Search

Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. We studied the duration of SP for diapause induction and the females’ ability to enter post-reproductive diapause at short day lengths in Drosophila montana strains from different latitudes in Europe, North America, and Japan. Our study shows that the females of this species have a life-long SP and that they retain an ability to switch between reproduction and diapause as a response to back-and-forth changes in day length for at least 3 months. D. montana strains from different latitudes showed high variation in females’ ability to enter post-reproductive diapause; females of the southern strains generally requiring longer time and/or lower temperature to enter this stage than those of the northern strains. Moreover, the proportion of females that switched to post-reproductive diapause in 3 weeks in short day conditions at 16 °C showed positive correlation with the critical day length (CDL) for diapause induction and the latitudinal and continental origin of the strains. Life-long SP increases females’ flexibility to respond to short-term changes in environmental conditions and enables reproducing females to switch to post-reproductive diapause when the days get shorter and colder toward the autumn. This ability can play a major role in species phenology and should be taken into account in theoretical and empirical studies on insect adaptation to seasonal variation.

Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. We studied the duration of SP for diapause induction and the females' ability to enter post-reproductive diapause at short day lengths in

Insect species with a wide distribution offer a great opportunity to trace latitudinal variation in the photoperiodic regulation of traits important in reproduction and stress tolerances. We measured this variation in the photoperiodic time-measuring system underlying reproductive diapause in Drosophila montana, using a Nanda-Hamner (NH) protocol. None of the study strains showed diel rhythmicity in female diapause proportions under a constant day length (12 h) and varying night lengths in photoperiods ranging from 16 to 84 h at 16°C. In the northernmost strains (above 55°N), nearly all females entered diapause under all photoperiods and about half of them even in continuous darkness, while the females of the southern strains showed high diapause proportions only in the circadian 24 h photoperiod. Significant correlation between the strains’ mean diapause proportions in ≥ 24 h photoperiods and critical day length (CDL; half of the females enter diapause) suggests at least partial causal connection between the traits. Interestingly, females of the northern strains entered diapause even in ≤ 24 h photoperiods, where the night length was shorter than their critical night length (24 h - CDL), but where the females experienced a higher number of Light:Dark cycles than in 24 h photoperiods. NH experiments, performed on the control and selection lines in our previous selection experiment, and completed here, gave similar results and confirmed that selection for shorter, southern-type CDL decreases female diapausing rate in non-circadian photoperiods. Overall, our study shows that D. montana females measure night length quantitatively, that the photoperiodic counter may play a prominent but slightly different role in extra short and extra long photoperiods and that northern strains show high stability against perturbations in the photoperiod length and in the presence of LD cycles. These features are best explained by the quantitative versions of the damped external coincidence model.

Insect species with a wide distribution offer a great opportunity to trace latitudinal variation in the photoperiodic regulation of traits important in reproduction and stress tolerances. We measured this variation in the photoperiodic time-measuring system underlying reproductive diapause in Drosophila montana, using a Nanda-Hamner (NH) protocol. None of the study strains showed diel rhythmicity in female diapause proportions under a constant day length (12 h) and varying night lengths in photoperiods ranging from 16 to 84 h at 16°C. In the northernmost strains (above 55°N), nearly all females entered diapause under all photoperiods and about half of them even in continuous darkness, while the females of the southern strains showed high diapause proportions only in the circadian 24 h photoperiod. Significant correlation between the strains’ mean diapause proportions in ≥ 24 h photoperiods and critical day length (CDL; half of the females enter diapause) suggests at least partial causal connection between the traits. Interestingly, females of the northern strains entered diapause even in ≤ 24 h photoperiods, where the night length was shorter than their critical night length (24 h - CDL), but where the females experienced a higher number of Light:Dark cycles than in 24 h photoperiods. NH experiments, performed on the control and selection lines in our previous selection experiment, and completed here, gave similar results and confirmed that selection for shorter, southern-type CDL decreases female diapausing rate in non-circadian photoperiods. Overall, our study shows that D. montana females measure night length quantitatively, that the photoperiodic counter may play a prominent but slightly different role in extra short and extra long photoperiods and that northern strains show high stability against perturbations in the photoperiod length and in the presence of LD cycles. These features are best explained by the quantitative versions of the damped external coincidence model.

