Development responses of symbiotic and aposymbiotic weevils Sitophilus oryzae L. (Coleoptera, curculionidae) to a diet supplemented with aromatic amino acids

DEVELOPMENT RESPONSES OF SYMBIOTIC AND APOSYMBIOTIC WEEVILS SITOPHfLUS OR YZAE L. (COLEOPTERA, CURCULIONIDAE) TO A DIET SUPPLEMENTED WITH AROMATIC AMINO ACIDS C. WICKFR and P. NAKIION Laboratoire

de Biologie.

INSA. 69621 Villeurbanne.

France

-Developmental times of symbiotx and aposqmbiotic btrains of the rice weevil .SI~C~JI/Z~/II,~ r~~~-_ut~were compared, in response to changes in concentration of phenylalanine or tyrosine in whole wheat flour pellets. Aposymbiotic insects were shown to require more aromatic amino acids than symbiotic insects. since a very low supply (O.i”,,) resulted in Faster growth (I l”,,). Incorporation results of [“HI-tyrosine during the larval and pupal stages indicated that total tyrosine Intake was lower ln aposymbiotic insects. but the mcorporation into the cuticle of both strains did not significantI> JitTcr. It is suggested that the slower growth rate of weevils without symbiotes is due. in part. to a lehs cficicnt utilization of exogenous tyrosine (in the food) and to a lack of endogenous orosine (supplied h! the .\ymbiotcs). Abstract-

INTRODUCTION MOST of the literature on endosymbiosis still remains descriptive and studies on the physiological and nutritional role of endocellular symbiotes have been developed only recently. In the weevil Sitophihs oryxc. symbiotes are in anatomical relation with the larval alimentary canal: the mycetome lies near the junction of the stomodaeum and the mesenteron (MANSOIIR, 1937: TIEGS and MURRAY. 1938). The larval mycetome degenerates during metamorphosis and in the imago. symbiotes are principally located at the apex of the female ovarioles (MANSOUR, 1930; NARDON. 1971). Yet, they can be found in the anterior mesenteric caeca in young adults of both sexes (NARL)ON and WICKER, 1981). In the genus Sitophiltrs. SCHNEIDER(1956) succeeded in obtaining aposymbiotic larvae by heat-treatment; these larvae, however. were able to grow only on sorghum and not on wheat. He inferred therefore the necessity of a growing-factor (factor ‘P’), that could be supplied by the symbiotes. The delayed growth of aposymbiotic larvae, observed by several authors (SCHNEIDER. 1956: BAKER and LUM. 1973; NARDON, 1973, 1976). also suggests that they lack something, compared with the normal insects. The larval mycetome does not communicate with the gut and bacteria probably play no digestive role: but the possibility of their secreting some growth factor into the haemolymph is not to be excluded. Otherwise. the symbiote-free imagos have less colour (SCHNEIDER. 1956: NARD~N and WICKER. 1981), as noted by most authors. BR(~KS and RICHARDS (19S5) observed the same phenomenon for B/urel/u (twmtnicu. Moreover if phenylalanine or tyrosine MJBSadded to the diet of those aposymbiotic

cockroaches. a darkening of the integument occurred. so that seven days later. symbiotic and aposymbiotic insects could be distinguished only with ditlicult? (HENRY and COOK. 1964). Therefore. it appeared interesting to invest&c the action of tyrosine and phenylalanine in S. OT+(C’. The nutritional studies related to hcmhiosls in the genus Sitophil~~ are rare because of the diticuhy in obtaining symbiote-free insects. As NAKIX)K (19731 was able to obtain aposymbiotic weevils b> keeping imagos at 35 C and X0”,, r.h. for about a month. several aposymbiotic strains are available in our laboratory. These have been mamtaincd on wheat and sorghum for several years. llntil the Xth genrration. aposymbiotic weevils showed ;I marked delay in growth. a diminished weight and a loss of fertility. Afterwards fertility and weight were hubj,cctcd to great variations but developmental time rcmalned constant. Then. while fertility appears to depend on scvcral (still uncontrolled) factors. it seems to he a constant rclationship between the harbouring of bymbiotch and the developmental time. The influence 01 tyrosine and phenylalanine on the darkening of the cuticle and the developmental time was studied in order to clarify :I possible role of symbiotes.

