Viral DNA in transformed cells: II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome

³²P-labeled adenovirus 2 DNA was treated with restricting endonuclease from Escherichia coli strain RY-13 (Yoshimori, 1972) (EcoRI) or restricting endonuclease from Hemophilus parainfluenzae (Hpa I) and the resulting fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete adenovirus 2 DNA were measured in the presence of DNA extracted from nine lines of adenovirus 2-transformed rat cells and from control cells. Six of the transformed cell lines contained viral DNA sequences homologous to two of the seven Hpa I⁴ fragments and to part of one of the six EcoRI fragments. From the order of the fragments formed by EcoRI and Hpa I on the adenovirus 2 map we conclude that these cell lines contain only the segment of viral DNA that stretches from the left-hand end to a point about 14% along the viral genome. Thus, any viral function expressed in transformed cells must be coded by this small section of viral DNA. The three remaining lines of adenovirus 2-transformed rat cells are more complicated and contain not only the sequences from the left-hand end of the viral DNA, but also other segments of the viral genome. However, no adenovirus 2-transformed rat cell contained DNA sequences homologous to the complete viral genome.     

α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

Lysosomal enzyme inactivation associated with defects in post-translational modification during development in Dictyostelium discoideum

The developmental accumulation of lysosomal alpha-mannosidase-1 activity in Dictyostelium discoideum is controlled at the level of de novo enzyme precursor biosynthesis. Aggregation-deficient mutants are defective with regard to the accumulation of alpha-mannosidase-1 activity beyond 8-16 h of development. We used enzyme-specific monoclonal antibodies to show that the activity defect in aggregation-deficient strains is not due to a lack of alpha-mannosidase-1-precursor synthesis or processing, or to preferential degradation of the mature enzyme protein. Instead, the defect is a result of enzyme inactivation: cells of aggregation-deficient strains contain significant amounts of inactive alpha-mannosidase-1 protein late in development. The alpha-mannosidase-1 inactivation phenotype is associated with a more general defect in lysosomal enzyme modification. A change in the post-translational modification system occurs during normal slime-mold development, as shown by differences in enzyme isoelectric point, antigenicity, and thermolability. We found that this change in modification does not occur in mutant strains blocked early in development. We propose a model in which pleiotropic mutations in early aggregation-essential genes can indirectly affect the accumulation of alpha-mannosidase-1 activity by preventing the expression of a developmentally controlled change in the post-translational modification system, a change which is required for the stability of several lysosomal enzymes late in development.     

Passage of molecules across the intercellular bridge between post-mitotic daughter cells

Living mitotic HeLa cells were microinjected with sodium fluorescein, fluorescein isothiocyanate (FITC)-labelled IgG, and Lucifer Yellow-CH to determine if the intracellular bridge and midbody forms a barrier to the migration of these molecules. All three fluorescent molecules were found to pass across the intercellular bridge. The post-mitotic cells maintained a molecular patency across the intercellular bridge that persisted for considerable lengths of time, extending into the next cell cycle.     

Observations on populations of the horseshoe crabLimulus (=Xiphosura) polyphemus

1. Marked populations of Limulus (=Xiphosura) polyphemus reveal that in Cold Spring Harbor, New York, they consisted of 10,000–18,000 adults in 1957 and 1961. The sex ratio in 1957 was about 4 males: 1 female. Pairs may remain attached for as long as 9 days. An undisturbed female may lay as many as 12,000 eggs in one nest.
2. The Cold Spring Harbor populations appear to be rather sedentary: none of the 1,000 animals marked on the north edge of the sandspit in 1961 were detected in the outer harbor either at Laurel Hollow Beach or the peninsula adjacent to the Cold Spring Harbor Yacht Club 500–800 meters from the tagging site (see Fig. 1), nor were they found in the small beach adjacent to the Biological Laboratory in the inner harbor. Similarly, none of the 300 animals marked at this last site were found at the north edge of the sandspit.
3. The phenotype of the compound eye varies from black to pigmentless. Samples observed in Cold Spring Harbor and in the Marine Biological Laboratory, Woods Hole, Massachusetts (separated by Long Island Sound and a distance of 150 miles) differ in the frequency of the various phenotypes scored, but the mode of inheritance of eye color remains obscure.
4. The available evidence indicates Limulus has considerable phenotypic variation in regard to body size, eye color, and other characters believed to be inherited, with the result that demes or physiological races are created. It is argued that the belief that this organism is stable and has not changed since the Triassic 200 million years ago has foundation only in regard to the pattern of the body of Limulus, but not in regard to its genotype. Limulus does not seem to be different from other organisms for which considerable genetic evidence is available, and thus the statement that DNA is fairly stable and has remained so for 200 million years is open to question.

