Nucleotide, nucleoside and base nutritional requirements of the mosquito Culex pipiens

J. Insccr Phjsrol., ‘(‘ Pergamon

Vol. 25. pp. 353 10 359.

Press Ltd.

1919.

Printed

0022-1910

79 0401-0353

~02.00 (1

in Grear Britain.

NUCLEOTIDE, NUCLEOSIDE AND BASE NUTRITIONAL REQUIREMENTS OF THE MOSQUITO CULEX PIPIEiVS R. H. DADD Division of Entomology and Parasitology. University of California. Berkeley, CA 94720. I-.S.A (Received

25 September

1978)

Abstract-Nucleic acid subcomponents needed to satisfy the dietary nucleic acid requirement of CU/KY were studied in growth experiments using synthetic media in which nucleosides. bases and alternative nucleotides were variously substituted in mixtures of 3 nucleotides (adenylic acid. thymidylic acid, and either cytidylic or uridylic acid) previously shown to be adequate replacements for whole nucleic acid. Any or all 3 nucleoGdes could be replaced by corresponding nucleosides without adverse effect. except that adenosine substitution moderately delayed pupation. All base substitutions were unsatisfactory: substitution of thymine for thymidylic acid allowed development to the adult stage but at a greatly reduced rate: smgle substitution of adenine, cytosine or uracil for the corresponding nucleotides allowed scarcely more development than in the total absence of nucleic acid derivatives. lnosinic acid or inosine were adequate substitutes for adenylic acid, but erotic acid or orotidine were ineffective in place of the pyrimldine ribonucleotides. cytidylic or uridylic acids. Deoxyadenylic acid could take the place of adenylic acid, though inefficienrly. but deoxycytidylic and deoxyuridylic acids were very poor replacements for the corresponding ribonucleotides. The minimal required nucleic acid derivatives thus appear to be a purine rlbonucleotide (adenylic or inosinic acids). a pyrimidine ribonucleoside (either uridine or cytidine), and the pyrlmidine deoxyribonucleoside. thymidine.

pipiens

INTRODUCTION

satisfactory mixtures of 3 nucleotides requirements to a more basic level.

LIKE vertebrates, most insects can synthesize the nucleotides needed as coenzymes or for the assembly of nucleic acids. However, several Diptera require dietary ‘nucleic acid or certain of its subcomponents for optimal rates of growth, though usually able to complete development from egg to the adult stage without it (DADD, 1970a, 1973; HOUSE, 1974). For a few Diptera, nucleic acid is essential for development to metamorphosis, and among these is the mosquito, Culex pipiens (DADD et al., 1973). Recently it was shown that a mixture of nucleotides could adequately replace dietary nucleic acid for C. pipiens (DADD and KLEINJAN, 1977). Of the 5 nucleotides in a mixture which first supported good development, guanylic acid proved unnecessary, adenylic and thymidylic acids were each required, and either uridylic or cytidylic acid was essential. With the corresponding nucleosides replacing all 5 nucleotides the growth rate of larvae was substantially reduced, although a majority developed to adults, but single replacement of any one nucleotide by the corresponding nucleoside scarcely delayed development. When the 5 corresponding bases were substituted together, all larvae died early in the first instar, probably because guanine, being insoluble and therefore in suspension, would have been differentially ingested to excess as a result of filter-feeding. Since guanylic acid, and hence also guanine, was eventually shown to be unnecessary, further attempts to define requirements below the level of nucleotides were put aside until a minimal nucleotide requirement was established. This paper describes experiments in which simpler compounds were substituted in 353

MATERIALS

so as to clarify

AND METHODS

The mosquito used in this work is a highly autogenous strain of Culex pipiens L. which has been maintained continuously in this laboratory since its acquisition for feeding behavioral studies (DADD, 1970b) from G. H. A. McClelland, ofthe University of California at Davis. It was originally collected from a sewage works at Dixon, Northern California. Detailed procedures for the surface sterilization of eggs, the preparation and dispensing of dietary media. and the aseptic allocation of newly-hatched larvae to culture tubes containing autoclaved dietary medium are given in a previous publication (DADD ef al., 1973). The basal medium, fully described elsewhere (DADD and KLEINJAN, 1976) is a solution of 20 amino acids, glucose, 8 water-soluble vitamins, 10 inorganic salts, with cholesterol in colloidal suspension and with a minute amount (0.05%) of agar to impart the slightly viscous texture needed for optimal liquid ingestion by the larvae (DADD, 1975). A full listing of nucleic acid components and their provenance is given in DADD and KLEINIAN (1977). In the earliest experiments reported here (those of Figs 2 and 3), 20 larvae per treatment were reared individually in 5 ml of medium per culture tube. It was subsequently found that development scarcely differed with up to 8 larvae per tube, so in all later experiments, the larvae were reared in pairs per tube of 5 ml of medium, still with 20 larvae per treatment. Tubes of larvae were kept in an incubator at 27’C. and

