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Summary Intestinal microbial communities from 362 anadromous Arctic char (Salvelinus alpinus) from the high Arctic Kitikmeot region, Nunavut, Canada, were characterized using high‐throughput 16S rRNA gene sequencing. The resulting bacterial communities were compared across four seasonal habitats that correspond to different stages of annual migration. Arctic char intestinal communities differed by sampling site, salinity and stages of freshwater residence. Although microbiota from fish sampled in brackish water were broadly consistent with taxa seen in other anadromous salmonids, they were enriched with putative psychrophiles, including the nonluminous gut symbiont Photobacterium iliopiscarium that was detected in >90% of intestinal samples from these waters. Microbiota from freshwater‐associated fish were less consistent with results reported for other salmonids, and highly variable, possibly reflecting winter fasting behaviour of these char. We identified microbiota links to age for those fish sampled during the autumn upriver migration, but little impact of the intestinal content and water microbiota on the intestinal community. The strongest driver of intestinal community composition was seasonal habitat, and this finding combined with identification of psychrophiles suggested that water temperature and migratory behaviour are key to understanding the relationship between Arctic char and their symbionts.

Fruit flies, Drosophila melanogaster, have been traditionally valued as a simple model system due to their easy and inexpensive culture, their relatively compact genome, and the variety of available genetic tools. However, due to similarities of their neurological and developmental pathways with those of vertebrates, Drosophila also offers advantages for developmental toxicity assays. The ability to distinguish the effects of a toxicant on adult females, males, and the developing offspring adds to the usefulness of this model. Here we describe key techniques to screen chemicals and other potential emerging toxicants such as nanoparticles on adult Drosophila female and male reproductive success. In addition, assessments of relative toxicity can be revealed by viability assays at each developmental stage from the embryo to the pharate, or preemergent, adult.

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.

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.

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.

The folic acid analog methotrexate (MTX), a competitive inhibitor of dihydrofolate reductase (DHFR), is used to treat a variety of cancers and autoimmune disorders. However, MTX also causes a wide range of toxic effects in healthy cells and is an established teratogen. Efforts to "rescue" the defects caused by MTX by administering a folate analog or by transgenic expression of a DHFR with an altered affinity for MTX have been attempted in a variety of mammals but limited protection was conferred. As a result, our understanding of the effect of MTX at the molecular genetic level remains incomplete and, in addition, continued mammalian sacrifice is not ideal. Due to the similarity of teratogenic effects produced by MTX in Drosophila melanogaster these insects were transformed with DHFR alleles to determine if rescue could be achieved. The resulting "MTX-resistant" flies were subsequently used to investigate changes in gene expression in response to MTX using semiquantitative reverse transcription PCR. The majority (12/14) of key transcripts that were affected in MTX-exposed females including transcripts involved in cell cycle, defense response, and transport were "rescued" in the "MTX-resistant" transgenic flies. These studies illustrate the utility of this invertebrate model for the investigation of molecular effects of MTX-induced teratogenicity, MTX-resistant DHFRs for gene therapy techniques, and teratogenic protection.

Some organisms that experience subzero temperatures, such as insects, fish, bacteria, and plants, synthesize antifreeze proteins (AFPs) that adsorb to surfaces of nascent ice crystals and inhibit their growth. Although some AFPs are globular and nonrepetitive, the majority are repetitive in both sequence and structure. In addition, they are frequently encoded by tandemly arrayed, multigene families. AFP isoforms from the mealworm beetle, Tenebrio molitor, are extremely potent and inhibit ice growth at temperatures below -5 degrees C. They contain a 12-amino acid repeat with the sequence TCTxSxxCxxAx, each of which makes up one coil of the beta-helix structure. TxT motifs are arrayed to form the ice-binding surface in all three known insect AFPs: the homologous AFPs from the two beetles, T. molitor and Dendroides canadensis, and the nonhomologous AFP from the spruce budworm, Choristoneura fumiferana. In this study, we have obtained the cDNA and genomic sequences of additional T. molitor isoforms. They show variation in the number of repeats (from 6 to 10) which can largely be explained by recombination at various TCT motifs. In addition, phylogenetic comparison of the AFPs from the two beetles suggests that gene loss and amplification may have occurred after the divergence of these species. In contrast to a previous study suggesting that T. molitor genes have undergone positive Darwinian selection (selection for heterogeneity), we propose that the higher than expected ratio of nonsynonymous-to-synonymous substitutions might result from selection for higher AT content in the third codon position.

