Photoperiodic induction of diapause in an insect is vitamin A dependent

Summary A photoperiodic response was found to be absent in larvae of the parasitoid waspApanteles glomeratus when its host (caterpillars ofPieris brassicae) was reared on a low-carotenoid artificial diet. Addition of vitamin A to the host's diet restored the response to short-day photoperiods in the wasp larvae, thus showing that vitamin A is essential for photoperiodic induction of diapause. Possibly vitamin A or a derivative of vitamin A functions as the photoreceptor pigment for the photoperiodic reaction in this species of insect.     

Endocrine-dependent expression of circadian clock genes in insects

Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tissue of an insect model species, the linden bug Pyrrhocoris apterus, are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the corpus allatum, an endocrine gland producing juvenile hormone. Our results provide the first experimental evidence for the effect of an endocrine gland on circadian clock gene expression in insects. Received 31 October 2007; received after revision 7 January 2008; accepted 9 January 2008 D. Dolezel, L. Zdechovanova: These authors contributed equally to this work.     

Chemical basis of pupal cannibalism in a caterpillar (Utetheisa ornatrix)

The mothUtetheisa ornatrix derives protection against predation from systemic pyrrolizidine alkaloids (PAs) that it sequesters as a larva from its foodplants (Leguminosae,Crotalaria spp.). We here show, in laboratory tests, thatUtetheisa deficient in body PA can make up for the chemical shortfall by cannibalizing pupae. We present evidence indicating that cannibalism in larvae is elicited not by hunger, but possibly by PA deficiency itself, and that in making cannibalistic choices larvae prefer PA-containing over PA-free pupae. PAs themselves, either in crystalline form or as additives to food items, proved phagostimulatory to larvae. In natureUtetheisa tend to pupate away from their foodplant, essentially out of reach of larval attack. The threat of cannibalism may have contributed to the evolution of this pupation behavior.Paper No. 102 of the series Defense Mechanisms of Arthropods; No. 101 is Eisner et al., Chemoecology (in press).     

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.     

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.     

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.     

Influence of oxygen concentration on development ofDrosophila melanogaster

Summary 1st, 2nd, and early 3rd instarDrosophila larvae are extremely sensitive to 100% O₂ or 75% O₂/25% N₂ (at atmospheric pressure) whereas eggs, late 3rd instar larvae, and pupae are relatively insensitive under our exposure conditions. Eclosing flies exposed to an O₂ enriched environment consistently possessed 2 eye abnormalities: dark eye color and altered eye shape.Supported by NSF-URP grant SP1782684 and a Summer Faculty Fellowship to Harry Nickla.     

Variations des taux d'ecdystéroïdes au cours du développement deBombyx mori; rapport entre ces variations et les phases de croissance et de morphogenèse

Summary During the development ofBombyx mori (monovoltin race) ecdysteroid levels were determined in oocytes, eggs, and haemolymph of larvae, and in the haemolymph of pupae. In haemolymph, the only RIA reactive materials are ecdysone and ecdysterone. In oocytes and eggs, other ecdysteroids are also detected. During larval instars, the ecdysteroid levels are low whereas they are very high during morphogenetic periods. During embryonic diapause, the ecdysone titer decreases during the cessation of morphogenesis.     

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.     

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.     

Enzymatic characterization of nine endoparasite species of small ermine moths (Yponomeutidae)

Summary Eight hymenopterous and 1 dipterous species, all endoparasitic in eggs, larvae, or pupae of small ermine moths (Yponomeuta) were investigated for their allozyme variation at 3–29 loci. The mean heterozygosity level of the hymenopterous species is one-third of that of the dipterous species. Zymogram patterns of the parasite larvae do not interfere with those of the host.     

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.     

Role of low ecdysteroid levels in the early last larval instar ofBombyx mori

The relationship between juvenile hormone and ecdysteroid titers during the early stage of the last larval instar of the silkworm,Bombyx mori, was examined in this study. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, 100% underwent supernumerary larval molting instead of metamorphosis. The application of juvenile hormone mimic during the early last instar did not induce supernumerary larval molting, but did delay metamorphosis. Temporal and quantitative ecdysteroid titer measurements revealed that in normal larvae the titers maintained very low levels (3–12 ng/ml) during the early stage of the last instar; however, in 20-hydroxyecdysone-treated larvae, levels ranging from 24 to 45 ng/ml were detected, and a major peak (246 ng/ml) was observed on day 6. These results show that very low ecdysteroid titer levels during the early stage of the last larval instar may play an important role in initiating decreases in juvenile hormone titers as well as in directing metamorphosis.     

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.     

Arylpyridyl-thiosemicarbazones: A new class of anti-juvenile hormones active against Lepidoptera

Summary A new class of anti-juvenile hormone agents is described. Active anti-juvenile hormone compounds were either diazine thiosemicarbazones or aryl substituted pyridyl thiosemicarbazones, synthesized from substituted benzaldehydes. While many analogs in these classes showed feeding and growth inhibition in a variety of insects, a select group caused formation of precocious pupal characteristics inAgrotis ipsilon (black cutworm) andHeliothis virescens (tobacco budworm) and black cuticle and precocious pupae inManduca sexta (tobacco hornworm). They were active only by diet incorporation. The symptoms of precocious development could be reversed by co-administration of a juvenoid. One of the active compounds was shown to inhibit juvenile hormone biosynthesis in vitro by corpora allata of the cockroachDiploptera punctata. However, none of the compounds were active inhibitors of purified chicken liver prenyl transferase.     

Cholinergic control of extracardiac pulsations in insects

Summary The autonomic nerve system which controls extracardiac pulsations of hemolymph pressure appears to be quite susceptible to cholinesterase inhibitors (physostigmine, organophosphorus insecticides). In pupae ofTenebrio molitor L. andGalleria mellonella L., the treatment induced large peaks in hemolymph pressure (over 4 kPa). The peaks were repeated at more-or-less constant, 30-s intervals for several hours or days. Removal of the pupal brain neither affected extracardiac pulsations nor influenced the specific responses to the investigated drugs or insecticides.     

Establishment of a colony of the mosquitoCuliseta longiareolata under laboratory conditions

Summary A colony ofCuliseta longiareolata was set up in the laboratory starting with more than 700 larvae and pupae collected from an old well. Procedures for successful establishment of a colony and laboratory maintenance of this mosquito are described.     

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.