R. H DADD

354

stages of development were recorded at suitable intervals, every day once pupal ecdysis had started, until adult emergence or death at an earlier stage. After excluding certain dead or non-growing 1st instar larvae from the raw data as outlined in DADD and KLEINJAN (1977) the following criteria of development were calculated for each treatment in an experiment: (a) the average number of ecdyses per starting larva until the death of all individuals; (b) the percentage of starters to become pupae; (c) the percentage of starters to become adult; (d) the average time, in days, to the pupal ecdysis. The best criterion of the amount of growth and development ultimately achieved by a treatment group is the average number of moults per starting larva, but this takes no account of rate of development. An index of developmental rate is provided by the average time to pupation. To incorporate both aspects of development into a single value it is convenient to calculate a Developmental Index for each treatment in an experiment, defined thus: average number of moults per starting larva x 10 Developmental Index = average days to pupation . When fewer than 15% (3 out of 20) starters pupate, an arbitrary divisor of 25 is used to generate the Developmental Index; this convention, discussed elsewhere (DADD and KLEINJAN,1977) is based on the fact that pupation times rarely exceeded 25 days under the conditions of these experiments. Most results are summarized graphically, primarily in terms of Developmental Indices. To discount the considerable variability in overall performances between batches of larvae used in replicate experiments carried out intermittently over a period of 2 yr, Developmental Indices for all treatments of an experiment are converted to percentages of the positive control treatment for that experiment and the

resulting values are plotted as bar graphs. Most experiments were performed thrice, and in the figures, barsfor the same treatment are grouped, with a letter on each bar identifying the experiment. As a further indication of performance, the percentage of starting larvae which became adult is shown above each treatment bar. The basal mixture of 3 nucleotides taken as the positive control in these experiments comprised 100 mg adenylic acid, 50 mg cytidylic acid and 50 mg thymidylic acid per 100 ml of dietary medium: this overall nucleotide concentration of 0.2% was chosen because it had previously been found that indistinguishably good results were obtained with the 5 nucleotide mixture at overall concentrations ranging between 0.1 and 0.4% (DADD and KLEINJAN,1977). Substitutions of nucleotides were made at these same levels, whereas nucleosides and bases were substituted at 60 and 40% by weight, respectively, of the corresponding nucleotide. Growth of C. pipiens is insensitive to wide variations in overall concentration of nucleotides, as shown by the data of Fig. 1 in DADD and KLEINJAN(1977). Dosage plots for individual nucleotides shown in Fig. 1 of this paper confirm that development is insensitive to substantial excesses above minimal requirements, and also suggest that balance between components of the nucleotide mixture is not critical. RESULTS Optimal concentrations for required nucleotides

Plots of Developmental Indices against serial concentrations for each required nucleotide are shown in Fig. 1. Over the period during which these assays were made, various modifications to the basal nucleotide mixture were introduced as ongoing study showed that reductions in overall nucleotide concentration and omission of certain nucleotides had

r

Thymldyllc

0

12525

5

IO 20

mg/lOO

40

IA 0’05

80 mg/lOOml

ml Concentrotlon

of vanable

I

I

I

I

I

I

I

2

4

8

16

mg/lOOml nucleotide

Fig. I. Developmental Indices for groups of C. pipiens reared in series of synthetic media in which single required nucleotides were provided at doubling concentrations. Each plot (or pair of plots in a few cases) represents a separate experiment, numbered I-14. The amounts of other nucleotides present at constant level throughout a concentration series are given in the following list, as mg per 100 ml of medium, with the symbols A, G. C, U and T as abbreviations for adenylic, guanylic, cytidylic, uridylic and thymidylic acids respectively. Expt 1: AlOO. E50, CSO, USO. Expt 2: G50, USO, C50, TlO. Expts 3 and 5: AlOO, TIO. Expt 4: A60, G40, U25, C25. Expt 6: CSO, TlO. Expt 7: AlOO. C50. Expt 8: G40, U25. C25. TlO. Expts 9 and 1I: U25. C25. TIO. Expts 10. 12 and 14: A60. U25, C25. Expt 13: U25. C25. TIO. and G40 when present.

