Effect of intercalating mutagens on crossing-over inDrosophila melanogaster females

Summary In order to evaluate the effect of several intercalating compounds on crossing-over inDrosophila melanogaster females, acridine orange, acriflavine, chloroquine, ethidium bromide and quinacrine were fed separately to larvae ofy ct f/+++ genotype. Our results show that acridine orange, acriflavine and ethidium bromide increase significantly the recombination frequency at thect-f region and support the view that, for intercalating agents, there is a relationship between clastogenic activity and female recombination induction.     

Genotoxic evaluation of ten carcinogens in theDrosophila melanogaster wing spot test

To provide further background data on the wing spot somatic mutation and recombination assay, 10 selected carcinogens (acetamide, acrylamide, benzo(a)pyrene, cyclophosphamide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) were tested in this assay. 72-h-old third-instar larvae, trans-heterozygous for 2 recessive wing cell markers:multiple wing hairs (mwh) andflare ³ (flr ³) were fed with 3 concentrations of each carcinogen during the rest of their development until pupation, and the genotoxic effects were measured as significant increases in the appearance of visible mutant hair clones on the adult wing blade. Our results show that 6 of the carcinogens tested produce significant increases in wing spot frequency, at least at one of the concentrations assayed. Benzo(a)pyrene, diethylstilbestrol, safrole and thiourea were the compounds that did not increase the incidence of mutant clones.     

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.     

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.     

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.     

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.     

Inhibitory effect of bacterial ubiquinones on the settling of barnacle,Balanus amphitrite

In an attempt to clarify the influence of marine bacteria on the settling of fouling invertebrate larvae, we screened for inhibitors, produced by marine bacteria, of settling by cyprids of the barnacle,Balamus amphitrite. We found that the culture broth ofAlteromonas sp. strain number KK10304, which was associated with the marine sponge,Halichondria okadai, effectively inhibited settling of the cyprids. Bioassay-guided isolation indicated ubiquinone-8 (1) as an effective inhibitor of cyprid settling. As ubiquinones are widely distributed in bacteria, several related compounds were also tested.     

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.     

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.     

Low allozyme and mtDNA variability in the island endemic speciesDrosophila sechelia (D. melanogaster complex)

Summary Genetic variability ofD. sechellia is investigated at both mitochondrial and nuclear levels. The results reveal the existence of a single main type of mtDNA with very few variants and a very low enzyme polymorphism. This situation is consistent with the small population size of this specialized species.     

Effects of a juvenile hormone mimetic,fenoxycarb, on post-embryonic development of the European corn borer,Ostrinia nubilalis Hbn.

Summary The effect of a juvenile hormone mimetic, fenoxycarb, Ro 13-5223, was tested on the larval instars of the European corn borer,Ostrinia nubilalis, by dipping or topical application. When larvae were treated in instars 2, 3 or 4, the duration of the fifth instar was modified. More permanent and fewer supernumerary larvae were obtained when treatment occurred in the early instars. This non-neurotoxic compound exhibited a strong dose-dependent juvenile hormone type of activity when it was applied to last instar larvae. Fenoxycarb prevented the onset of pupation and produced supernumerary larvae and intermediates. Permanent larvae were obtained if fenoxycarb was applied on day 0 or day 1 of the last instar. The use of such a JH mimetic in the understanding of endocrine control of diapause is discussed.     

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.     

Evolutionary and ecological implications of cardenolide sequestration in the monarch butterfly

Summary Monarch butterflies sequester cardenolides from their larval host plants in the milkweed genusAsclepias for use in defense against predation. Of 108Asclepias species in North America, monarchs are known to feed as larvae on 27. Research on 11 of these has shown that monarchs sequester cardenolides most effectively, to an asymptote of approximately 350 g/0.1 g dry butterfly, from plants with intermediate cardenolide contents rather than from those with very high or very low cardenolide contents. SinceAsclepias host plant species are distributed widely in space and time across the continent, monarchs exploit them by migration between breeding and overwintering areas. After overwintering in central Mexico, spring migrants east of the Rocky Mountains exploit three predominantAsclepias species in the southern USA that have moderately high cardenolide contents. Monarchs sequester cardenolides very effectively from these species. First generation butterflies are thus well protected against predators and continue the migration north. Across the northern USA and southern Canada most summer breeding occurs on a fourthAsclepias species and in autumn most of these monarchs migrate back to Mexican overwintering sites. The ecological implications of this cycle of cardenolide sequestration for the evolution of monarch migration are discussed.     