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.

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.

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.

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTXcells were ~1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTXcells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing ~400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.Key words: dihydrofolate reductase, methotrexate, drug resistance, point mutation.

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.

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.

We have previously identified a Thr- and Cys-rich thermal hysteresis (antifreeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times the freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was undertaken for other antifreeze types. Many active proteins were observed, but all appeared to be isoforms of the THP that differed in their number of 12-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitutions, and N-linked glycosylation. Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tightly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated with the need to rapidly increase gene product in response to climate change. A three-dimensional model is proposed for the fully disulfide-bonded structure of T. molitor THP, which can accommodate addition or deletion of 12-amino acid repeats. The structure is a beta-helix that places most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol.

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.

Using Drosophila as a host, we have examined the effect that the presence of an intron has on the accumulation of a processed transgene mRNA. To provide a model system that was free from major position effects, two fish antifreeze protein (AFP) transgenes were arranged in divergent transcriptional orientation from a central Drosophila yolk protein promoter/enhancer region and introduced into the flies by P-element transformation and/or mobilization. In this way the organization of both the structural genes and the promoter elements mimicked their natural arrangements. When one member of the fish AFP transgene pair had its single 180 bp intron deleted, there was a 2- to 11-fold (average 5-fold) decrease in its mRNA level compared to that generated from the control gene containing the fish AFP intervening sequence. When the deleted intron was replaced by a 70 bp intervening sequence originating from the yolk protein 1 gene, mRNA accumulation was restored to its original level. Even for the streamlined genome of Drosophila, where the intron number and size are generally reduced compared to mammals, the presence of an intervening sequence appears to facilitate mRNA accumulation.

In winter flounder, the levels of type I antifreeze protein (AFP) and its mRNA vary seasonally by as much as 1000-fold. Elevated levels in the fall are prompted by the loss of long day-lengths, while higher spring temperatures correlate with AFP clearance. We have investigated the role of temperature on AFP accumulation using transgenic Drosophila melanogaster by expressing multiple AFP genes under control of the heat-inducible hsp70 promoter. AFP and AFP mRNA persisted far longer in flies reared at 10 degrees C compared to 22 degrees C. This difference appears to be mediated by cold-specific mRNA stability since no such temperature effect was observed with either an endogenous heat-inducible mRNA or a constitutively expressed mRNA.

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse alpha-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species.

In the last two decades, nanoparticles (NPs) have found applications in a wide variety of consumer goods. Titanium dioxide (TiO(2)) and silver (Ag) NPs are both found in cosmetics and foods, but their increasing use is of concern due to their ability to be taken up by biological systems. While there are some reports of TiO(2) and Ag NPs affecting complex organisms, their effects on reproduction and development have been largely understudied. Here, the effects of orally administered TiO(2) or Ag NPs on reproduction and development in two different model organisms were investigated. TiO(2) NPs reduced the developmental success of CD-1 mice after a single oral dose of 100 or 1000 mg/kg to dams, resulting in a statistically significant increase in fetal deformities and mortality. Similarly, TiO(2) NP addition to food led to a significant progeny loss in the fruit fly, Drosophila, as shown by a decline in female fecundity. Ag NP administration resulted in an increase in the mortality of fetal mice. Similarly in Drosophila, Ag NP feeding led to a significant decrease in developmental success, but unlike TiO(2) NP treatment, there was no decline in fecundity. The distinct response associated with each type of NP likely reflects differences in NP administration as well as the biology of the particular model. Taken together, however, this study warns that these common NPs could be detrimental to the reproductive and developmental health of both invertebrates and vertebrates.