MATERIALS

AND

METHODS

Weevils were raised at 27.5 C and 75“,, r.h. (L.AVIOLETTEand NARDON. 1963). A symbiotic strain and the aposymbiotic strain which was obtained from this later were used for these experiments. For the nutritional experiments. a technique for rearing S. Or),XJ~ on ‘artificial grains’ was developed. After four gen-

102 I

C. WICKER and

1027

erations on these pellets. insects were able to lay eggs and to grow normally (developmental time did not differ in these pellets from that in wheat kernels). In each experiment, one lot of about two-hundred insects was used as parents: these imagos were sampled two weeks after their emergence and placed on 100 g of a test diet for two days. The number of imaginal emergences was then noted each day and development time was defined as the period from the oviposition to the emergence from the pellets.

The pellets are made by kneading whole wheat flour with distilled water and agar (2”,), keeping the conditions as aseptic as possible (by using sterile water and clean instruments). About 300 pellets were made out of 1OOg of flour. The pellets were kept at room temperature for drying for about 3 days. After that time, the moisture content of the pellets, determined by means of a CHOPIN incubator, was too low (about 9”J to encourage fungus growth. The pellets were kept at 27.5 C and 75”,, r.h. for about two weeks before use. during which time the moisture content increased to about 14”,,.

One batch of insects of each strain was reared on pellets supplemented with 3,5-C3H]-tyrosine (I pmole per 100 g diet: specific activity: 5 Ci/mi!limo!e) from the egg until adult emergence. The insects were removed (from 0 to 12 hr after emergence) and transferred to non-labelled pellets for two hours after which time labelled diet was completely eliminated from the gut, since the gut transit is less than two hours. as showed by experiments with coloured pellets (unpublished data).

Each weevil was homogenized with distilled water (0.5 ml) and centrifuged at 1500 g for IO min. The pellet was washed twice in water in order to remove as much non-cuticular material as possible. After the second washing, the observation of the pellet under a phase-contrast microscope revealed only few trachea! and muscular tissue residues which remained bound to the cuticle. Each sample (0.5 ml) was mixed with 4.5 ml scintillation liquid (Unisolve) and counted for 2 min.

Amino acid analyses were performed with a Technicon automatic analyser. according to the procedure of DELOBEL and BONNOT (1976) on protein hydrolysates (6N HCl; 105°C; 24 hr). RESULTS &ff;cr

of the

amino

acids

on dereloprnental

P.

NARDON

aposymbiotic strain at the 0. I”, concentration cannot be explained. Other results obtained on sorghum and wheat indicated a slight increase of the fertility at this concentration. It must be mentioned that our experimental procedure is only adapted to the determination of the mean developmental time. The oviposition period (2 days) is not sufficient to establish fertility. Presently no conclusions concerning fertility can be drawn from these experiments. When the phenylalanine concentration was increased to 0.5”,, (Table 2). faster development of the symbiotic strain was observed. This heightened effect diminished when the amino acid level was increased, but remained significant. even at 1.5”,,. For the aposymbiotic strain on the contrary. the acceleration decreased above 0.5”,,; the l.5”,, concentration resulted in a slight but significant (P = 0.05) delay in the rate of growth. When the aromatic amino acids were added to sorghum pellets instead of wheat pellets. the same effects were observed: a growth acceleration which became lower at 0.5 and O.‘Y,, and a delay of growth at l.S’,, (unpublished data). Inc’ofyorc~fio~~01 iclhrl imo rhc~ cxficiv

DISCUSSION It is well established that sclerotization and melanization of insects require large amounts of tyrosine (KARLSON et [II.. 1962: WIRTZ and HOPKINS, 1974: BRUNET. 1980). In Sitophihs oryxc. contrary to what was observed in cockroaches (HENRY and CME. Table 1. The effect of tyrosine and phenylalanine (0.1”~ on the mean development time of symbiotic and aposymbiotic S. or ww iI