     

Phytophthora species isolated from alpine and sub-alpine regions of Australia,including the description of two new species; Phytophthora cacuminis sp. nov and Phytophthora oreophila sp. nov

Plant deaths had been observed in the sub-alpine and alpine areas of Australia. Although no detailed aetiology was established, patches of dying vegetation and progressive thinning of canopy suggested the involvement of root pathogens. Baiting of roots and associated rhizosphere soil from surveys conducted in mountainous regions New South Wales and Tasmania resulted in the isolation of eight Phytophthora species; Phytophthora cactorum, Phytophthora cryptogea, Phytophthora fallax, Phytophthora gonapodyides, Phytophthora gregata, Phytophthora pseudocryptogea, and two new species, Phytophthora cacuminis sp. nov and Phytophthora oreophila sp. nov, described here. P. cacuminis sp. nov is closely related to P. fallax, and was isolated from asymptomatic Eucalyptus coccifera and species from the family Proteaceae in Mount Field NP in Tasmania. P. oreophila sp. nov, was isolated from a disturbed alpine herbfield in Kosciuzsko National Park. The low cardinal temperature for growth of the new species suggest they are well adapted to survive under these conditions, and should be regarded as potential threats to the diverse flora of sub-alpine/alpine ecosystems. P. gregata and P. cryptogea have already been implicated in poor plant health. Tests on a range of alpine/subalpine plant species are now needed to determine their pathogenicity, host range and invasive potential.     

Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth,Cydia pomonella

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (T_m) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites.     

Reprint of: Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth,Cydia pomonella

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (T_m) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites.     

Feeding impairs chill coma recovery in the migratory locust (Locusta migratoria)

Low temperature causes loss of neuromuscular function in a wide range of insects, such that the animals enter a state known as chill coma. The ability to recover from chill coma (chill coma recovery time) is often a popular phenotype to characterise chill tolerance in insects. Chill coma in insects has been shown to be associated with a decrease in haemolymph volume and a marked increase in [K⁺], causing dissipation of K⁺ equilibrium potential and resting membrane potential. High potassium diet (wheat) has also previously been shown to increase haemolymph [K⁺] in Locusta migratoria leading to sluggish behaviour. The present study combined these two independent stressors of ion and water homeostasis, in order to investigate the role of K⁺- and water-balance during recovery from chill coma, in the chill sensitive insect L. migratoria. We confirmed that cold shock elicits a fast increase in haemolymph [K⁺] which is likely caused by a water shift from the haemolymph to the muscles and other tissues. Recovery of haemolymph [K⁺] is however not only reliant on recovery of haemolymph volume, as the recovery of water and K⁺ is decoupled. Chill coma recovery time, after 2 h at −4 °C, differed significantly between fasted animals and those fed on high K⁺ diet. This difference was not associated with an increased disturbance of haemolymph [K⁺] in the fed animals, instead it was associated with a slowed recovery of muscle [K⁺], muslce water, haemolymph [Na⁺] and K⁺equilibrium potential in the fed animals.     

Insect antifeedant elemanolide lactones from Vernonia amygdalina

Chemical investigation of insect antifeedants from the bitter tasting leaves of Vernonia amygdalina by the application of semi-preparative reversed     

Effect of cysteine on stability and toxicity to aphids of a cyclic hydroxamic acid from gramineae

Toxicity of DIMBOA, the major cyclic hydroxamic acid in maize extracts, to the aphid Schizaphis graminum, was decreased by addition of cysteine to the insect diet. The ld₅₀ for DIMBOA on aphids was, after 24 hr, 2.1 and 0.9 mM in diets with and without added cysteine, respectively. DIMBOA decomposed 1.5 times faster in diets or buffer with added cysteine. Decomposition products of DIMBOA (4 mM) in insect diets with or without added cysteine were not toxic. It is suggested that the observed variations in toxicity of DIMBOA are a consequence of differences in its rate of disappearance from the diet.     

