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The action of three distinct recombinant antifreeze proteins (AFPs) as methane hydrate inhibitors was examined using a recently-developed reactor. Compared with traditional approaches, this reactor uses minimal reactant volumes and short experimentation times to assess phase equilibria, kinetics and morphology of a hydrate system in a single experiment (3-in-1). Two of the recombinant AFPs are considered highly active with respect to the inhibition of ice: ‘Maxi’, a fish AFP, and a beetle AFP (TmAFP). The third protein from a grass, is classified as a low activity AFP (LpAFP). ‘Maxi’, an AFP that has not been tested previously as a hydrate inhibitor, slowed hydrate growth rates up to an order of magnitude compared to pure water. TmAFP and LpAFP also exhibited kinetic inhibition, but were less effective than ‘Maxi’. In the presence of AFPs, hydrate films were thinner and showed a single growth mechanism compared to multiple crystal growth mechanisms observed in control experiments. The addition of TmAFP generated large irregular hydrate halos that propagated outside the original water boundary. Halo propagation was somewhat less prominent with LpAFP, and was not observed with ‘Maxi’. Although, none of the AFP’s showed thermodynamic inhibition properties, ‘Maxi’ appeared to form clusters of hydrate which remained metastable in the liquid–vapour region.

Low-temperatures pose extreme challenges to crops causing significant economical impacts. Frosts are responsible for more than 30% of weather-related insured crop losses in some temperate climate jurisdictions, but are particularly devastating for small holdings and communities reliant on a bountiful harvest. Low-temperatures are also frequently accompanied by other abiotic and biotic stresses, including pathogen attacks. Some pathogens have sub-zero temperature optima, while others leverage low-temperatures to promote freezing at high sub-zero temperatures by way of ice-nucleating proteins in order to access intracellular nutrients. To survive low-temperatures and the attendant risks, various plant species have evolved complex and intricate signaling networks, molecular mechanisms, and physiological changes, in addition to symbiotic relationships with microbiota. Enhancing low-temperature survival and pathogen-induced freezing tolerance in cold susceptible, agriculturally significant crops is an attractive area of research with immense translatable value to all aspects of society. This area of research will be particularly important in our near future as climate change increases the unpredictability of frosts, particularly in the spring and autumn. Against this backdrop, the world population continues to grow while arable land remains finite and wealth inequality exacerbates food poverty. In this review, we examine plant (i) low-temperature stress, (ii) cold acclimation responses, particularly in crops (iii) antifreeze proteins, and (iv) frost-associated pathogens. Lastly, we suggest integrated approaches to improve crop frost tolerance.

Titanium dioxide (TiO2) nanoparticles (NPs) are used as an additive (E171 or INS171) in foods such as gum, candy and puddings. To address concerns about the potential hazardous effects of ingested NPs, the toxicity of these food-grade NPs was investigated with a defined model intestinal bacterial community. Each titania preparation (food-grade TiO2 formulations, E171-1 and E171-6a) was tested at concentrations equivalent to those found in the human intestine after sampling 1-2 pieces of gum or candy (100 250 ppm). At the low concentrations used, neither the TiO2 food additives nor control TiO2 NPs had an impact on gas production and only a minor effect on fatty acids profiles (C16:00, C18:00, 15:1 w5c, 18:1 w9c and 18:1 w9c, p < 0.05). DNA profiles and phylogenetic distributions confirmed limited effects on the bacterial community, with a modest decrease in the relative abundance of the dominant Bacteroides ovatus in favor of Clostridium cocleatum (-13% and +14% respectively, p < 0.05). Such minor shifts in the treated consortia suggest that food grade and nano-TiO2 particles do not have a major effect on human gut microbiota when tested in vitro at relevant low concentrations. However, the cumulative effects of chronic TiO2 NP ingestion remain to be tested.

An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27 °C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4 °C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP-INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae.