Amino acid

Days to emergence*

Symbiotic

246 463 283

None phenylalanine tyrosine

31.6 + 0.1 31.7 & 0.1 31.9 i 0.1

Aposymbiotic

137 76 40

None phenylalanine tyrosine

45.9 f 0.4 41.0 f 0.4 41.0 + 0.6

Strain time

When tyrosine or phenylalanine was added at the same concentration (0.1”;) to the diet. the same effect on mean development time was obtained (Table 1). While no significant difference in the growth rate of symbiotic insects could be detected, a significant acceleration ( 10.6%) occurred for the aposymbiotic weevils (P < 0.001). The diminished fertility of the

(7X& 3)

The total radioactivity appeared about 40’,, lower in the aposymbiotic weevils than in the symbiotic insects. The difference was significant at 0.01 probability level. On the other hand. the incorporation of tyrosine (and its metabolites) into the cuticle was always higher in the symbiotic strains (23”,,). but the difference was not significant according to statistic analysis. Given our experimental procedure, label in the emerging imago mainly, if not exclusively. came from alimentary tyrosine (or tyrosine-derived metabolites) which was accumulated in the larva! stages and used at metamorphosis for the regeneration of new tissues and especially the secretion of imagina! cuticle. This result favours the view that aposymbiotic weevils have a smaller tyrosine pool but incorporate a larger part of the pool into their integument, so that the cuticular radioactivity was about the same in both strains.

,I = number of insects. * Means i S.E.

Development

responses

Table 2. The effect of phenylalanine

Concentration ” 0

Strain Symbiotic

Aposymbiotic

and aposymbiotic

II 283 479 416 35.1

31.7 28.9 29.’ 30.7

0.1 0.5 0.9 I.5

40

422 197 66

41.0 41.9 44.9 47.63

and aposymbiotic

Acceleration of growth rate

Days to emergence* 0.1 0.1 0.3 0.3

- I.0 + 8.6 +7.5

IO.6 & 0.5 * 0.7 It 1.4

+ 10.6

& k i f

IO3

weevils

on the growth rate of symbiotic strains of S. or~xr

0.1 0.5 0.9 1.5

* Means + SE. t Means are significantly 0.05 level. t Means are significantly 0.0’1 and 0.001 levels. 1964). the administration

of symbiotic

r-test 1.x 20.0:

16.6: 5.1:

+ 1.9

5.8: 11.7: 2.1t 2.4t

+x.6 + 2.2 - 3.‘)

different

from controls

lacking

added

phenylalanine

at the

different

from controls

lacking

added

phenylalaninc

111

the

_

of phenylalanine

or tyrosine

in the diet did not perceptibly modify colouring of the concentration symbiote-free insects, whatever used and in spite of the preferential incorporation of tyrosine-derived metabolites into the integument. Aromatic amino acid levels did not appear to be a limiting factor for the pigmentation of the weevils. The lighter colour of aposymbiotic insects could be due to some change in the enzymatic system involved in pigmentation. On the other hand. the concentration of aromatic amino acids was shown to be of great importance for the development time in our experiments. BAKER (19791. studying the requirements for the csscntial dietary amino acids of S. orF-_ne larvae. could not demonstrate a strict reqmrement for phcn:lalanine: the elimination of phenylalanine and tyroslne from the diet did not prevent development of the insects. which wah only 13”,, delayed. BAKER indicated that contamination of the cornstarch by phenylalanine may have been possible, but even in this case it should be noted that the dietary requirement for phen!lalanine seems especially low in S. or.r:trc~.

Table 3. Incorporation

Nevertheless. phenylalanine is an essential ammo acid ror larval development in most insects (I)ALIII. 1973). Tdwliwt~ CO&WH. for example. did not develop on a cornstarch-based diet when phenylalanine was omitted (TAYLOR and Mrr~rcr. 1966). Our experiments showed that a bery low supplementation of free phenylalanine or tyrosine (Cl”,,) did not influence the growth rate of symbiotic weevils but enhanced that of aposymbiotic ones (10.6”~. These preliminary data suggest that the requirements for aromatic amino acids in symbiote-free and symbiotecontaining insects differ qualitatively and quantitatively. The supply of tyrosine or phenylalanine by the symbiotes may be hypothesized. In f&t. the problem appears to be more complex. According to BLCHIU’FR(1965). insects acquire symbiotic organisms in order to supply nutritional deliciences. However. the wheat flour used for the production of pellets was shown to contain U..W’,, tvrosine and 0.57”,, phenylalanine. These conccntratks are not negligible and in Lict. alloy the tlewlopmcnt of several indicating