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.

The widespread distribution of insects over many ecological niches is a testimony to their evolutionary success. The colonization of environments at high latitudes or altitudes required the evolution of biochemical strategies that reduced the impact of cold or freezing stress. This review focuses on our current interests in some of the genes and proteins involved in low temperature survival in insects. Although the most widespread form of protection is the synthesis of low molecular weight polyol cryoprotectants, proteins with intrinsic protective properties, such as the thermal hysteresis or antifreeze proteins are also important. These have been cloned and characterized in certain moths and beetles. Molecular techniques allowing the isolation of genes differentially regulated by low temperatures have revealed that heat shock proteins, cold stress proteins, membrane protectants, as well as ice nucleators and other less well characterized proteins likely also play a role in cold hardiness.

The transfer of chimaeric plasmids to Drosophila melanogaster cell lines has been examined as a system for investigation of the hormonal regulation of the genes coding for D. melanogaster yolk polypeptide 1 (YP1) and Locusta migratoria vitellogenin B (VgB). Constructs containing promoters and putative 5'-regulatory sequences from these genes, ligated to bacterial chloramphenicol acetyltransferase (CAT) coding DNA, were transfected into Drosophila Kc (Kc-H) and S3 cells, and transient expression of CAT was assayed. Activity was expressed both from the homologous promoter of pYP1CAT and from the heterologous locust promoter of pVgCAT at comparable levels. In S3 cells, with calcium phosphate-mediated transfer of pYP1CAT there was a twofold induction of CAT activity after the addition of 10(-6) M ecdysterone, but no hormonal stimulation was noted when the polycation polybrene was used to achieve transfection. For Kc cells, calcium phosphate was ineffective for transfection, and after transfection with polybrene neither pYP1CAT nor pVgCAT was induced by the juvenile hormone (JH) analog methoprene. It is concluded that S3 cells may be useful for investigating the molecular basis of gene regulation by ecdysteroids, but conditions suitable for the analysis of JH action have not yet been established.

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9- cis -retinoic acid (9- cis -RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9- cis -RA and all- trans -RA with high affinity (IC 50 = 61.2–107.7 nM; K d = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9- cis -RA, in addition to all- trans -RA. We estimate that whole embryos contain 3 nM RA, including 9- cis -RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9- cis -RA to its RXR is established relatively early in evolution and embryonic development.

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 μM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 μM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 μM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.Key words: dihydrofolate reductase, Drosophila melanogaster, methotrexate.

Antifreeze proteins (AFPs) are encoded by approximately 17 genes in the spruce budworm, Choristoneura fumiferana. Northern analysis using 6 different cDNA probes showed isoform-specific patterns that varied during development. Transcripts for the majority of isoforms were most abundant in the second instar overwintering stage, but some were also detected in first instar and even in egg stages. In situ hybridization using riboprobes corresponding to two 9 kDa protein isoforms showed differential AFP expression even in second instars; CfAFP10 RNA was detected in all tissues, but CfAFP337 RNA distribution was more limited. Two genomic regions encoding three AFP genes have been isolated. Presumptive regulatory regions conferred transcriptional activity when placed upstream of a luciferase reporter sequence and transfected into a C. fumiferana cell line. The CfAFP2.26 core promoter is an 87 bp sequence containing a TATA box, whereas the CfAFP2.7 core promoter is a 76 bp sequence with both a TATA box and CAAT box, which directed higher reporter activities when tested in vitro. Reporter activity was not enhanced with five different hormones, although lower activities were observed with all intron-containing constructs. AFP message half-life, as assessed using reporter assays, was not appreciably influenced by isoform-specific-3'UTRs. These studies successfully demonstrate the temporal and spatial diversity of AFP expression encoded by this small gene family, and underscore the complexity of their regulation.

