Involvement of the period gene in developmental time-memory: effect of the perShort mutation on phase shifts induced by light pulses delivered to Drosophila larvae.

Phases of circadian locomotor activity rhythms of adult Drosophila reared in constant darkness have been shown to be set by a light stimulus delivered as early as the first-instar larval stage. This implies that a circadian clock functions continuously throughout postembryonic development. The clock genes period (per) and timeless (tim) are expressed cyclically in the larval central nervous system of Drosophila, and daily oscillations of per expression persist throughout metamorphosis in a group of cells, which gives rise to the pacemaker cells underlying locomotor activity rhythms of adults. Therefore, PER and TIM cyclings in these neurons may be responsible for the phenomenon of "larval time-memory." In the absence of any evidence for the involvement of these genes in such a developmental clock, and because circadian-pacemaker functions are underanalyzed in terms of the functions during development, the authors tested the time-memory of a fast-clock period mutant. They show that dark-reared perS mutant individuals as well as wild-type flies can be entrained as larvae and that a brief light pulse given to such entrained larvae can induce phase shifts in animals of either genotype. However, the direction and magnitude of phase shifts were different between wild type and perS, suggesting that a clock under the control of period gene participates in the regulation of developmental time-memory. The authors show that the relevant clock can be entrained by two light input pathways, one involving the phospholipase C encoded by the norpA gene, the other mediated by the blue-light receptor cryptochrome. Phase shifts of molecular oscillations during the larval stage were smaller than those measured by adult behavior, suggesting molecularly transient responses during development.

Molecular and behavioral analysis of four period mutants in Drosophila melanogaster encompassing extreme short, novel long, and unorthodox arrhythmic types.

Of the mutationally defined rhythm genes in Drosophila melanogaster, period (per) has been studied the most. We have molecularly characterized three older per mutants-perT, perClk, and per04-along with a novel long-period one (perSLIH). Each mutant is the result of a single nucleotide change. perT, perClk, and perSLIH are accounted for by amino acid substitutions; per04 is altered at a splice site acceptor and causes aberrant splicing. perSLIH exhibits a long period of 27 hr in constant darkness and entrains to light/dark (L/D) cycles with a later-than-normal evening peak of locomotion. perSLIH males are more rhythmic than females. perSLIH's clock runs faster at higher temperatures and slower at lower ones, exhibiting a temperature-compensation defect opposite to that of perLong. The per-encoded protein (PER) in the perT mutant cycles in L/D with an earlier-than-normal peak; this peak in perSLIH is later than normal, and there was a slight difference in the PER timecourse of males vs. females. PER in per04 was undetectable. Two of these mutations, perSLIH and perClk, lie within regions of PER that have not been studied previously and may define important functional domains of this clock protein.

Effects of Fertilizer and Pesticides on Soybean Growing in Heterodera glycines-infected Soil.

A nematicide, dibromochloropropane; the fungicides benomyl and maneb; an insecticide, oxydisulfoton; the herbicides trifluralin, linuron, and dinoseb; and fertilizers were applied to Heterodera glycines-infested soil. A resistant soybean cultivar alone produced the highest yield in one test, and its yield was not affected by application of pesticides or fertilizer. In two tests the cultivar that was supposed to be resistant was not. Application of nematicide alone resulted in higher yields of the susceptible cultivar, compared with the untreated check, in only one of three tests. Various combinations of pesticides also resulted in higher yields, and in all cases the nematicide was included. Pesticides and fertilizer must be used with discretion on soybean.

Resistance in Commercial Soybean Cultivars to Six Races of Heterodera glycines and to Meloidogyne incognita.

Soybean cultivars grown in pots in the greenhouse were tested for resistance by inoculation with Meloidogyne incognita or one of six races of Heterodera glycines. Selected cultivars were tested against each nematode isolate. The numbers of cultivars tested against each H. glycines race and the numbers resistant and (or) moderately resistant were as follows: Race 2 - 114 tested, 1 resistant and 9 moderately resistant; race 3 - 170 tested, 56 resistant and 17 moderately resistant; race 4 - 89 tested, 1 resistant and 13 moderately resistant; race 5 - 106 tested, 4 moderately resistant; race 6 - 95 tested, 10 resistant and 25 moderately resistant; race 14 - 81 tested, 2 resistant and 10 moderately resistant. No cultivar was resistant to all races. Meloidogyne incognita was tested on 139 cultivars; 50 were resistant.

Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster.

The pero1 and the pers mutations in Drosophila melanogaster, which seem to eliminate or speed up, respectively, the clocks underlying biological rhythmicity, were mapped to single nucleotides. Chimeric DNA fragments consisting of well-defined wild-type plus mutant DNA subsegments were constructed, introduced into flies by germ-line transformation, and assayed for biological activity. These experiments localized both pero1 and pers to a 1.7-kilobase DNA fragment that is mostly coding DNA. Sequencing of this subsegment from each mutant showed that pero1 is completely accounted for by a nonsense mutation in the third coding exon of a 4.5-kilobase RNA transcribed from this locus. The pers mutation is also a single nucleotide substitution, in the fourth coding exon, which results in a serine-to-asparagine substitution in the per gene protein product. The functional significance of these changes is discussed with reference to the phenotypes of the two mutations.

Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: overlapping genomic fragments that restore circadian and ultradian rhythmicity to per0 and per- mutants.

P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.

Morphometric and serologic comparisons of a number of populations of cyst nematodes.

Thirty-five populations of Heterodera glycines and populations of 15 other Heterodera, Globodera, and Punctodera species were studied morphometrically and some were compared serologically. There was a wide range of each measurement within each nematode population. Except for one soybean cyst nematode population from Indiana, which was a tetraploid and considerably larger than the others, morphometric measurements overlapped. In a discriminant function comparison most of the populations were closely grouped but at least three were rather distinctly separated. Morphometrically H. fici, H. cruciferae, H. schachtii, and H. trifolii were closely associated with H. glycines. Serology indicated a close relationship between H. glycines, H. lespedezae, H. trifolii, H. schachtii, and the Heterodera sp. from Rumex, while H. betulae appeared to be more distantly related.

Infra-species Variation in Reactions to Hosts in Heterodera glycines Population.

Eighteen hosts were inoculated with each of four races of Heterodera glycines. A discriminant function analysis of the reactions of these races to these hosts demonstrated that these races could be separated but not consistently. Then 33 H. glycines populations collected from 13 states and five obtained from Japan were tested on differential hosts. The number of variants discriminated within these 38 populations depended on the number of differentials and the rating system used. When five differentials were used with a (+) or (-) rating system there were six "races," but when 13 differentials were used with a (+) or (-) system there were 25 physiological groups. If an index rating system was used there were 36 groups. Apparently H. glycines is a very variable species and delineation of races varies with criteria chosen.

Development of Heterodera glycines pathotypes as affected by soybean cultivars.

The reproductive abilities of four races of Heterodera glycines were compared on soybean cultivars by using single cyst or mass inoculations. Progeny transfers were used to determine changes in reproduction of H. glycines. Reproduction of all races (1, 2, 3, 4) was best on 'Lee' and poorest on P1 88788. The size of females produced and numbers of eggs/female of the different races varied with the cultivar. Races 1 and 3 appeared to contain low populations of Races 2 or 4. Races 2 and 4 were best selected by a series of transfers on 'Pickett' soybean.

The Effect of Temperature and Moisture on the Survival of Heterodera glycines in the Absence of a Host.

Soybean-cyst nematode larvae survived in water up to 630 days, depending on incubation temperature. Most larvae were killed when ice crystals formed in water, and all died after 1 day at 40 C. At temperatures of 0, 4, 8 and 12 C, larvae survived for the duration of the experiments (630 days). From 16 to 36 C, survival was inversely correlated with temperature. In naturally infested soil, nematode survival was similax but more extended and related to moisture level. Larvae survived 7-19 months in flooded soil, 29-38 months in dry soil, and for 90 months in soil maintained near its field capacity.

Life Cycle, Host Range, and Reproduction of Heterodera betulae.

Heterodera betulae, a cyst-forming nematode, originally recovered from roots of river birch in Arkansas, appears to have a limited host range. Of 80 plant species in 20 families tested, only five species of Betula and Cleome spinosa supported reproduction. The minimum time for a complete life cycle was 52 days at 28 C. No reproduction occurred at 31 C or above, and development was very slow below 20 C. Successful population propagations from single larvae demonstrated that males were not necessary for reproduction.