There is concern that waste waters containing manufactured metal nanoparticles (NPs) originating from consumer goods, will find their way into streams and larger water bodies. Aquatic invertebrates could be vulnerable to such pollution, and here we have used fruit flies, Drosophila melanogaster, as a model invertebrate, to test for the effect of NPs on fitness. Both copper NP and microparticle (MP)-containing medium slowed development, reduced adult longevity and decreased sperm competition. In contrast, ingestion of silver resulted in a significant reduction in developmental success only if the metal particles were nanosized. Ag NP-treatments resulted in reduced developmental success as assessed by larval and pupal survival as well as larval climbing ability, but there was no impact of silver on adult longevity and little effect on reproductive success. However, Cu NPs generally appeared to be no more toxic to this invertebrate model than the bulk counterpart. The impact of silver ingestion in larvae was further investigated by 454 pyrosequencing of the 16S rRNA genes of the midgut flora. There was a striking reduction in the diversity of the gut microbiota of Ag NP-treated larvae with a rise in the predominance of Lactobacillus brevis and a decrease in Acetobacter compared to control or Ag MP-treatment groups. Importantly, these experiments show that perturbation of the microbial assemblage within a metazoan model may contribute to Ag NP-mediated toxicity. These observations have implications for impact assessments of nanoparticles as emerging contaminants.

The performance of polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap) as kinetic gas hydrate inhibitors in saline solutions and with heptane was evaluated using high pressure microdifferential scanning calorimetry, as well as with a new apparatus, consisting of two high pressure stainless steel crystallizers. Although PVP and PVCap were found to prolong natural gas hydrate induction time in saline solutions, nucleation was followed by catastrophic hydrate crystal growth. PVP was found to be more effective in this case, since this hydrate growth was modestly slower. The addition of n-heptane to the natural gas in the system created a 4th phase. This resulted in increased induction time and a slowing of hydrate growth relative to the gas mixture. Unexpectedly, in the presence of n-heptane, addition of kinetic hydrate inhibitors (KHIs) decreased induction time, but catastrophic growth did not occur. Here PVCap was more effective than PVP in both prolonging the induction time and decreasing the rate of hydrate crystal growth. Once formed, however, hydrate decomposition took longer and proceeded in two steps in the presence of n-heptane. This observation has profound applications on the use these KHIs under ocean field conditions. In the case of hydrate blockages, our observations that hydrate dissociation started later with the KHIs and complete dissociation took longer could have far reaching economic implications for industry. © 2013 Elsevier Ltd. All rights reserved.

Despite numerous applications for functionalized carbon nanotubes (fCNTs) in consumer products, such as electronics, and food packaging, as well as their development as drug delivery vehicles, the consequence of their uptake by living systems has been understudied. In particular, the impact of fCNTs on early development of different species is largely unknown. Here we investigated the effect of ingested hydroxyl-fCNTs on reproduction and development in two model organisms: Drosophila and CD-1 mice. While fCNTs had no measurable impact on Drosophila, a single oral dose of fCNTs (10 mg/kg) administered to pregnant CD-1 dams during organogenesis significantly increased the number of resorptions and resulted in fetal morphological and skeletal abnormalities. The observed difference between the responses of these two models likely reflects their physiology and/or differences in administration. This research underscores the need to examine the effects of fCNTs on reproductive health and development before the opportunities for maternal exposure by fCNTs increase further.

Antifreeze proteins (AFPs) share two related properties: the ability to depress the freezing temperature below the melting point of ice (thermal hysteresis; TH); and the ability to inhibit the restructuring of ice into larger crystals. Since the 'hyperactive' AFPs, which have been more recently discovered, show an order of magnitude more TH than previously characterized AFPs, we have now determined their activities in ice restructuring inhibition (IrI) assays. IrI activities of three TH-hyperactive AFPs and three less TH-active AFPs varied over an 8-fold range. There was no obvious correlation between high TH activity and high IrI activity. However, the use of mutant AFPs demonstrated that severe disruption of ice-binding residues diminished both TH and IrI similarly, revealing that that the same ice-binding residues are crucial for both activities. In addition, bicarbonate ions, which are known to enhance the TH activity of AFPs, also enhanced their IrI activity. We suggest that these seemingly contradictory observations can be partially explained by differences in the coverage of ice by TH-hyperactive and non-hyperactive AFPs, and by differences in the stability of AFP-bound ice under supercooled and recrystallization conditions.

Pipeline blockage by gas hydrate is a serious problem in petroleum industry. Recently, low-dosage inhibitors have been developed. In particular, poly(N-vinylcaprolactam) (PVCap) is a stronger inhibitor than poly(N-vinylpyrrolidone) (PVP). In this study, PVCap was also found to have stronger inhibition activity compared to PVP, but it was less effective during reformation of hydrate. To understand the mechanism, the adsorption of PVCap and PVP on silica, a common nucleating agent, was examined using quartz crystal microbalance with dissipation factor observation function. The results reveal that PVP forms a loose film on silica whereas PVCap forms a relatively more rigid and compact film. However, most of the PVCap film could be rinsed off. These results help explain the different inhibition activities of PVCap and PVP.