The inhibition activities of two antifreeze proteins (AFPs) on the formation of tetrahydrofuran (THF) clathrate hydrate have been tested. AFPs from fish (wfAFP) and insect (CfAFP) changed the morphology of growing THF hydrate crystals. Also, both AFPs showed higher activities in inhibiting the formation THF hydrate than a commercial kinetic inhibitor, poly(vinylpyrrolidone) (PVP). Strikingly, both AFPs also showed the ability to eliminate the "memory effect" in which the crystallization of hydrate occurs more quickly after the initial formation. This is the first report of molecules that can inhibit the memory effect. Since the homogeneous nucleation temperature for THF hydrate was measured to be 237 K, close to that observed for ice itself, the action of kinetic inhibitors must involve heterogeneous nucleation. On the basis of our results, we postulate a mechanism for heterogeneous nucleation, the memory effect and its elimination by antifreeze proteins.

Methotrexate (MTX), a synthetic folate analog, is a tight-binding inhibitor of dihydrofolate reductase (DHFR), a key enzyme for the biosynthesis of purines, thymidylate, and several amino acids. As a consequence, MTX decreases titres of reduced folates, interferes with DNA synthesis, and results in the arrest of rapidly proliferating cells, making it a drug of choice for the treatment of a variety of cancers and auto-immune disorders. MTX is also a known teratogen in all higher animals tested, but there is little information about the effects of this drug on invertebrates. Here we show that MTX has little effect on the survival of Drosophila melanogaster adult flies, but severely diminishes female fecundity. Reduced oviposition, coupled with aberrant egg morphologies, resulted in near sterility of MTX-treated females. Rare surviving progeny showed developmental abnormalities including larval tumors, and bristle, wing, eye, and leg defects. To determine if these phenotypes could be attributed solely to DHFR inhibition, microarray analysis was undertaken and included MTX-treated females, ovaries, and cell line samples. Genes encoding transcripts that were perturbed by the drug were verified using quantitative real-time RT-PCR. Many of these genes were involved in cell cycle regulation, signal transduction, transport, defense response, transcription, or various aspects of metabolism. These studies show that MTX treatment has multiple targets and, in addition, provides a new invertebrate model for the study of teratogenesis.

Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

Antifolates, such as methotrexate, are used to inhibit dihydrofolate reductase (DHFR), an enzyme essential for the biosynthesis of thymidylate, purines, and several amino acids. DHFR sequences corresponding to mutations found in a methotrexate resistant Drosophila S3 cell line (L30Q), a methotrexate resistant fly population (K31P, Q134K), as well as predicted in silico (L22R) were expressed in Chinese Hamster Ovary (CHO) cells. The L30Q and L22R DHFRs both conferred resistance to methotrexate. L22R DHFR provided approximately 200-fold resistance to methotrexate when compared to wild-type Drosophila DHFR allowing CHO(L22R) cells to divide in 10 microM methotrexate, a level of resistance not previously observed in any mammalian system. Constructs using this substitution in combination with other Drosophila DHFR specific residues would make excellent candidates for gene therapy and genetic markers in the treatment of certain human disorders.

We review a novel computational model for the study of crystal structures both on their own and in conjunction with inhibitor molecules. The model advances existing Monte Carlo (MC) simulation techniques by extending them from modeling 3D crystal surface patches to modeling entire 3D crystals, and by including the use of “complex” multicomponent molecules within the simulations. These advances makes it possible to incorporate the 3D shape and non-uniform surface properties of inhibitors into simulations, and to study what effect these inhibitor properties have on the growth of whole crystals containing up to tens of millions of molecules. The application of this extended MC model to the study of antifreeze proteins (AFPs) and their effects on ice formation is reported, including the success of the technique in achieving AFP-induced ice-growth inhibition with concurrent changes to ice morphology that mimic experimental results. Simulations of ice-growth inhibition suggest that the degree of inhibition afforded by an AFP is a function of its ice-binding position relative to the underlying anisotropic growth pattern of ice. This extended MC technique is applicable to other crystal and crystal–inhibitor systems, including more complex crystal systems such as clathrates.