Reproductive diapause is a primary mechanism used by arthropods to synchronize their life cycle with seasonal changes in temperate regions. Our study species, Drosophila montana, represents the northern insect species where flies enter reproductive diapause under short day conditions and where the precise timing of diapause is crucial for both survival and offspring production. We have studied clinal variation in the critical day length for female diapause induction (CDL) and their overall susceptibility to enter diapause (diapause incidence), as well as the temperature sensitivity of these traits. The study was performed using multiple strains from four latitudinal clines of the species – short clines in Finland and Alaska and long clines in the Rocky Mountains and the western coast of North America – and from one population in Kamchatka, Russia. CDL showed strong latitudinal clines on both continents, decreasing by one hour per five degrees decline in latitude, on average. CDL also decreased in all populations along with an increase in fly rearing temperature postponing the diapause to later calendar time, the effects of temperature being stronger in southern than in northern population. Female diapause incidence was close to 100% under short day/low temperature conditions in all populations, but decreased below 50% even under short days in 19°C in the southern North American western coast populations and in 22°C in most populations. Comparing a diversity of climatic data for the studied populations showed that while CDL is under a tight photoperiodic regulation linked with latitude, its length depends also on climatic factors determining the growing season length. Overall, the study deepens our understanding of how spatial and environmental parameters affect the seasonal timing of an important biological event, reproductive diapause and helps to estimate the evolutionary potential of insect populations to survive in changing climatic conditions. peerReviewed

The work has been supported by the Academyof Finland to A.H. (project 267244) and Natural Environment Research Council (NERC) funding (NE/J020818/1 to M.G.R.; NE/L501852/1 to R.A.W.W.). The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity. Publisher PDF

Enzyme gene variability has been studied by starch gel electrophoresis in 20 Finnish vendace (Coregonus albula) populations. Eighteen populations originate from fresh water and two from brackish water. Fourteen populations spawned in the time typical for the species, i.e. in the autumn, four in the winter and two in the spring. Genotype-environment interactions were studied with regard to several hydrological and spatial variables. Genetic polymorphism in vendace is relatively high for a fish. About half of the enzyme loci studied were polymorphic and the mean heterozygosity per locus per individual is 0.08. The degree of polymorphism is related to the metabolic function of enzymes. The average genetic distance between populations, based on five polymorphic loci, is 0.036 (ranging from 0.001 to 0.245) and 0.016 based on all loci studied. The genetic distances indicate that the winter- and spring-spawning populations are the most differentiated. They differ also very much from each other. This indicates that each one of them has arisen independently from populations spawning in autumn. The change in spawning time can be attributed to selection operating on zygotes and juveniles. This selection is due to adverse winter oxygen conditions associated with dystrophy and warm water. The genetic differentiation observed between populations may be due to natural selection or genetic drift or both.

The genic variability at twenty loci in marginal, submarginal and central populations of Drosophila subobscura has been studied by starch gel electrophoresis. The samples from marginal populations orginate from Finland, the submarginal ones from Sweden and Denmark, whereas flies collected from France and northeastern Italy are taken to represent central populations. The average degrees of genic polymorphism are undifferentiated over the distribution area of D. subobscura. Most loci have fairly uniform allele frequencies in all populations. Local differences have been found at six loci. These fluctuations represent adaptations to local environmental differences.

