-supplemented diets. That fourth instar larvae whose diets were similarly changed did develop normal pigmentation within 48 hr suggests that carotenoids may have to be present before the last larval ecdysis for incorporation into cuticular pigments. Acknowledgements-The author wishes to express his appreciation to the Bristol Laboratories, Syracuse, New York, who kindly furnished the kanamycin sulphate used in these experiments, and to his laboratory assistants, Miss LESLIE LUBY and Mr. GREGORYPALMER. The research was supported by the Faculty of Science and Mathematics, State University of New York College of Arts and Science, Plattsburgh, New York. REFERENCES CARLSONS. D., STFZVESH. R., III, VANDEBERGJ. S. and ROBBINSW. E. (1967) Vitamin A deficiency: Effect on retinal structure of the moth Manduca sexta. Science, Wash. 158, 268-270. COHEN C. F. and BARKERR. J. (1963) Vitamin A content and spectral response of house flies reared on diets with and without a vitamin A source. J. cell. camp. Physiol. 62, 43-47. CROMARTIE R. I. T. (1959) Insect pigments. A. Rev. Ent. 4, 59-76. DADDR. H. (1961) Observations on the effects of carotene on the growth and pigmentation of locusts. Bull. ent. Res. 52, 63-81. DAHLMAND. L. (1969) Cuticular pigments of the tobacco hornworm (Manduca sexta) larvae: Effects of diet and genetic differences. J. Insect Physiol. 15, 807-814. GILMORED; (1961) The Biochemistry of Insects. Academic Press, New York. GILMORED. (1965) The Metabolism of Insects. W. H. Freeman, San Francisco. GOODWINT. W. and SRISUKHS. (19.51) Biochemistry of locusts-V. The green pigment of the hemolymph and integument of solitary locusts (Locusta migratoria migratorioides, R. & F., and Schistocerca gregaria, Forsk.). Biochem. J. 48, 199-203. HACKMANR. H. (1952) Green pigments of the hemolymph of insects. Archs Biochem. Biophys. 41, 166-174. HOWDENG. F. and HUNTER-JONESP. (1958) An artificial diet for the laboratory rearing of locusts. Nature, Lond. 1527-l 528. OHNISHI E. (1959) Pigment composition in the pupal cuticles of two colour types of the swallowtails, Papilio xuthus L. and P. protenor demeirius Cramer. J. Insect Physiol. 3, 132-145. RIDDIFORLI L. M. (1967) Antibiotics in the laboratory-rearing of Cecropia silkworms. Science, Wash. 157, 1451-1452. RIDDIFORD L. M. (1968) Artificial diet for Cecropia and other saturniid silkworms. Science, Wash. 160, 1461-1462. VAN DER GEEST L. P. S. (1968) Effects of diets on the haemolymph proteins of larvae of Pieris brassicae. J. Insect Physiol. 14, 537-542.