Drosophila melanogaster , a freeze intolerant and cold shock sensitive insect, was transformed with the hyperactive insect antifreeze protein gene (AFP) from the spruce budworm, Choristoneura fumiferana . Transformation P-element constructs (pCasper) were made with CfAFP 337 isoform DNA using a strong constitutive promoter, Actin 5c. This is the first report of insect AFP used to transform another insect. Properly folded active insect AFP was only detected when signal sequences were used to target proteins to the endoplasmic reticulum for secretion into the hemolymph. The 18 residue Drosophila binding protein signal sequence (BiP) constructs resulted in transformed fly lines with significantly higher AFP expression in hemolymph than when the native C. fumiferana AFP signal sequence was used. The resultant transgene fly lines have the highest levels of thermal hysteresis, 0.8 °C, seen for any engineered Drosophila . Despite the high level of expression, even higher than some overwintering fish with natural levels of endogenous AFP, the transformants did not display any cold shock resistance compared to controls or low AFP expressing lines. These results indicate that insect AFP alone cannot protect Drosophila from cold shock and may not be useful for Drosophila cryopreservation.

A complementary deoxyribonucleic acid (cDNA) and the corresponding gene segment encoding a member of the 70-kDa heat shock protein (Hsp70) family have been cloned and sequenced from Locusta migratoria, the African migratory locust. These animals are noted for their thermotolerance, which can exceed temperatures of 50 degrees C. Conceptually translated, the sequence shows a 654-residue protein with theoretical molecular weight of 71.4 kDa, which more closely resembles the mammalian Hsp70 (84-85% similarity) than Hsp70 from other insects, with approximately 75% similarity to the sequence from the fruit fly. Comparisons of cDNA and genomic sequences show that the gene contains 2 introns, a 245-bp intron located in the 5' untranslated region and a 91-bp intron in the coding region. Transcript abundance, as estimated by Northern blot analysis and reverse transcription-polymerase chain reaction, shows that heat shock treatment (45 degrees C for 3 hours) does not elevate hsp70 messenger ribonucleic acid levels in fat bodies or in neural tissues. Immunological assays of Hsp70 show that the protein is constitutively expressed, with a modest, approximately 2-fold induction after a 3-hour heat shock in fat body preparations. Although this sequence could be an hsc70 rather than an hsp70, it was the only cDNA isolated from heat-shocked tissue. Whatever the formal designation, such modest induction and constitutive expression may be ideally suited as an adaptation to the locust's chronic exposure to heat shock temperatures and the consequent demand for chaperone proteins.

High–resolution three–dimensional structures are now available for four of seven non–homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice–binding site of the AFP has been defined by site–directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice–binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic—more so than that portion of the protein exposed to the solvent. Surface–surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts.

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 beta-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-Delta-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 beta-helical structure and hence the size of its ice-binding face.

The freeze-intolerant insect, Choristoneura fumiferana (spruce budworm), produces multiple antifreeze protein (AFP) isoforms for protection during the overwintering stage. We now report the cloning of AFP genes from insects; Afp-Lu1 encodes a approximately 9-kDa AFP isoform, and Afp-Iu1 encodes a approximately 12-kDa AFP isoform. Both CfAFP genes have similar structures with a single 3- to 3.6-kb intron interrupting the coding region. The second exon of an additional CfAFP gene, 2.7a, encoding a new approximately 9-kDa isoform, was found 3.7 kb upstream of Afp-Lu1 and demonstrates that some AFP family members are linked in tandem. This gene appears to encode an AFP with 68-76% identity to previously isolated CfAFPs. With its eight Cys residues necessary for disulfide bonding and five perfectly conserved 'Thr button' (Thr-Xaa-Thr) ice-binding motifs, it can be modeled as a functional AFP. Southern blot analysis shows that there are approximately 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies. Transcript accumulation from Afp-Lu1 and Afp-Iu1 (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer. Contrary to expectations, this differential expression demonstrates that CfAFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures.