A partially palindromic 15-nt. sequence upstream from a juvenile hormone-regulated gene (jhp21) was previously identified in the African migratory locust, Locusta migratoria. This sequence was proposed as a juvenile hormone (JH) response element (JHRE), and a protein that bound to it, as a transcription factor (TF). A yeast strain was constructed containing four tandem copies of the JHRE and after transfection with a cDNA library made to fat bodies from vitellogenic females, yeast one-hybrid experiments yielded sequences for four putative binding proteins. One of these sequences, corresponding to a transcript that was present in fat body irrespective of JH stimulation, encodes a 35kDa protein. This was designated tfp1 and appears to have a leucine zipper motif and a lipid-binding motif. Recombinant tfp1 bound to JHRE in electrophoretic mobility shift experiments and addition of tfp1 antibody in the binding reaction resulted in the disappearance or shift of TF. We suggest that JH induces the association of pre-existing proteins, including tfp1, to form an active complex, which binds to the JHRE upstream from jhp21 and regulates its transcription.

Not surprisingly, in the spruce budworm, Choristoneura fumiferana, antifreeze protein (AFP) gene expression is most abundant in the second instar, overwintering stage. However, low level RNA and protein expression was also found in the sixth instar larvae, a summer stage. In situ hybridization further confirmed the presence of AFP mRNA in sixth instar midgut tissues. Sequencing of cDNAs corresponding to ‘‘summer-expressed’’ transcripts revealed an isoform that was not apparent in a cDNA library made to second instar larvae. Although similar to AFP cDNAs obtained from overwintering larvae, this AFP-like isoform (CfAFP6) has two Cys substitutions. Since AFPs from this species fold into a b-helix that is stabilized by disulfide bonds, it was of interest to determine if this summerexpressed isoform had AFP activity. No thermal hysteresis activity was found when CfAFP6 was cloned and expressed in E. coli, even after in vitro denaturation and refolding. As well, there was no activity detected when the sequence of a known, active isoform was changed to mimic the Cys substitutions in CfAFP6. Since CfAFP6 does not appear to contribute to freeze resistance, its apparent absence in the overwintering second instar should not in itself be considered curious. r 2006 Elsevier Ltd. All rights reserved.

The ecdysone receptor(1), which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths. It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts. Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here. Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation. After expression in E. coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A. Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains. In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts. Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III. Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face.

Although juvenile hormone (JH) has essential roles in insect development and reproduction, the molecular mechanisms of gene regulation by JH remain an enigma. In Locusta migratoria, the partially palindromic 15-nt sequence, GAGGTTCGAGA/TCCTT/C, found upstream of a JH-induced gene, jhp21, was designated as a putative juvenile hormone response element (JHRE). When JH-deprived adult female locusts were treated with the active JH analog, methoprene, a fat body nuclear factor that bound specifically to JHRE appeared after 24 h. Binding exhibited a preference for an inverted repeat with GAGGTTC in the left half-site, a single nucleotide spacer, and a right half-site in which some variation is acceptable. Binding to JHRE was abolished by phosphorylation catalyzed by a C-type protein kinase present in the nuclear extracts. The DNA-binding protein is thus believed to be a transcription factor, which is brought to an active state through the action of JH and then participates in the regulation of certain JH-dependent genes.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a β-roll domain with eight loops of 14–15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein’s inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins, but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coil. NMR analysis and secondary structure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeating structure and presumptive disulfide bond arrangement.

The gene encoding the glycolytic enzyme, triosephosphate isomerase (TPI; EC 5.3.1.1), is a favourite model for molecular evolutionists who either subscribe to the theory that introns co-evolved with the ancestral gene, the introns early view, or alternatively, that introns are more recent immigrants. The discovery of an intron in the TPI gene of Culex mosquitoes at a site which was predicted by proponents of the intron early school supported that theory. More recently, the discovery of additional intron sites in several eukaryotes was presented as evidence supporting the introns late school. We have found the `Culex intron' in two closely related mosquitoes, but not in two more evolutionary primitive Dipterans, suggesting that, if it is an `ancient intron', loss may be more frequent than that supposed by the intron late school. In addition, we have found that three introns punctuating the TPI gene from the Lepidopteran, Heliothis, appear to be ancestrally related and may be the result of transposable element insertion, 50–90 million years ago. It is argued that both opposing schools in the intron debate be reconciled — some introns may have been early and certainly others have arrived subsequent to the appearance of the TPI gene.