Drosophila littoralis is a latitudinally widespread European species of the Drosophila virilis group. The species has ample genetic variation in photoperiodism (adult diapause) and circadian rhythmicity (pupal eclosion rhythm), with adaptive latitudinal clines in both of them. The possible common genetic basis between the variability of photoperiodism and circadian rhythms was studied by a long-term crossing experiment. A northern strain (65 °N) having long critical day length (CDL = 19.9 h) for diapause, early phase of the entrained rhythm in LD 3:21 (ψLD3:21= 12.3 h), and short period (τ= 18.8 h) of the free-running rhythm for the eclosion rhythm was crossed with a southern strain (42 °N) having short CDL (12.4 h), late eclosion phase (ψLD3:21= 20.2 h), and long period (τ= 22.8 h). After 54 generations, including free recombination, artificial selection, and genetic drift, a novel strain resulted, having even more “southern” diapause and more “northern” eclosion rhythm characteristics than found in any of the geographical strains. The observed complete separation of eclosion rhythm characteristics from photoperiodism is a new finding in D. littoralis; in earlier studies followed for 16 generations, the changes had been mostly parallel. Evidently, the genes controlling the variability of the eclosion rhythm and photoperiodism in D. littoralis are different but closely linked. To test for the possible gene loci underlying the observed geographical variability, the period gene was studied in 10 strains covering all the known clock variability in D. littoralis. The authors sequenced the most suspected Thr-Gly region, which is known to take part in the adaptive clock variability in Drosophila melanogaster. No coding differences were found in the strains, showing that this region is not included in the adaptive clock variability in D. littoralis.

The Aethes rubigana complex is shown to comprise four closely related species in the Palearctic region: Eurasian Aethes cnicana (Westwood, 1854), European A. rubigana (Treitschke, 1830), Asiatic A. citreoflava V. I. Kuznetsov, 1966, and A. kyrkii, here described as a new species from northern Finland. A. kyrkii is morphologically most close to A. cnicana, but enzyme electrophoresis revealed complete genetic isolation between them. Descriptions are given of the three species occurring in Europe, and their respective biologies are outlined. Statistical analyses of traits of the forewing are given. The taxonomic status of A. arcticana (Brandt, 1937) is revised.

Pupal eclosion rhythms were studied in seven strains of C. costata originating between latitudes 43°N in Japan to 69°N in Finland. The strains represented the following types of diapause: larval obligatory, larval photoperiodic (critical daylengths from 14 h in the south to 21 h in the north), and no photoperiodic diapause. The eclosion rhythmicity of all strains was weak in all diel photoperiods. The free-running rhythms of eclosion were also of low amplitude. Three other species of Chymomyza were studied for comparison: Chymomyza distincta and C. fuscimana with a larval diapause and C. caudatula with an adult diapause. Only C. caudatula had the rhythmic eclosion that drosophilids have in general. We conclude that the exceptionally weak eclosion rhythmicity in C. costata is an adaptation for eclosion at any permissive time of day around midsummer. The weak rhythmicity of eclosion in C. costata would thus the phenotypic plasticity, an adaptation to the exceptional phenological timing of the species. There was no parallel variation between circadian eclosion rhythms and photoperiodism.

PCR screening with primers specific for the T-, M-, and O-type P element subfamilies was performed to investigate the interspecific distribution in 18 species and to reconstruct the phylogenetic history of the various types within the obscura species group. T-type elements occur in D. ambigua, D. tristis, D. obscura, D. subsilvestris, and D. eskoi. In the genomes of D. subobscura, D. madeirensis, and D. guanche they are present in the form of terminally truncated T-type derivatives. The wide distribution suggests that the T-type subfamily had a long evolutionary history in the obscura lineage. In contrast, the patchy occurrence of M- and O-type elements can be ascribed to four independent events of horizontal invasion of different lineages. The cladogenesis of the obscura group was investigated using a partial sequence of the Adh gene as a marker. In contrast to earlier findings, the position of D. eskoi had to be revised. D. eskoi appears as the closest relative of the D. ambigua clade, whereas D. tsukubaensis is the sister taxon of the species pair D. bifasciata/D. imaii. This result is in good accordance with the P element data, where high sequence similarity (95%) was found among the T-type elements of D. eskoi and those of D. ambigua and D. tristis.