I

Base nutritional

reqmrments

of the mosquito

little or no adverse effects. Thus, the basal nucleotide mixtures for these experiments were various, as noted in the legend for Fig. 1. Towards the end of the series, certain other modifications were made in the basal medium, mostly for precautionary reasons suggested by concurrent studies of nutrients other than nucleotides. Most of these changes (omission of glutamine, substitution of agarose for agar. an increase in sodium chloride) had no evident effect, but one change in the basal diet for the final assay of thymidylic and adenylic acids (Expts 13 and 14), supplementation with animal lecithin, consistently improved development. The plots for cytidylic and uridylic acids in Fig. 1 show that very little development occurred with both these nucleotides lacking. Development was progressively improved with increasing concentrations of either nucleotide up to 20 mg/lOO ml and was maximal for all higher concentrations up to 80 mg] 100 ml. The two experiments, 3 and 5, in both of which uridylic and cytidylic concentration series were directly compared, suggest quantitative as well as qualitative equivalence for these nucleotides. The plots for thymidylic acid concentration in Fig. 1 indicate optimal development for a minimal concentration of between 2 and 4 mg/lOO ml. The several repetitions of this assay were prompted by this surprisingly low minimal requirement, and also by the fact, contrary to previous experience, that in some experiments a majority of larvae developed to adults in treatments without thymidylic acid. The detailed records show that percentages of adults obtained for treatments lacking thymidylic acid were 0, 34, 40, 47, 60 and 89, values which are, except for the first experiment, substantially higher than the adult percentages of 8, 8, 10, 13, 14 and 20 for thymidylicdeficient diets used in the previous study of DADD and KLEINJAN (1977). Thymidylic acid greatly in excess of

No AGCUT nuclelcs

ACT

AC!

ACT

Cu/u.r prpi~~.c

3’5

the concentrations used in these assays was harmless; basal medium for the experiments reported in the following section of this paper contained 50 mg/lOO ml, and 100 mg/ 100 ml has been used with no apparent ill effect. The plots for adenylic acid in Fig. 1 show that development was optimal with concentrations in the range of 40-80 mg/lOO ml. As for the thymidylic acid assays, there were some anomalous findings compared with previous experience. The detailed records show that treatments lacking adenylic acid entirely gave higher percentages of adults than in previous work; for the experiments summarized in Fig. 1 these percentages were 29,36,37,56,60.71 and 80, whereas for adenylate-free diets in DADD and KLEINJAN (1977) the percentages of adults were 0. 0, 0, 0, 10, 15 and 29. While accumulating concentration data for adenylic acid it was noticed that the highest adult percentages for adenylate-free treatments occurred in experiments for which the basal nucleotide mixture lacked guanylic acid, and in Fig. 1 this is reflected in the relatively high Developmental Indices for the guanylate-free series at zero or low concentrations of adenylic acid. When this difference was first suspected it was tested for by including an adenylate-free, guanylate-supplemented treatment in an otherwise guanylate-free series (Expt 1 l), the result being a reduced percentage of adults and a reduced Developmental Index for this supplemented treatment. In the final adenylic acid assay (Expt 13 of Fig. l), two identical dosage series were directly compared, one with and one without a standard concentration (40 mg/lOO ml) of guanylic acid; for concentrations of adenylic acid above 10 mg/lOO ml there was little difference in development whether guanylic acid was present or not, but for 5 mg/lOO ml or zero adenylic acid, development was markedly inferior with guanylic acid present, extremely so for zero adenylic acid. Thus it appears that the

qCT

As!

PC!

!4!!T

LIC!