Three malathion carboxylesterase (MCE) phenotypes have been described previously in Lucilia cuprina adults: low, intermediate, and high. The MCE specific activities of adults with the intermediate and high phenotypes are 21- and 33-fold higher than those with the low phenotype, but the high phenotype is almost 1000-fold more resistant to malathion than either of the other phenotypes. Here we show that MCE activity also peaks in Day 4 larvae from three lines representative of these phenotypes. The MCE activity of the low-line larvae is only 2.7- and 4-fold lower than those of the intermediate and high-line larvae, respectively. The relatively high MCE activity of the low-line larvae is largely explained by their crop activity. Assays in the presence of esterase inhibitors reveal three distinct types of MCE activity across the three lines and two developmental stages. Activity in the low-line adults is not completely inhibited by paraoxon and is classified as a subclass II carboxylesterase. The MCE activities in the larvae of the low and intermediate lines and adults from the intermediate line are inhibited by low concentrations of paraoxon, classifying them as subclass I carboxylesterases. The MCE activities in the high-line larvae and adults are also classified as subclass I carboxylesterases, but they are more sensitive to inhibition by triphenyl phosphate than those in the other two lines. These data suggest that MCE in the malathion-resistant high line may be structurally different from the MCEs in the malathion-susceptible intermediate and low lines.

Resistance to malathion in a strain of the Australian sheep blowfly is due to a 10-fold increase in malathion carboxylesterase (MCE) activity relative to a more susceptible strain. MCE was purified to apparent homogeneity from these two strains and was shown to be a monomer of 60,500, with a pI of 5.5 in both strains. Purified MCE from both populations had identical Km and Vmax values for the hydrolysis of malathion as well as for three other esterase substrates. Similarly, the kinetics of inhibition by several inhibitors were the same for the MCE from each strain. These data therefore suggest that resistance to malathion is due to a quantitative rather than a qualitative change in the MCE of the two strains. Estimation of the total MCE content in each strain showed that the resistant blowflies had nine times more MCE than the more susceptible insects. Although blowfly MCE showed greater specificity for naphthyl esters over malathion, it nevertheless hydrolyzes malathion faster than any other esterase yet isolated from an insect. This is in sharp contrast to previously studied insect strains in which organophosphate resistance has been attributed to large increases in nonspecific esterases that show very slow or no hydrolysis of the insecticides.

Adult flies of Drosophila melanogaster were examined for their tolerance to direct (cold-shock) and indirect chilling (chilling) injury. An exponential increase in mortality with low temperatures occurs. Chilling tolerance (T ch ) at 4 or 0°C was 6.3 days or 37.91 h for 50% survival (LT 50 ), respectively, while the cold-shock tolerance (LT 50 of T cs ) at −7°C was only 1.5 h. The increase in cold-shock tolerance (T cs ) at −7°C by acclimation at 4, or 0°C was rapid: the LT 50 increased about 50% with only 2 h of treatment in comparison with the highest LT 50 obtained by acclimation. An equilibrium point (T eq ), the optimal acclimation time for maximal survival of chilling and/or cold-shock, was calculated from the survival data of experiments designed to assess T ch and T cs . The T eq was found to be the half time of the acclimation period which resulted in 90% mortality at the chilling temperature (LT 90 of T ch ). Selection for tolerance to cold-shock or chilling over several generations resulted in a significant increase in T cs or T ch , respectively. Higher contents of glycogen and total proteins were observed in the cold selected lines than in the control line. Exposure to chilling temperatures eventually depleted the energy reserves, but the highest concentrations of triacylglycerols and proteins were observed at the acclimation time with the highest T cs . This suggests that systemic regulation in these energetic reserves may be an important part of the cold-hardening mechanisms to cope with the fluctuations of low temperatures causing cold-shock or chilling injury.