of [3H]-tyrosine

;I

larger

strains

tyrosin-pool

in the in

into entire insect and cuticle

Symbiotic strain II = 20 Weight (mg) Total label in insects* (disintegrations,? min:mg) Label in cuticles (disintegrations;2 m&cuticle) (disintegrations, 2 min;mg insect) Label in cuticles/total label in whole insects (“,Jt

aposymbiotic

Aposymbiotic strain ,I = 20

l-te51

’ ” + _.-_ _ 0.01

l.YY & 0.06

2939 + 402

2375 i- 1’5

1730 + 109 555 + 51

YY5 + 106 199 + 50

l.S-lb 0.7Q

14.x + 1.0

10.6 + I.2

?.h$

3.71:

All values are means + SE. * Total label is obtained by the addition of the label in the cuticle and the label m the remaining part of the insect. t The ratio is calculated for each insect. using the same values as reported above. $ Means are significantly different at the 0.001 level. # Means are not significantly different.

lahoratc~ry. Hl the <\n;biotic

C. WI(.L;EK and P. NARDON

1024

insects, our experiments led to the conclusions that the limiting factor in aposymbiotic insect growth is essentially to be found at the level of metabolism rather than food. Two metabolic levels can be mentioned: the digestive protein assimilation and the tyrosine re-utilization from its stored form. Indeed it is known that tyrosine can be stored, notably in the fat body (PRICE. 1972) as dipeptides (BODNARYK, 1978: COLLETT, 1976; DELOBELand BONNOT, 1976) or glucosides (KRAMER rt ctl.. 1980). This allows us to understand that a continuous supply of exogenous free tyrosine preferentially favours aposymbiotic weevils, if the lack of symbiotes deprive them of any endogenous source and moreover if they are deficient in their digestion or (and) tyrosine release. Later studies will be carried out to clarify this point. and experiments are presently being run with defined artificial diets. If phenylalanine or tyrosine are limiting factors of the growth rate of aposymbiotic weevils. other factors will probably interact. In the experiments reported. the addition of these free amino acids in the diet could not restore the development of the aposymbiotic strain to the level of the symbiotic strain (a 30’!,, difference persisted). Other deficiencies could be involved (amino acids, vitamins. etc.). This could explain why an increase in the phenylalanine concentration led to a great nutrient imbalance and thus resulted in an inhibition of the development. at a IS”,, concentration, only in the aposymbiotic weevils. Experiments are being carried on to identify the various elements of that imbalance. .4c~no~~irdyt,,tten1, We thanh our colleagues G. BONB. DI-LOWL,for providing the amino acids analy-

NOT and

sis on the whole-wheat

flour.

REFERENCES BAKER

J. E. (1979) Requirements of the essential amino acids of larvae of the rice weevil. Encir. Ent. 8. 451-453. BAKER J. E. and LUM P. T. M. (1973) Development of aposymbiosis in larvae of Siruplzilm or~~xw by dietary treatment with antibiotics. J. storrd Prod. Rtcs. 9, 241-245. BODNARYK R. P. (1978) Structure and function of insect peptides Adr. It~wct Physiol. 13. 69-132. BRINKS M. A. and RICHARDS A. G. (1955) Intracellular symbiosis in cockroaches-l. Production of aposymbiotic cockroaches. Biol. Bull. 109, 22-39. BRVNET P. C. J. (1980) The metabolism of the aromatic ammo acids concerned in the cross-linking of insect cut]cle. insect Biochrrrr. IO, 467.-500. BLICHNEK P. (1965) Endo.s~rnhiosis 01’ Atlirnals with Plant Mi~oorganisrm. J. Wileyylnterscience, New York. COLLETT J. 1. (1976) Peptidase-mediated storage of amino acids in small peptides. Insr~r Biochmrn. 6, 179-185.