Fig. 2. Relative Developmental Indices for group of C. pipiens reared on synthetic media with nucleosides variously substituted for corresponding nucleotides in a satisfactory mixture of 3 nucleotides. Three replicate experiments, J, K, and L. were carried out, and bars for the same treatment of each experiment are grouped and lettered in white to indicate the experiment. For each experiment all actual Developmental Indices were converted to percentages of a control treatment (ACT) for that experiment, and these percentages are plotted as bars. The number above each bar gives the percentage of adults obtained for that treatment. Abbreviations for nucleic acid components are as follows: A, G, C. U and T denote adenylic. guanylic, cytidylic. uridylic and thymidylic acids respectively; a, c and f denote the nucleosides adenosine. cytidine and thymidinc. IV.254

R. H.

356

requirement for adenylic acid is exacerbated guanylic acid IS present.

when

Substituents in satisfactory mixtures of 3 nucleotides Data from three experiments to compare diets in which nucleosides were variously substituted in a satisfactory mixture of 3 nucleotides are summarized graphically in Fig. 2. In each experiment the treatment lacking all nucleic acid components supported little development, while treatments using the original mixture of 5 nucleotides gave marginally better Developmental Indices than the 3 nucleotide mixture. With thymidine substituted for thymidylic acid, or cytidine for cytidylic acid, development was virtually unaffected, but with adenosine replacing adenylic acid, Developmental Indices were less, by 10-30x, than for the 3 nucleotide control. All multiple substitutions involving the replacement of adenylic acid by adenosine resulted also in appreciably reduced Indices, whereas the double substitution of cytidine and thymidine (leaving just one nucleotide, adenylic acid) had no adverse effect on development. It should be emphasized that whatever the nucleosides substituted, development ultimately was good, indicated by the generally high percentages of adults obtained with all nucleoside-substitution treatments. The detailed records show that the lower Developmental Indices for treatments involving adenosine substitutions were essentially the outcome of slower growth to pupation. A group of experiments in which corresponding bases were substituted for nucleotides is presented in Fig. 3. With thymine in place of thymidylic acid a majority of larvae eventually became adult, but the detailed records show that time to pupal ecdysis was greatly protracted, and this largely accounts for the co. 30”” reduction in Developmental Indices for this treatment as compared with the 3 nucleotide control. Substitution of either adenine or cytosine for their corresponding nucleotides reduced development almost to the very low levels for the treatment entirely lacking in nucleic acid components, with very few

AGCUT

No nuclelcs

ACT

AC@

DADD

larvae reaching pupation. All multiple substitutions of bases similarly rendered the diet ineffective, and in some cases (e.g. substitution of adenine and cytosine, or of adenine, cytosine and thymine) appear detrimental in comparison with the total absence of nucleic acid components. Thus, though thymine may be inefficiently utilized as a replacement for thymidylic acid, adenine and cytosine are virtually ineffective as substitutes for their corresponding nucleotides. The results shown in Figs. 4 and 5 derive from three replicate experiments, each of 20 treatments, which, for convenience, are presented in two parts. In Fig. 4, a group of treatments deals with substitution of the corresponding nucleoside or base for uridylic acid in a satisfactory mixture of 3 nucleotides containing uridylic rather than cytidylic acid. Developmental Indices and adult percentages are all high for the two treatments using mixtures of 3 nucleotides (with cytidylic and uridylic acids, respectively) and for the two corresponding nucleoside-substituted treatments, with cytidine and uridine respectively. With the base, uracil, substituted for uridylic acid, Indices are low, and though about a quarter of the larvae eventually became adult, the time to pupation was double that for satisfactory diets. Thus, the pattern of effects for uracil-containing components is essentially similar to that for the cytosine-containing components dealt with in Figs 2 and 3. Also presented in Fig. 4 are data for treatments involving the substitution of inosinic acid or inosine for adenylic acid. Previously (DADD and KLEINJAN, 1977) it was mentioned, but without supporting data, that inosinic acid was an adequate substitute for both adeny!ic and guanylic acids in the original mixture of 5 nucleotides. The present results show that with either inosinic acid or inosine substituted for adenylic acid, development is good, though perhaps marginally inferior. It may be noticed that in the first of the three experiments of Fig. 4, all larvae died without growth in the two treatments using inosinic acid substitutions. This abrupt demise can be accounted to lithium toxicity. Since the preliminary tests of inosinic acid

A@T

@CT

Am

@C@

6Z@T

@@il

Fig. 3. Relative Developmental Indices for groups of C. pipiens reared on synthetic media with bases variously substituted for corresponding nucleotides in .a satisfactory mixture of 3 nucleotides. Three replicate experiments, M, N and 0, were carried out, and bars for the same treatment of each experiment are grouped and lettered in white, to indicate the experiment. Relative Developmental Indices as described for Fig. 2. Additional abbreviations for nucleic acid components are as follows: circled a, circled c and circled t denote adenine. cytosine and thymine, respectively. Other abbreviations as for Fig. 2.