Azedarachol,a steroid ester antifeedant from Melia azedarach var. japonica

A new steroid ester, azedarachol, from the root bark of Melia azedarach has been identified as an antifeedant against a Japanese insect pest and the structure has been assigned as 2α,3α,16β-trihydroxy-5α-pregnane 20R-methacrylate.     

The chemistry of thujone: I. Synthesis of insect juvenile hormone analogs via wittig coupling

Syntheses of the C₈ and C₁₀ olefinic units cis- and trans-5-ethyl-1-iodo-hex-4-enes and cis- and trans-7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed.     

Chitin and chitinase: A kinetic model

A model is described for the action of insect molting chitinase on chitin microfibrils in cuticle. The model reconciles the disparate structures proposed for chitin in the literature. It also accounts for the kinetic characteristics of molting fluid chitinase insofar as known from in vitro studies, viz. positive co-operativity of possibly three catalytic sites, complexity, and processivity. These have hitherto been difficult to account for in vivo, given the arrangement of chitin in anhydrous microfibrils in arthropod cuticle.     

Low temperature thresholds: are chill coma and CT(min) synonymous?

The effects of sub-lethal low temperatures on insect physiology and behaviour are important determinants of insect activity including foraging, mating, and predation avoidance. A substantial body of research seeks to relate the temperatures at which these activities are compromised to both, climatic conditions at species range limits and underlying physiological processes. The interpretation of this research is complicated by confusion in the names and definition of the responses measured and their associated temperature thresholds. The development of the nomenclature and explanations of the underlying physiological causes are reviewed in order to elucidate the correct sequence of responses/thresholds and associated terminologies. The results of this analysis indicate that: (1) chill coma is a clearly defined, reversible physiological state characterised by the absence of electrophysiological activity. (2) The onset of chill coma begins when low temperatures begin to impair insect behaviour and physiology, and is punctuated by a series of behavioural and/or physiological thresholds or responses. These include the temperatures at which (i) spontaneous movements cease, (ii) coordination is lost to the degree that locomotion becomes impossible, and (iii) chill coma is entered. (3) Confusion has arisen because (a) the term ‘onset of chill coma’ has been used to describe all three of these responses/thresholds and (b) the term CT_min has entered the insect literature from the vertebrate literature. These issues are discussed and a potential solution is proposed to provide clarity and consistency in the future literature.     

Bacterial genera isolated from field-collected (Diapausing) and laboratory-reared cotton boll weevils, Anthonomus grandis (Coleoptera: Curculionidae)

Little data exist on the bacterial flora of the cotton boll weevil, Anthonomus grandis, which is a common insect pest to cotton farmers in the United States. This investigation determined the total numbers of aerobic and anaerobic bacteria in diapausing field-collected boll weevils and active adult laboratory-reared boll weevils. Identifications were made of aerobic genera isolated from field-collected and laboratory-reared boll weevils that had previously been surface sterilized. The genera found, in order of decreasing frequency, in the field-collected boll weevils were Lactobacillus, Erwinia, Flavobacterium, Enterobacter, and Pseudomonas. The genera found, in order of decreasing frequency, in the laboratory-reared boll weevils were Lactobacillus, Pseudomonas, Streptococcus, Erwinia, Enterobacter, and Flavobacterium, Lactobacillus was the most frequently found genus in both groups.     

Leaf wax of Triticum aestivum

Leaf waxes from spring wheat varieties Selkirk and Manitou contain hydrocarbons (6%, 10%), long chain esters (14%, 13%), free acids (5%, 8%), free alcohols (19%, 21%), β-diketone (16%, 20%), hydroxy β-diketones (8%, 10%), unidentified gum (29%, 16.5%) and minor amounts of diol diesters, glycerides and aldehydes. The major hydrocarbon is nonacosane and major esters are octacosyl esters of C₁₄–C₃₂ acids but C₂₀ and C₂₂ alcohol esters of trans 2-docosenoic and tetracosenoic acids are also present (Selkirk 20%, Manitou 10% of total esters). Previously unknown trans 2-docosen-1-ol is present as an ester (Selkirk 5%, Manitou 2.5% of total esters). Free acids are C₁₄–C₃₂ acids and trans 2-docosenoic and tetracosenoic acids (Selkirk 30%, Manitou 9% of free acids). Octacosanol is the principal free alcohol. Hentriacontane-14,16-dione is the β-diketone and the hydroxy β-diketones are a 1:1 mixture of 8- and 9- hydroxyhentriacontane-14,16-diones.     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.