DADD R. H. (1973) Insect nutrition: current developments and metabolic implications. A. RN. EM. 18, 381 420. DELOBEL B. & BONNOT G. (1976) Presence de [i-alanyltyrosine chez le dipthe tachinaire Pkr\,ut, c,uudufu Rond. Importance relative des acides ammks libres, peptidiques et prot&ques. Ann. Zoo/. Ecoi. Arritrr. 8, 493-497. HENRY S. M. and Cook: T. W. (1964) Amino acid supplementation bv symbiotic bacteria in the cockroach. Contr. Boycr Thon;p.&, IN. 22, 507. KARLSON P.. SEKERIS C. E. and SEI(ERI K. E. (1962) Zum Tyrosinstoffwechsel der Insekten-VI. Identifizierung von N-Acetyl-3,4-dihydroxy-fl-phenylgmin (N-acetyldopamln) als Tyrosinmetabolit. HopprGrjGr’\ Z. phniol. Chew. 327, 86-94. KRAMER K. J.. HOIXINS T. L.. AHM~U R. F.. MUELLER D. and LoohHAbvr G. (1980) Tyrosine metabolism for cuticle tanning in the tobacco hornworm. Mlr&~c~u sc~t(l (L.) and other Lepidoptera: identification of b-o-glucopyranosyl-O-L-tyroslne and other metabolites. Arch. Bierrluw. Biopk>h. 205, 146 155. LAVIOLETTEP. and NARDON P. (1963) Action des rayons ; du cobalt 60 sur la mortalit et la fertlliti- des adultes d’un charancon du riz. Buli. Biol. 97. 306 333. MANSXIR K. (1927) The development of the larval and adult midgut of Colundru or~~w: the rice weevil. Q. JI ,,tic,ro.\c,. .%i. 71. 313-352. MANSOUK K. (1930) Preliminary studies on the bacterLal cell mass (accessory cell-mass) of Culundrrr oryxr: the rice weevil. Q, Jl rtricrosc~. Sci. 73, 421-436. NARDON P. (1971) Contribution g I’Ctude des symbiotes ovariens de Sitr)phil~r.s .~rsnhii. Localisation. histochimie et ultrastructure che7 la femelle adulte. C.r. hchtl. S&c. .4cad. Sci.. Ptrris D272, 2975-2978. NARUON P. (1973) Obtention d’une souche asymbiotique chez le charanqon Sitophihcs strstrkii: difft-rentes mCthodes et comparalson avcc la souche symbiotique d’orlginc. C.r. IIC+M/. SPtrnc. 1cud. SC;.. Pork D277, 98 I -984. NARD~N P. (1976) Un aspect parttculier de d&renciation sexuelle: cas des insectes possCdant des symbiotes i transmission maternelle. Bull. Sot.. -_oo/. Fr. 101. 33-38. NARDON P. and WIC~~K C. (1981) La symbiose chez le genre Sitophilus (Coliopttre curculionide). Principaux aspects morphologiques. physiologiques et 8PnPtiques. Ann. Biol. 20, 327-374. PKI(.I- G. M. (1972) Tyrosine metabolism in the larva of the blowfly. Crd/iphorrr c,r-!,t/rrocepll~i(u. Imrcr Bio~lrtwr. 2. 175-185. SCHNEIDER H. (1956) Morphologische und experimentelle Untersuchungen iiber die Endosymbiose der Korn und Reis-kafer (C‘ulundru yrcnturici und C. or~:Liv). Z. Morph. 6ikol. Tiertz 44, 555-625. TAYLOR M. W. and MFDIC’I J. C. (1966) Ammo acid requirements of grain beetles. J. Nutr. 88, 176-180. TIEGS 0. W. and MURRAY F. V. (1938) Embryonic development of Cl~ltrnLfrrr or~‘;cre. Q. JI n~icrcw. Sci. 80, 159 284. WIRTZ R. A. and HOPKINS T. L. (1974) Tyrosine and phenylalanine concentrations in haemolymph and tissues of the american cockroach. Pc~riplmrtrc cmwrxwm (L.) during metamorphosis. J. Imwct Pl~ysiol. 20, 1143-1154.