Base nutritional

AGCUT

No nuclexs

requirements

ACT

AUT

of the mosquito

ACT

Au_T

Culur

A@T

pipiens

ICT

IyT

357

LCT

Fig 4. Relative Developmental Indices (defined for Fig. 2) for groups of C. prpiens reared on synthetic media with cytidine. uridine and uracil substituted for corresponding nucleotides and with inosinic acid or inosme substituted for adenylic acid. Bars for the same treatments of three replicate experiments. P. R and S. are grouped and lettered in white to indicate theexperiment. Numbers above bars give the percentages of adults which developed for each treatment. Abbreviations additional to those given for previous Figs are as follows: u and circled u denote uridine and uracil, respectively: I and i denote inosinic acid and inosine respectively.

referred to in DADD and KLEINJAN (1977) a fresh sample of the chemical as the lithium rather than the sodium salt had been obtained, and was used for experiment P, whereas the two subsequent experiments (Q and R) reverted to the use of inosinic acid sodium salt, with consequent good growth. Also previously reported without supporting data was the finding that erotic acid and orotidine, precursors in the generalized scheme for pyrimidine nucleotide biosynthesis (ADAMS et al., 1976), were ineffective as substitutes for uridylic or cytidylic acids (DADD and KLEINJAN,1977). In Fig. 5, the very poor Developmental Indices and low percentages of adults for treatments in which either erotic acid or orotidine were substituted for uridylic or cytidylic acids (the

interchangeable pyrimidine nucleotides) confirms their inutility. The remaining treatments of Fig. 5 deal with the replacement of single ribonucleotides by deoxyribonucleotides, it having been shown previously (DADD and KLEINJAN, 1977) that a mixture of 5 deoxyribonucleotides, though allowing substantial development, was poor compared with the standard mixture of 4 ribonucleotides plus thymidyhc acid. The bar graphs of Fig. 5 show much variability between experiments for treatments with 5’-deoxyadenylic acid substituted for adenylic acid in both the cytidylic acid- and uridylic acid-containing mixtures of 3 nucleotides. In all cases the detailed records show little adverse effect on development up to pupation, but

87

No nucleic5

ACT

AUT

AoT

A@T

dACT

dAUT

AdCT

AdUT

dAdCdUT

Fig. 5. Relative Developmental Indices for groups of C. pipiens reared on synthetic media with erotic acid or orotidine substituted for the pyrimidine ribonucleotide (uridylic or cytidylic acids), and with various deoxyribonucleotides substituted for corresponding ribonucleotides. The groups of three (sometimes two) bars refer to the corresponding treatments of three replicate experiments P. R and S (white lettering). which are part of the same experiments dealt with in Fig. 4. Additional abbreviations: [iii and o denote erotic acid and orotidine respectively; dA. dC and dU denote deoxyadenylic. deoxycytidylic and deoxyoridylic acids respectively.

R. H.

358

whereas 60% or more of larvae pupated for both treatments in all experiments, very few adults emerged for the cytidylic acid-containing treatment of experiment R, or for the uridylic acid-containing treatments of both experiments R and S. Thus, deoxyadenylic acid appears to be utilized to a considerable degree in place of adenylic acid, but with markedly reduced efficiency, which becomes manifest at the end of pupal development as a variable but substantial failure of adult emergence. With either deoxyuridylic or deoxycytidylic acids substituted for the corresponding ribonucleotides (uridylic or cytidylic acids), development was very little better than in the total absence of nucleic acid components, though in all pertinent treatments of all experiments a few individuals pupated and became adults after protracted larval development. Similarly, with a mixture of 4 nucleotides entirely in the deoxy form, development was only slightly better than without nucleic acid components.