The expression of fusion gene constructs containing Drosophila regulatory sequences and the structural portions of fish antifreeze protein genes have been examined by transfer into Drosophila melanogaster using P elements. A fusion gene, containing the enhancer, promoter, and cap site of the yolk polypeptide 1 gene, joined in the 5'-untranslated region to the structural portion of the winter flounder type I antifreeze gene, was transcribed in mature female transformants to give an mRNA of the predicted size, but no antifreeze protein was detected by Western blotting. When the same antifreeze protein gene was fused to a Drosophila hsp 70 gene regulatory region and placed downstream of the yolk polypeptide gene enhancer, appropriate expression of mRNA was directed by both gene regulatory elements. However, a translation product from this mRNA was only observed under heat shock conditions and was present at low levels. It is suggested that type I antifreeze mRNA, with its high content of alanine codons and their grouping into clusters of up to seven in a row, is poorly translated when in competition with other host mRNAs. In agreement with this hypothesis, a fusion gene construct between the yolk protein gene regulatory region and two type III antifreeze protein genes produced sub-mmolar concentrations of antifreeze protein in mature females from each of several transgenic lines analysed. The type III antifreeze protein does not have an imbalanced amino acid composition or sequence irregularities, and may be an appropriate choice for conferring freeze protection to frost-susceptible hosts by gene transfer.

We have accomplished gene transfer into embryos of Locusta migratoria, the African migratory locust. Freshly oviposited eggs were injected with circular or linear plasmids containing the Drosophila hsp70 promoter and the choramphenicol acetyltransferase (CAT) reporter gene (hsp-cat), or with circular plasmid containing the Drosophila copia promoter fused to CAT (copia-cat). Southern blot analysis showed that the hsp-cat plasmid persisted extrachromosomally for at least 8 days after injection. There was no evidence for plasmid replication. Transient expression from the introduced promoters was determined by monitoring CAT enzyme activity. After injection of hsp-cat, activity was detected at varying levels in 6–8% of day 3 and day 9 embryos. Embryos injected with copia-cat, assayed on day 3, had a greater frequency but no higher level of expression. The described gene transfer system is promising for analysis of other promoters, including those of Locusta.

After 5 days at the restrictive temperature (29.5°C) adult Drosophila females heterozygous for the dominant temperature-sensitive mutation, L(3)3DTS, have an ecdysteroid level of about half that in mutant females at 22°C and subsequently become completely sterile due to the inviability of progeny embryos. The lethal phase of progeny from mutant females varies depending upon the length of time DTS-3 females are kept at a sublethal temperature of 27°C. Thus, the DTS-3 mutation shows a maternal effect, and a deficiency of ecdysteroids or ecdysteroid-induced gene products may be responsible for progeny lethality. This lethality cannot be attributed to a deficit in the products of the hormonally-regulated yolk polypeptide genes however, since yolk polypeptide mRNA and protein levels are not reduced in DTS-3 females at the restrictive temperature.

1. 1. NADP-Malic enzyme (NADP-ME; l -malate:NADP + oxidoreductase (decarboxylating) EC 1.1.1.40) from Drosophila melanogaster has a mol wt of 266,000 and a subunit mol wt of 67,250 ± 3080. The amino acid composition of Drosophila NADP-ME is closely related to the NADP-ME's from pigeon liver, chick liver, rat liver and E. coli . 2. 2. Drosophila NADP-ME exhibits major malate oxidative decarboxylase and oxalacetate decarboxylase activities and minor pyruvate and oxalacetate reductase activities. 3. 3. The isoelectric point for Drosophila NADP-ME is 5.1 and the pH optimum for the oxidative decarboxylation of l -malate is 7.9. 4. 4. The malate oxidative decarboxylase activity of Drosophila NADP-ME is inhibited by NADPH, oxalacetate and ATP, but not pyruvate; the reverse reaction is inhibited by l -malate and NADP + . Drosophila NADP-ME is insensitive to N -ethylmaleimide at concentrations that strongly inhibit chick liver NADP-ME. 5. 5. Drosophila NADP-ME and chick liver NADP-ME are rapidly inactivated by palmityl-CoA, a process that is slowed by NADP + and Mn ⇔ .

Drosophila melanogaster larvae contain two isozymes of leucine aminopeptidase that are detectable on starch gels. The more anodally migrating isozyme, LAP A, is first detected 12 hr after oviposition and is a soluble enzyme localized to the haemolymph. The specific activity of the other isozyme, LAP D, is highest in young larvae and associated with cell membranes, especially from the midgut and Malpighian tubules. It is probable that these two enzymes are both functionally and spatially unique in larvae of D. melanogaster .