The adult eclosion rhythms of two Japanese strains of Chymomyza costata were studied in diel thermoperiods of different duration and at different average temperatures. One of the strains had the 'wild-type' photoperiodic larval diapause and the other was a mutant strain lacking the photoperiodic response. At constant temperatures the wild-type strain had weakly rhythmic eclosion in diel photoperiods while the mutant strain was arrhythmic. Free-running rhythms could scarcely be observed at all. The amplitude of the rhythm of both strains was much higher in diel temperature cycles than in corresponding light-dark cycles, and generally higher in continuous darkness than in continuous light. When the average temperature under entraining conditions was lowered, the rhythmicity increased and the median of the eclosion peak was displaced to later hours in the entraining cycle. Both strains were rhythmic at the lowest temperatures, i.e. near 10 degrees C. At low temperatures the majority of the eclosions occurred during the high temperature phase or light phase of the entraining cycle. Although the rhythm started well in the entraining temperature cycles, the subsequent free-running rhythm in constant conditions lasted for only 2-3 days. We concluded that the exceptionally weak rhythmicity of eclosions and the relative importance of temperature cues are adaptive traits which make it possible for this northern species to respond directly to favourable but unpredictable changes in its environment. Copyright 1997 Published by Elsevier Science Ltd. All rights reserved

Reproductive diapause, and its correct timing, plays an important role in the life cycle of many insect species living in a seasonally varying environment at high latitudes. In the present paper we have documented variation in the critical day length (CDL) for adult reproductive diapause and the steepness of photoperiodic response curves (PPRCs) in seven clinal populations of Drosophila montana in Finland between the latitudes 61 and 67°N, paying special attention to variation in these traits within and between cline populations. The isofemale lines representing these populations showed a sharp transition from 0% to 100% in females' diapause incidence in the shortening day lengths, indicated by steep PPRCs. The mean CDL showed a clear latitudinal cline decreasing by 1.6h from North to South regardless of the age of the lines, variation within the populations (i.e. among lines) in this trait being up to 3h. The steepness of the PPRCs correlated with the age of the line and this trait showed no clear latitudinal cline. Further studies on a large number of lines from one D. montana population confirmed that while maintaining the flies in diapause preventing conditions in the laboratory has no effect on CDL, older lines had steeper PPRCs. High variation in CDL within and between D. montana cline populations is likely to be heritable and provide a good potential for the evolution of photoperiodic responses. Information on genetic variation in life-history traits, such as diapause, is of utmost importance for predicting the ability of insects to survive in seasonally changing environmental conditions and to respond to long term changes in the length of the growing period e.g. by postponing the timing of diapause towards shorter day length and later calendar date.

The frequency of multiple insemination was studied in a boreal Drosophila montana population using mother-offspring data for a sex-linked allozyme locus. Mating with respect to the marker studied was random. In crowded laboratory cultures the heterozygous offspring had higher viability than homozygotes, but no deviations from Hardy-Weinberg proportions were found in natural populations. In multiply sired progenies the males did not contribute equally to the progeny, but the proportion sired by the second male was 0.76. The estimated frequency of multiple inseminated females was 1.19 +/- 0.31, indicating that practically all the females carried sperm of at least two males. The estimate is the largest ever reported in natural populations of Drosophila. However, the interspecific comparisons may not be relevant, because the frequency of multiple insemination does not necessarily reflect the real lifetime frequency of multiple matings. The effects of local ecology and life history characters, e.g., the uniform age structure and the temporal patterning of matings, on the high degree of detected multiple inseminations are discussed.