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.     

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.     

The sensitive period for yellow phenocopy induction inDrosophila melanogaster

Summary Yellow phenocopies ofDrosophila melanogaster were produced by raising larvae on -DMT contaminated media. Using a survivorship test, the sensitive period for phenocopy induction was found to occur during the third larval instar of development, with increased survivorship at 1% -DMT compared with lower concentrations. It was also found that treatment with -DMT significantly slowed development. These findings are related to the relevant morphological and behavioral developmental pathways and to phenocopy induction.Publication No. 35 from the Evolutionary Genetics Laboratory, University of Auckland.Acknowledgments. We thank the New Zealand Entomology Society for a grant from their Anniversary Fund to R. D. Newcomb and the University Grants Committee, Research Grant No. 394.597 to D.M. Lambert.     

The prey consumption and prey preference of the larvae of the mosquitoCulex (Lutzia) raptor on the larvae ofCulex quinquefasciatus

The maximum consumption of the larvae of the pest and vector mosquitoCulex quinquefasciatus by the predatory mosquitoCulex (Lutzia) raptor was studied at various instars of both the predator and the prey. The prey preferences of the predator when given lavae of different instars were also investigated. The IVth instar of the predator consumed the maximum number of Ist instar and the maximum biomass of IVth instar larvae of the prey. Instars I and II of the predator preferred the Ist of the prey; instars III and IV of the predator preferred instars II and III of the prey respectively.The predator consumed an average of 157.1 larvae during its whole larval period, when each instar of the predator was given its preferred instar of the prey.     

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.     

Ecdysteroid titers during larval reproduction of the dipteran insectHeteropeza pygmae

Summary Ecdysteroid titers were measured in whole-body extracts of pedogenetically reproducing larvae of the dipteran insectHeteropeza pygmaea and in the dietary fungus. The titers are very low in the first 3 days of larval growth, but increase during the last 2 days. The level of 20-hydroxyecdysone is then significantly higher than that of ecdysone. Measurements of the titers in the fungus gave no conclusive results.Supported by the Swiss National Science Foundation, grants No. 3.218-0.77 to D.F.W. and 3.714-0.80 to B.L. We thank Xiang-Xiong Zhu for help with the RIA and Mrs G. Rhyner for technical assistance. Gifts of ecdysone antiserum from Dr J. D. O'Connor and of 23,24-[³H₂]-ecdysone from Dr J. Koolman are also gratefully acknowledged.     

Hotspots of homologous recombination

Homologous recombination occurs at higher than average frequency at and near hotspots. Hotspots are special nucleotide sequences recognized by proteins that promote, directly or indirectly, a rate limiting step of recombination. This review focuses on two well-studied examples, the Chi sites of the bacteriumEscherichia coli and the M26 site of the fission yeastSchizosaccharomyces pombe. Chi, 5 G-C-T-G-G-T-G-G 3, is recognized by the RecBCD enzyme, which nicks the DNA near Chi and produces a 3-ended single-stranded DNA tail; this tail is a potent substrate for homologous pairing by RecA and single-stranded DNA binding proteins. M26, 5 A-T-G-A-C-G-T 3, is recognized by a heterodimeric protein and stimulates, by an as-yet-unknown mechanism, meiotic recombination at and near theade6 gene. Additional hotspots in bacteria, fungi, and mammals enhance recombination directly or indirectly via a variety of mechanisms. Although hotspots are widespread among organisms, the biological role of their localized enhancement of recombination remains a matter of speculation.