Several genomic clones were isolated from a Drosophila library screened with cDNA prepared from abundant adult female mRNA. Cytoplasmic dot hybridizations have shown that the genes in all of these clones are expressed only in posteclosion (stages 8-14) follicles. One set of overlapping clones (lambda 20, lambda 28, and lambda 30) was localized by in situ hybridization to 66D, a previously described locus for chorion genes. Restriction mapping demonstrated that these clones contained chorion genes which had been isolated previously. Another clone, lambda 7, was mapped to chromosomal region 26A. This clone carries genes that hybridized to mRNA species similar or identical in size to the known chorion genes encompassed by lambda 28. Furthermore, one of these genes shows homology to the 66D chorion locus, apparently with the s18-1 gene. R-loop and S1-nuclease mapping indicated that lambda 7 contains two genes of 700-800 base pairs in length. Dot hybridization of cytoplasmic RNA from egg chambers demonstrated that these genes are expressed predominantly during stages 9 + 10, the time of vitelline membrane synthesis. Analysis of DNA extracted from embryos and various female tissues by dot hybridization showed that lambda 7 sequences are not amplified in the mature ovary. These results suggest that the two genes carried by lambda 7 and derived from region 26A may code for protein components of the vitelline membrane. In addition it appears that some evolutionary relatedness exists between one of these genes and a member of the chorion multigene family.

The role of esterases in malathion resistance in Culex tarsalis has been investigated. When larvae of a resistant and a sensitive strain were placed in water containing [ 14 C]malathion, malathion penetrated to give initially similar internal levels. With resistant mosquitoes, after 15 min the internal malathion concentration decreased to low levels while the monoacid degradation products accumulated in the larvae and were excreted into the surrounding water, whereas in susceptible larvae the internal malathion level stayed high and was lethal. It is suggested that the decrease in internal malathion and the resulting resistance were caused by an active malathion carboxylesterase in the resistant strain. A specific assay for malathion carboxylesterase with [ 14 C]malathion showed 55 times more activity in resistant than in susceptible larvae, whereas when general esterase activity was assayed with α-naphthyl acetate only 1.7 times the activity was found. Analyses by starch gel electrophoresis showed a peak of malathion carboxylesterase, 60-fold higher from resistant than from susceptible larvae, in a gel zone which did not stain for general esterase activity. General esterases that did not hydrolyze malathion showed different electrophoretic patterns in the two populations, which are likely due to the nonisogenic character of the strains. These results show that use of a specific assay and the demonstration of degradation of malathion in vivo are essential for assessment of the contribution of esterase activity to the malathion-resistant phenotype in mosquito populations.

A genetic analysis of an array of mitomycin-induced rearrangements in immature Drosophila oocytes is reported. Induced aberrations were recovered representing detachments of the compound- X chromosome, Y chromosome fragments, X chromosome loss and mosaicism. The spectrum of rearrangements induced by mitomycin C was very similar to that induced by X-ray treatment of immature oocytes. This work suggests that mitomycin C has two modes of action. The drug is radiomimetic for it induces the types of aberrations recovered after X-irradiation. Mitomycin C also seems to have a delayed effect which is reflected in the relatively high recovery of mosaics.

The hormone ecdysterone induces a characteristic sequence of changes in puffing activity in the salivary gland chromosomes of Drosophila melanogaster. A few puffs are induced very rapidly by the hormone and a larger number are only active after a lag period of several hours. To study the interrelationship of the activities of these "early" and "late" puffs, genotypes aneuploid for two early puffs have been constructed. In the duplication genotype the early puffs are active for less time than in the euploids while in the deficient genotype they are active for a longer period. Under appropriate assay conditions duplication of the early puffs results in a greater and more rapid response of some, but not all, late puffs to the hormone. Deletion of the early puffs results in a delayed response of the same late puffs. These data support the idea that the early puffs are autoregulated and that their products control activity at some late puff sites.

An isozyme of leucine aminopeptidase (LAP D) in Drosophila melanogaster larvae was localized in the midgut cells, midgut lumen, the peritrophic membrane and the lumen contents of the hind intestine by histochemical methods. Leucine aminopeptidase activity in larvae increased in response to increased concentrations of dietary protein. The implications of a LAP D dependence on a secretagogue stimulus are discussed.