Populations of Drosophila littoralis are known to be latitudinally highly variable in photoperiodic adult diapause and pupal eclosion rhythm. Phenotypic correlations between the two time-measuring systems among the strains from different latitudes are, however, weak. In the present study, two differing strains were crossed reciprocally in order to search for causal (genetic) correlations between the two traits in the strains. Segregation in the F2 generations showed that variation in each trait was based on a few variable loci only. In the F2, flies having different eclosion times also differed in their diapause. This association was not complete and could have been due to genetic linkage between the traits. For that reason, the hybrid generations were raised for eight generations more to allow recombination between the traits. In F8, selection against diapause was started in the lines by raising them in a light-dark cycle of 15:9, where only females of the southern type reproduce. After eight selected generations, the lines were studied for the traits. Diapause was completely of the southern type, and the eclosion rhythm had also changed in parallel. The change in the phase of the free-running rhythm was not complete. From the present experiment, and from earlier knowledge of the geographical variation in D. littoralis, I conclude that the same pacemaker that is seen in the eclosion rhythm could also participate in daylength measurement for diapause. However, there are also noncorrelated variable parts in the measuring systems of both traits, which may mask the correlated variation.

Lakovaara, S. (Department of Genetics, University of Oulu, SF-90100 Oulu 10, Finland), Saura, A., Lankinen, P., Pohjola, Liisa and Lokki, J. The use of isoenzymes in tracing evolution and in classifying Drosophilidae. Zool. Scr. 5 (3–4): 173–179, 1976.—Studies on the genetic constitution of natural populations of organisms have shown that these populations are extensively polymorphic at gene loci coding for enzymes. Much of this polymorphism detected by electrophoresing enzyme proteins is, however, not useful in classifying organisms, even though in general species differ from each other more than populations within a species. Good diagnostic characters are monomorphic species-specific genes and their products. Finding these characters requires a thorough study of many populations of the species being studied. Once these characters have been found they can be used in assigning single individuals into different taxa. Electrophoresis of enzymes and allozyme differences between species can also be used in tracing the evolution of organisms. On the basis of allozymes a new phylogeny is proposed for the Drosophila obscura species group. Traditionally this group has been divided into two subgroups, obscura and affinis. Genetic characters divide this species group into three evolutionary lineages. The first lineage comprises the Eurasian obscura subgroup, the second the American affinis subgroup and the third the American obscura subgroup. D. alpina and D, helvetica, which belong to the obscura and affinis subgroups, respectively, are not appreciably related to any other species.

The genetic variability at 20 enzyme loci in natural populations of Otiorrhynchus salicis Ström was studied by starch gel electrophoresis. Altogether 135 weevils were analyzed. The samples originated from a diploid bisexual population in Austria, from four triploid parthenogenetic populations in the Carpathian mountains, and from three triploid parthenogenetic populations in central Sweden. Altogether 16 different genotypes were found in triploid parthenogenetic populations. Two major types, comprising 39 out of the 76 parthenogenetic individuals, occur both in Scandinavia and in central Europe. The less frequent types can be derived from these through mutations. O. salicis is a flightless insect, which has been assumed to have overwintered the Würm glaciation in icefree refugia in Scandinavia. The overall genetic similarity found in the material suggests that the parthenogenetic race spread to its isolated Scandinavian area in postglacial times.

The time measuring system ofDrosophila littoralis strains originating between 40–70° N was found to be highly variable and latitude dependent. The critical daylength for photoperiodic adult diapause varied from 12 h or no diapause response in the south to 20 h in north. The median timing of pupal eclosion rhythm varied correspondingly from 21 h to 12 h from lights off in LD 3∶21, and the period of free-running rhythm of eclosion from 24 h to 19 h. The phase of the free-running rhythm was also variable, and correlated with the phase of the entrained rhythm. Latitudinal variation in the entrained rhythm of eclosion and in diapause is adaptive, leading to eclosion early in the morning and to overwintering at the adult stage. In some strains with a late phase of eclosion, strong transient cycles were seen following the transition from LL to DD. A total damping of the free-running eclosion rhythm within 2–7 days was common to all strains. This damping was more pronounced in the northern strains. The phase and period of eclosion rhythms were statistically independent. Diapause was not correlated with any parameters of the eclosion rhythm in the analysis. Diapause may still be influenced by the period of the eclosion rhythm, even though its minor contribution may be masked by a more variable, eclosion rhythm independent system in the determination of diapause.