DISCUSSION These studies extend and refine a previous investigation (DADD and KLEINJAN, 1977) which showed that the dietary nucleic acid requirement of C. pipiens could be satisfied by adenylic acid (a purine ribonucleotide), thymidylic acid (a pyrimidine deoxyribonucleotide), and either cytidylic or uridylic acid (pyrimidine ribonucleotides). In this earlier study it was mentioned that inosinic acid could effectively replace both adenylic and guanylic acids in the original mixture of 5 nucleotides, but that neither erotic acid nor orotidine could take the place of a pyrimidine ribonucleotide (uridylic or cytidylic acid). These findings are here substantiated on the basis of substitution experiments with a satisfactory mixture of 3 nucleotides. The present work narrows the ultimate requirements of C. pipiens for nucleic acid components to the level of nucleosides. Most larvae developed to adults with all nucleotides replaced by the corresponding nucleosides. and only with the substitution of adenosine for adenylic acid was development somewhat retarded. Base substitutions, on the other hand, were all unsatisfactory. Though many larvae became adult with thymine in place of thymidylic acid, development was very slow, and with adenine, cytosine or uracil substituted for their corresponding nucleotides, most larvae failed to reach pupation. Taking into account that deoxyadenylic acid was an inefficient substitute for adenylic acid, and that deoxycytidylic and deoxyuridylic acids were ineffective substitutes for the corresponding ribonucleotides, the irreducible minimum of nutritionally required nucleic acid components for C. pipiens appears to comprise a purine ribonucleotide (inosinic or adenylic acid), a pyrimidine ribonucleoside (cytidine or uridine) and the pyrimidine deoxyribonucleoside, thymidine. With reference to the generalized biosynthetic pathways discussed in standard texts (e.g. ADAMS et al., 1976), it appears that purine nucleotide synthesis in C. pipiens must be deficient at some point(s) on the pathway to inosinic acid, but with little or no

DADD

impediment to the derivation of all necessary purine riboand deoxyribonucleotides from inosine-5’monophosphate or from inosine itself. Also. on the assumption that orotidine would be readily phosphorylated, pyrimidine nucleotide biosynthesis appears to be blocked at the final step from orotidine5’-monophosphate to uridylic acid, and in any case at least beyond erotic acid. Further, the dietary need for thymidylic acid or thymidine indicates a weak or absent thymidylate synthetase pathway. These restrictions aside, all necessary transformations from uridylic acid to other pyrimidine nucleotides, both ribo and deoxyribo, seem sufficiently active to offer no limitations on rate of development. Bearing in mind that nucleosides were effectively utilized by C. pipiens and that most of the nucleotides used in this work were the 3’(2’)-monophosphates rather than the 5’phosphates which are central to the general scheme for nucleotide transformations, it may be concluded that modifications in nucleotide phosphorylation freely occur. Although nucleic acid bases are not precursors in the main pathways of either purine or pyrimidine nevertheless in many nucleotide biosynthesis, organisms a number of so-called ‘salvage’ pathways allow bases to be incorporated into nucleotides (ADAMS et al., 1976). The ability of several Diptera to utilize certain bases in complete or partial replacement of a nucleic acid requirement must depend upon such salvage pathways, several of which have indeed been documented in Drosophila cell lines (WYSS. 1977). Examples of dietary base utilization are documented for Cochliomyiu hominivorax (GINGRICH. 1964), Musca domestica (BROOKS and FRAENKEL, 1958) and Drosophila melanoguster (HINTON, 1956; SANG. 1957; BURNET and SANG, 1963), though it should be noted that the extensive Drosophila literature records many adverse effects of dietary bases on development (EL KOUNI and MARCH, 1977). By contrast, neither bases nor nucleosides could substitute for nucleotides as partial replacements for the nucleic acid requirement of Agriu ujjinis (HOUSE, 1964). In C. pipiens, salvage mechanisms for adenine, cytosine and uracil appear to be absent or feeble, since these bases were ineffective dietary substitutes for corresponding nucleotides or nucleosides. There may, however, be a weak salvage pathway for thymine, since its substitution for thymidylic acid allowed substantial, though markedly delayed, adult development. Most previous studies with Diptera found that although dietary nucleic acid was necessary for an optimal rate of larval growth it was not essential for development to the adult state (HINTON ef al., 1951: HINTON, 1956,1959; SANG, 1957; HOUSE and BARLOW, 1957; BROOKED and FRAENKEL, 1958; ROYES and ROBERTSON,1964; FALK and NASH. 1972). suggesting that requisite pathways for de nova nucleic acid biosynthesis must be available, but with some so weak as to be rate-limiting in a type of insect notable for exceptionally rapid larval growth. However, dietary nucleic acid (or certain components thereof) is essential for the complete development of C hoininivorux (GINGRICH, 1964) and for some strains and mutants of D. melunoguster (GEER, 1964; NORBY, 1971; VYSE and SANG, 1971; FALK and NASH, 1974; NABUIB and NASH, 1976). Culexpipiens comes into this

