Occurrence of retinal and 3-hydroxyretinal in a possible photoreceptor of the silkworm brain involved in photoperiodism

Summary Deficiency of dietary carotenoid and vitamin A caused an absence of photoperiodic response of diapause induction in the silkworm,Bombyx mori, and an addition of vitamin A to the diet restored the response. By high pressure liquid chromatography (HPLC) a possible photoperiodic receptor of the silkworm brain was found to contain both retinal and 3-hydroxyretinal which are chromophores of insect visual pigments. These pieces of evidence suggest that a retinoid protein might function in the photoperiodic response of the silkworm.     

Threshold and quantitative photoperiodic responses exist in an insect

The photoperiodic response for the induction of adult diapause and that for the determination of nymphal body coloration were compared inPlautia stali Scott (Heteroptera: Pentatomidae). The former was an all-or-none response with a distinct threshold, but the latter was a graded response which varied quantitatively with the photophase duration. The photoperiodic clock in this species is discussed.     

Changes in patterns of ecdysteroid secretion by the ring gland ofDrosophila in relation to the sterol composition of the diet

Summary Brain-ring glands fromDrosophila larvae reared on a defined diet containing campesterol (24-methyl-cholesterol) as the major sterol, secreted-in addition to ecdysone-a compound identified previously as a 24-methyl analogue, 20-deoxy-makisterone A. Using ergosterol or cholesterol as the sterol component of the diet, only ecdysone was detectable in cultures of brain-ring glands.     

Circadian rhythmicity is involved in photoperiodic time measurement in the aphidMegoura viciae

The photoperiodic clock in the vetch aphidMegoura viciae is generally accepted to be based on a non-circadian mechanism or hourglass, as no evidence has been found for the involvement of the circadian system in the photoperiodic response. By using a recently-devised protocol which discriminates between single and repeated night length measurement, we demonstrate here that long-night measurement inMegoura is executed in a repetitive way, and thus that its photoperiodic clock is based on a circadian oscillator after all. However, it is also apparent that the determination of short nights is not repetitive.Dedicated to our dear friend, the late Professor A. D. (tony) Lees who, sadly, died before we had a chance to discuss the data reported here.     

Two mechanisms in the biological clock ofPieris brassicae L.: an oscillator for diapause induction; an hour-glass for diapause termination

Summary Resonance experiments for photoperiodic termination of pupal diapause demonstrated thatPieris brassicae uses a night-measuring hour-glass mechanism. In previous work the same resonance technique for diapause induction revealed that photoperiodic time-measurement is a function of the circadian system. For the first time in a living organism it has been shown that the biological clock operates by means of an oscillator for photoperiodic onset of a phenomenon and according to an hour-glass system for photoperiodic termination.     

Cryopreservation of parasites

Summary In this review, advances in cryopreservation of helminth parasites are reported. Our own studies demonstrate that metacestodes ofEchinococcus multilocularis can be maintained in a viable state for at least 1–2 years by appropriate deep-freezing and storage in liquid nitrogen. Infective larvae of the nematodeToxocara canis cryopreserved for 1 week in liquid nitrogen were maintained after thawing in vitro in a chemically defined medium for 35 weeks. Although motility of previously deep-frozen larvae was reduced they produced secretory/excretory antigens of similar immunodiagnostic quality as those from unfrozen larvae. Whereas infective larvae of several species of trichostrongylids can be easily cryopreserved, the infective larvae of the cattle lungworm,Dictyocaulus viviparus, and muscle larvae ofTrichinella spiralis are more sensitive to damage by subzero temperatures. Therefore, survival rates after cryopreservation are low, but improvement of the cooling schedules appears to be feasible. It is concluded that cryopreservation of certain stages of helminth and protozoan parasites is a useful technique for long-term storage of defined isolates, which can contribute considerably to reducing the number of experimental animals usually required for serial passages.     

Primitive actimoterigian fishes can synthesize ascorbic acid

Amphibians and reptiles evolved with the capacity to synthesize ascorbic acid. Some higher vertebrates, like bats, guinea pigs, primates, and humans have lost the microsomal enzyme gulonolactone oxidase, and in cases of ascorbic acid deficiency suffer from symptoms of scurvy. The question of whether the capacity to synthesize ascorbate is also present in lower vertebrates could throw light on the evolution of this pathway. In order to find out whether ascorbic acid synthesis took place in two primitive Actinopterigian fish, the paddlefish (Polydon spathula) and the white sturgeon (Acipenser transmontanus) were fed with a scorbutogenic diet or diet(s) supplemented with a graded level of ascorbic acid. We found no growth depression nor external symptoms of scurvy, which would be pronounced in modern bony fishes (Teleostei) under similar conditions. The tissue level of ascorbate in both these primitive species indicated that vitamin C in intestine and liver is not depleted when fed a scorbutogenic diet. Gulonolactone oxidase activity was found in the kineys of the Actinopterigian fishes. Thus, I question the accepted evolutionary pathway for ascorbic acid biosynthesis in lower vertebrates and suggest that the modern bony fishes,Teleostei, lost their ability to express the gulonolactone oxidase genes after they had separated during the Silurian from their common ancestor with the coelacanths (Latimeria) and Dipnoi.     