The approximate amount of variability at the gene level has now been known for about ten years. This knowledge is mainly based on the technique of gel electrophoresis of proteins and of enzymes in particular. It has been difficult, however, to demonstrate an unequivocal correlation between the physiological function of an enzyme and its elec-trophoretically detectable variants. Once a physiological difference has been established, it may be assumed to have an influence on the adaptive norm of an individual. There are, however, numerous difficulties with this approach. E.g. the substitution of a single amino acid for another may be thought to have an infinitesimally small effect on the total function of the enzyme molecule and the fitness of its bearer.

SUMMARYThe genetic variability at 16 enzyme loci in 52 Scandinavian triploid parthenogenetic populations of the beetleAdoxus obscurushas been studied by starch-gel electrophoresis. The overall genotypes of different beetles have been compared with each other and with a sample from a diploid bisexual population from Canada. Eighty per cent of the parthenogenetic beetles have the same overall genotype. The remainder belong to six genotypes, three of which are found in only one population. The variability within and between parthenogenetic populations ofA. obscurusis much lower than in other comparable parthenogenetic insects studied by us. This is interpreted to be a consequence of the efficient migration of the genotypes with the highest fitness, leading to a replacement of less-adapted genotypes.A. obscurusis a flying insect, whereas the other parthenogenetic insects studied by us are sluggish flightless forms.

The study of enzyme polymorphism in natural populations of different species of Drosophila has lately been one of the main subjects of population genetics (Ayala and Powell, 1972; Lakovaara and Saura, 1971; Lewontin and Hubby, 1966; O'Brien and MacIntyre, 1969; Prakash, 1973a, b; Richmond, 1972; Saura, 1974; Saura et al., 1973; Yang et al., 1972; and others). These Drosophila studies constitute by far the most extensive data now available on the genic polymorphism in a single genus. The general result of the studies on the genic polymorphism in natural populations of Drosophila has been the detection of a rather constant degree of heterozygosity and slightly fluctuating allele frequencies from population to population of a given species. Most investigators doing experimental work on natural populations hold that the electrophoretic variants detected by them are subject to natural selection, i.e. the polymorphisms at enzyme loci are maintained by different forms of balancing selection (e.g. the studies cited above). Gillespie and Kojima (1968) and Kojima et al. (1970) first suggested that physiological function of enzymes may be correlated with the degree of polymorphism at the corresponding loci. The argument is important-in fact, it may well become far more important and straightforward in proving or disproving the significance of selection than the often frustrating efforts to prove conclusively anything concerning e.g. the effects of migration and environment on the genetic makeup of populations. The hypothesis of Gillespie and Kojima states that glucose metabolizing enzymes are in general less variable than other enzymes. This seems to hold true for Droso phila, but it need not be universally true. Data from vertebrates and other insects do not always agree (Frydenberg and Simonsen, 1973; Lokki et al., 1975). Pooling of data froni different organisms may easily lead to confusing results. In evolutionary studies of proteins, gel electrophoresis has a single advantage over the more accurate amino acid sequencing. By electrophoresis it is possible to observe with little effort the variability in thousands of individuals. In this study we utilize data collected by screening hundreds of thousands of flies belonging to the family Drosophilidae for electrophoretically detectable variants of a-glycerophosphate dehydrogenase. This enzyme (a-GPDH, L-glycerol-3-phosphate NAD oxidoreductase, E C 1.1.1.8.) is coded by the aglycerophosphate dehydrogenase (aGPDH) locus. The most important function of aGPDH in the metabolism of an adult fly is its function in the flight muscle, where the soluble, NAD-dependent enzyme together with a mitochondrial, particle-bound and flavine-linked oxidase comprises the so-called a-glycerophosphate cycle (e.g. Hansford and Sacktor, 1971). This cycle produces energy for flight through electron transfer in the mitochondria. The two other functions of a-GPDH are maintenance of the balance between oxidized and reduced NAD in the cytoplasm for glycolysis, and supplying glycerophosphate for synthesis of lipids (e.g. O'Brien and MacIntyre, 1972a, b).