Base nutritional

requirements

latter category of Diptera for which certain components of nucleic acid are stringently essential. A pyrimrdine nucleoside is absolutely essential. for little de\elnpment beyond the middle larval stages occurred without either cytidine or uridine. Thymidine and adenylic acid are required for optimal performance, but it is debatable whether they are absolutely essential. since many larvae could eventually develop to adults in the absence of either from the diet, arguing some biosynthetic capability. It is surprising that the requirement for adenylic acid appears more stringent in the presence of dietary guanylic acid. Since this latter IS not required in the diet, being derived from adenylate (or inosinate) that may be present, it would be expected to spare the adenylate requirement. The reverse seemed to be the case, and this might suggest that if there is a limited capability for purine nucleotide biosynthesis, it is inhibited by high levels of one ot its ultimate end products, guanylic acid. Though the work reported here shows that excellent growth of C. pipirns can be achieved with a mixture comprising only adenylic acid, thymidine and either cytidine or uridine. for routine use in subsequent nutritional studies a mixture providing all five bases wax retained. for precautionary reasons. Based on the concentration studies for individually required nucleotides. the composition of the mixture in mg per 100 ml of dietary medium, is as follows: adenylic acid 60: guanylic acid 40: cytidylic acid 25; uridylic acid 25; ;md thymidine IO. Used in diets supplemented with a ilpid factor recently found necessary for adult viability JII~ KI II\J-\\. 1978). this mixture has (DIl)l) proved $atisfdctory for supporting two successive ~enic generations of C’ pipienx

I( k,l,,~~,/[,rly~,r,~~,~~/.\ -It 1sa pleasure to acknowledge the mellcult,uv technical assistance of Ms. J. E. KLEINJAU m the c\pcrlnlental w(lrk.

REFERENCES ALIAMSR

L. P.. BI:KDOK R. H.. CAMPBELL A. M. and S\n L! II R. M. S. ( 1976) The Biochcmistty oj rhe Nuckic .AC/C/,A(Ed. by DAVIDSON). 8th ed. Academic Press. New 1 IIrk BKOO&IS V J. and FRAL\;K~L G. (1958) The nutrition of the I;II-\ rl 01’ the hnuselly. .&lus~~trclr>nrc~.tri~ L. Pl~ysiol. Zoo/. 31, XX-223. BLIKX~TB. and SANG J. H. (1963) Dietary utilizationofDNA and its derivatives by Drosophila mcdanogaster (Meig.). J /I/ ,i’(‘, P/r I..FiO/ 9. 553-561. DSIX) K. H. (1970a) Arthropod nutrition. In Chemid Zoo/o~~ (Ed. by FL(IRRIK M. and SCHEER B. T.). Vol. 5. Academic Press. New York. D~Ix) R. H. (1970b) Comparison of rates of ingestion of particulate sollds by Culr.~ pip/ens larvae: phagostimulant eflcct of water-soluble yeast extract. Enromologia. c.x\p. uppl. 13, 407-409. DAIII) R. H. (1973) Insect nutrition: current developments and merabolic implications. A. Rel,. Ent. 18, 381-420. 1),41)1) R. H (lY75) Ingestion of colloid solutionc by filter-