Ecdysis inhibition inAcrolepiopsis assectella larvae by digitonin: antagonistic effects of cholesterol

Summary Digitonin has a toxic effect onAcrolepiopsis assectella, inhibiting development and molting of young larvae fed on a semi-synthetic diet. This effect depends on concentration. It does not occur when cholesterol is added to the diet.Acknowledgment. We thank Mme Annie Heinzmann for technical assistance.     

Induction of cyst formation by low temperature in the dinoflagellateGonyaulax polyedra Stein: Dependence on circadian phase and requirement of light

Encystment, which at a temperature of 15°C is photoperiodically controlled inGonyaulax polyedra, can also be induced by a decrease of temperature, from 20 to 10 or 8°C in the absence of photoperiodic signals. The cyst-inducing capacity of the decrease in temperature depends on the circadian phase: in constant light, the maximum of sensitivity was found at the beginning of subjective night. In a light/dark cycle, however, cyst formation was reduced during dark phase, indicating that light is required for the process of encystment. A similar light dependence was seen in the effect of the physiologically occurring cyst inducer 5-methoxytryptamine, but not in the encystment response to the protonophores monensin and nigericin.     

Origin and identification of bacteria which produce kairomones in the frass ofAcrolepiopsis assectella (Lep., Hyponomeutoidea)

The volatiles used by the parasitoidDiadromus pulchellus to find its host, the leek moth, are produced by the bacteria developing in the frass of the host larvae. The origin and the nature of these bacteria were investigated. Samples were taken from healthy leeks and from infested leeks in the field, as well as from the frass of larvae reared in the laboratory either on the host plant or on an artificial diet. The various species of bacteria identified were cultured in the presence of precursors of leek sulphur volatiles and their volatile emissions were analysed.Klebsiella oxytoca and variousBacillus, common decomposers of plant matter, were the principal species producing active volatiles which were alkyl disulphides.     

Brain factor fromGalleria mellonella (Lepidoptera) stimulating silk gland activity

Brain extracts from day 1–4 last instar larvae ofGalleria mellonella (Lepidoptera) stimulate RNA synthesis in cultured silk glands from day 3 last instar larvae. When the fibroin-synthesizing posterior parts of silk glands were incubated for 3 h in vitro in the presence of brain extract (0.1 brain equivalent), [³H]-uridine incorporation into RNA was stimulated more than twofold. The stimulating effect of brain extract showed a dose response relationship. It is suggested that the heat-resistant and protease-sensitive brain factor is a peptide.     

Detection of protease inhibitors in the hemolymph of resistantAnticarsia gemmatalis which are inhibitory to the entomopathogenic fungus,Nomuraea rileyi

Summary A complex of protease inhibitor activities has been detected in the hemolymph of the 6th instarAnticarsia gemmatalis larvae that are resistant to infection by the fungusNomuraea rileyi. A site-specific serine protease inhibitor extracted fromA. gemmatalis hemolymph inhibits both the germination ofN. rileyi conidia and subsequent germ tube development.     

Mixed life cycles in the transitional zone between voltinisms in the fall webworm,Hyphantria cunea

The fall webworm,Hyphantria cunea Drury, was introduced from North America into Japan at half a century ago. At present, the populations north of 36° N are bivoltine and those south of 36° N are trivoltine. In the present study, the life cycle in the transitional zone between voltinisms was clarified in Tsukuba (36.1° N). When fourth instar larvae of the second generation were collected in the field and maintained in the laboratory, the incidence of pupal diapause increased when the sampling date was delayed from 16 August to 5 September. The fact that some developed without diapause inidcates the occurrence of a trivoltine life cycle in this locality, where a bivoltine life cycle is predominant. The critical photoperiod for diapause induction at 20 and 25°C was 14 h 13 min and 14 h 10 min, respectively. The photoperiodic response may explain the rapid increase in diapause incidence in late summer. In laboratory-reared diapause pupae, the time and period of chilling (5°C) greatly influenced the time required for adult emergence at the final incubation temperature of 25°C. However, diapause pupae chilled for a sufficiently long period developed to adulthood rapidly, irrespective of the conditions before chilling. Therefore, the difference in timing of entering diapause between pupae of the second and third generations would not result in their temporal reproductive isolation in the following spring. It is concluded that the mixed voltinism in Tsukuba is not a result of a mixture of genetically distinct bivoltine and trivoltine populations, but a result of the phenotypic plasticity in a genetically rather homogeneous population.     

Chronic mercury vapor poisoning of aphids

Summary Exposure of green peach aphids,Myzus persicae (Sulz.), to an atmosphere containing mercury vapor resulted in a curtailment ofembryogenesis and larviposition by adults, and in the development by larvae and adults of a cuticular darkening of their legs, head capsule, antennae, cornicles and cauda. Mortality of affected larvae resulted from molting difficulties, particularly by last-instar alatiform female and male larvae. Greenbugs,Schizaphis graminum (Rond.), and pea aphids,Acyrthosiphon pisum (Harr.), responded to mercury vapor exposure in similar ways.     

Cyclohexadienones-insect growth inhibitors from the foliar surface and tissue extracts ofSenecio cannabifolius

Dewaxed leaf surface extracts of 12 plants from Hokkaido, prepared by dipping fresh leaves in chloroform for 3 min, were used in a choice leaf-disk bioassay against larvae of the tobacco cutwormSpodoptera litura. Activity was found only in the extract ofSenecio cannabifolius, a very successful weed in Hokkaido. Individual fractions of the extract, however, were not active. Incorporation of the individual fractions of the surface extracts as well as fractions of the methanolic extracts of the leaf residue into an artificial diet fed to neonateS. litura led to the isolation of ethyl (1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl) acetate, the major surface compound, as the active principle. This compound was also present in the methanolic extract of the leaf residue together with methyl (1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl) acetate, which had the same growth inhibitory effect on the larvae. The presence of these compounds in the foliar surface and tissue suggests a defensive role against herbivores.     

In vitro metabolism of the trichothecene 4-monoacetoxyscirpenol by fungus- and non-fungus-feeding insects

Summary The metabolism of the trichothecene 4-monoacetoxyscirpenol by intact gut tissue was determined in the fungus-feeding Nitidulid,Carpophilus hemipterus (L.) and the non-fungus-feeding caterpillars, the fall armyworm,Spodoptera frugiperda (J. E. Smith) and the corn earworm,Heliothis zea (Boddie). The primary metabolite was the hydrolysis product scirpentriol. The amount of metabolism by theC. hemipterus larvae was ca 10 times that of the caterpillars on a per mg protein basis, suggesting metabolic adaptation for feeding on fungi that may contain mycotoxins.Acknowledgment. The authors wish to thank S. Taylor for technical assistance.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Detection of toxic metabolites in the hemolymph ofBeauveria bassiana infectedSpodoptera exigua larvae

Highly active metabolites have been detected in the hemolymph of the lepidopteranSpodoptera exigua infected with the mycopathogen,Beauveria bassiana. A combination of phenyl sepharose and CM ion exchange chromatography was utilized to extract the active metabolites from infected hemolymph samples. The active in vivo metabolites, having a molecular mass greater than 10 KDa, were thermolabile and were inactivated by proteinase K. These metabolites were characterized by their ability to disrupt metamorphosis, killing treated larvae at the wandering or pupal stage. Additionally, injection ofS. exigua larvae with active samples caused a reduction in the number of filopodial-producing hemocytes. The biological activities and biochemical properties suggest that novel compounds are produced duringB. bassiana mycosis.     

Spermatogenesis is extraordinarily accelerated in metamorphosis-arrested larvae of a salamander,Hynobius retardatus

Laboratory experiments were conducted to induce neoteny inHynobius retardatus, which had been reported to propagate in larval forms like axolotl. A large number of newly hatched larvae were reared in an aqueous solution of thiourea (TU) and sodium perchlorate (SPC) in order to arrest the metamorphosis. Gonadal development in the metamophosis-arrested larvae was compared with that in normally metamorphosing and metamorphosed controls. Metamorphosis-arrested male larvae produced morphologically mature spermatozoa approximately 4 months after hatching, when the gonads in the controls began to differentiate into testes, or to show the premeiotic proliferation of germ cells. Possible endocrine controls of these phenomena are discussed.     

Analysis of the relationship between age of larvae at mutagen treatment and frequency and size of spots in the wing somatic mutation and recombination test inDrosophila melanogaster

The relationship between the induction of mutant clones and the time of mutagen treatment was studied in the somatic mutation and recombination test (SMART) in wing cells ofDrosophila melanogaster. Larvae trans-heterozygous for the recessive marker mutations multiple wing hairs (mwh) and flare (flr) were produced. Batches of these larvae were then treated with mutagen at different ages spanning all three larval instars. Methyl methanesulfonate was fed acutely for 2 h by immersing the larvae in a solution of the mutagen mixed with powdered cellulose. Wings of the surviving adult flies were mounted and scored for the presence of spots. The frequency and size of single and twin spots were recorded separately. Twin spots are produced exclusively by mitotic recombination, whereas single spots can results from various types of mutational and exchange events. There exists a clear correlation between time of induction and frequency as well as size of the single spots. In young larvae only few but very large spots are induced, whereas in older larvae the frequencies are considerably increased but the sizes are smaller. The twin spots show a different relationship. Practically no twin spots are found in very young and in very old larvae. The results demonstrate that in the wing spot test the optimal age of the larvae for mutagen treatment is 72 h.     

Thermoperiodically induced diapause in a mite in constant darkness is vitamin A dependent

Summary By rearing the predacious miteAmblyseius potentillae in a daily temperature cycle in constant darkness it could be shown that diapause may be thermoperiodically induced. When the same experiments were performed using diets without vitamin A it appeared that vitamin A is necessary to achieve a state of reproductive diapause in this mite.2 February 1987