of the mosquito

C‘U/~,.\ piplens

feeding mosqtuto larvae: reiationshlp

359 to viscosity. .I ‘_yp

Zoo/ 191. 395-406. DADD R. H., GOMEL I. and NAMBA M. (1973) Requirement for ribonucleic acid In a semi-synthetic larval diet for the mosquito Cu1e.v prpirns J med Enr 10. 47-52. DADD R. H. and KLEINJAK J. E. (I 976) Chemically defined dietarq media for larvae of the mosquito CU/~‘.‘I pipicvu (Diptera: Culicidae): Effects of colloid texturlzers. J n~cd Enr 13. 281-791. DADD R. H. and KL~I\JA? J E. (1977) Dtetary nucleotide requirements of the moh~uito. C‘U/~> t p/pi~~.\ .I /n.vcac,! Pl?WiO/ 23, 333-34 1 DADE R H. and KLI,IXJA%J. E. (1Y78) An essential nutrient for the mosquito C&J\- ptpirnt associated with certain animal-derived phosphohpids. .drlrr. (‘,I, .SfJl,. ‘lm. 71, 794-800. FALK D R. and ~\~ASH D. i IY72) The search for auxotrophic mutant5 in Drruophilu nwlunopusrw In III.SC,C,IcrnLl Mirf, Nutririr~rt (Ed. by ROIXUGL:L~ J. C.I. pp. IS-31, North Holland. Amhtcrdam. FALK D R. and NASH D. ( 1974) Sex-hnked auxotrophic and putative auxotrophic mutant\ of Dnwphrlrr mc~ltmoyus~er Gmrtrl~.r 76. 755-766 GEEK B. W. ( lY64) Inheritance of the dIetar) rlbonuclelc acid requlremrnt of Drowphrl~r mc~ltmr~posrcr Gcwrrc~\ 39, 7X7-796. GINCKI( H K. E. (lY6-I) NutrItional studies on screw-worm larvae with chemicalI> defined media. .Inn GUI .SO~, .Im 57, 35 t-360. HINTOK T. (1956) Nucleic acid utllizatlon by Dn~~+rlrr PIIwIoI Zoi~l 29, X-26. HINTO\ T. (lY59) Mi\cellaneous nutrItional variations. environmental and genetic. in Drosoph~ltr .-1nu .Y 1’ .duc/ SC,; 77, 366-372. HINTO~ T.. EUIS J. F. and N~YES D. T. 11951) An adenine requirement m a strain of Drosophila Prt~t, .Ytr/ .lwd su i s .?I 37 , -‘)r?Y+ Hoist’ H. L. (1964) Effect5 of dietic nucleic acids and components on growth of .d~qriodfffrnr.5(Fallen) (Diptera: Sarcophagidae). Gun J Zvul 42, 801-806. HOUSE H L. (1974) Nutrition. In The Phwiolog~~ of In.twrs (Ed. by Rot ~11.11 M.). Vol. 5. 2nd ed. Academic Press. New 1’ork. Ho~sr H. L. and BARLOW J. S. (1957) Effects ofnucleIc acids on larval growth. pupation and adult emergence ol P.~ru~/~~.sur~ophr,~u q/finis (Fall. 1. lVorure. Lord 180, 44. EI. Koc11 M. H. and NASH D. (1977) Survival of Drosophila r,lrlurfr?gcr.,rc,r lar\ ae on deiined medium supplemented with naturally occurrmg nucleosides and nucleic acid bases. J hsrc, Phj,.sid 23, 327-33 I. NORBY S. (I 97 1 I A spcclfic requirement for pyrimidines in of Dro.soph~lu mrlanogastc~r rudimentary mutants Hwrdirru 66. 205-3 I4 NAGLTB F. N. M. and NASH D. (1976) Nucleoslde auxotrophy in Dro.rophi/o: An autosomal locus yielding mutants supplemented by purine and pyrimidine ribonucleosides !\I&(, ,gr,l_ Gcnratrc,s 147, 13-21. Ro’r~s W. V. and RO!XRTSO\ F. W. (1964) The nutritional requirements and growth relations of different species of Drusophila J t’\-p Zoo/ 156, 105-135. SANG J. H. (lY57) Utihzation of dietary puiines and pryimldmeb by Dro.cop/trlu mc~ltrnrrgu.~rw Proc H SK. Edinh. 1B/ 66. 33Y-359. VYSF E. R. and SANG J. H. (lY71) A purlne and pyrlmldme requiring mutant oi Drorophilrr nlcluno,gusrrr Gmer Rcs 18, 117-1’1. Wr’ss C. (lY77) Purme and pyrimidine salvage in a clonal ~rr,.~~,/~lfil~rcell Ilnt‘. .I Itl\<,<,l PhI~.rl~~l. 23, 739-747.