THE origin and function of introns in eukaryotic genes has provoked considerable debate since their discovery in 1977. Central to this issue are studies on the highly conserved enzyme, triosephosphate isomerase (TPI, EC 5.3.1.1). The 'introns early' argument suggests that introns are as old as the genes themselves and that the apparent correlation of many of the intron sites in plant, animal and fungal TPI genes with the boundaries of modules1 is evidence of the assembly of ancient proteins by exon shuffling2–4. In contrast, the 'introns late' view holds that ancient genomes contained few if any introns; introns were inserted into pre-existing genes during the last billion years5,6. We have found that the TPI gene from the mosquito, Culex tarsalis, contains an intron in a unique position that was predicted by W. Gilbert2 and the exon shuffling hypothesis.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation., Author Summary Vitellogenesis, a hormonally-regulated process for the synthesis of yolk proteins by the fat body or liver and their sequestration in developing oocytes, takes place in all oviparous animals except mammals. Polyploidy has also been implicated in hormone-regulated development and reproduction. Although juvenile hormone (JH) is known to regulate polyploidy in insect models and also plays a pivotal role in stimulating insect vitellogenesis, the molecular mechanisms remain poorly understood. In the migratory locust, Locusta migratoria, vitellogenesis is dependent on JH, and JH stimulates DNA replication and increases ploidy in the fat body. Here, we show that a JH-receptor complex comprised of Methoprene-tolerant (Met) and a steroid receptor co-activator activates the transcription of two mini-chromosome maintenance (Mcm) genes, Mcm4 and Mcm7. Knockdown of Mcm4 or Mcm7 via RNAi can phenocopy JH-deprivation and Met-depletion, resulting in reduced ploidy, blocked vitellogenin (Vg) expression, as well as arrested oocyte maturation and ovarian growth. This study provides evidence that JH acts through its receptor on the Mcm machinery to replicate the genome of fat body cells in preparation for the massive synthesis of Vg and possibly other proteins required for oocyte maturation and egg production.

There is increasing evidence that heat shock (HS) has long-term effects on electrophysio- logical properties of neurons and synapses. Prior HS protects neural circuitry from a subsequent heat stress but little is known about the mechanisms that mediate this plasticity and induce thermotolerance. Exposure of Locusta migratoria to HS conditions of 45°C for 3 h results in thermotolerance to hitherto lethal tempera- tures. Locust flight motor patterns were recorded during tethered flight at room temperature, before and after HS. In addition, intracellular action potentials (APs) were recorded from control and HS motoneurons in a semi-intact preparation during a heat stress. HS did not alter the timing of representative depressor or elevator muscle activity, nor did it affect the ability of the locust to generate a steering motor pattern in response to a stimulus. However, HS did increase the duration of APs recorded from neuropil segments of depressor motoneu- rons. Increases in AP duration were associated with protection of AP generation against failure at subse- quent elevated temperatures. Failure of AP generation at high temperatures was preceded by a concomitant burst of APs and depolarization of the membrane. The protective effects of HS were mimicked by pharmaco- logical blockade of IK 1 with tetraethylammonium (TEA). Taken together, these findings are consistent with a hypothesis that HS protects neuronal survival and function via K 1 channel modulation. © 2001 John

The cloning of pyrroline 5-carboxylate reductase from Drosophila melanogaster was accomplished by cDNA complementation of an Escherichia coli proline auxotroph. The corresponding P5cr gene is tightly clustered with three other expressed coding regions. A bidirectional promoter, an overlapping 3'UTR and an intraintronic sequence may all be found in only 4.3 kb, making this the most densely clustered region of unrelated genes in any eukaryote.

To facilitate studies on the hormonal control of development in the migratory locust, Locusta migratoria, we have undertaken the cloning of cDNAs for nuclear hormone receptors. Sequences obtained by polymerase chain reaction (PCR) showed homology with receptor family members including the ecdysteroid receptor (EcR). A cDNA clone corresponding to the EcR fragment includes an open reading frame of 1622 nucleotides, predicting a 59 kDa protein showing clear homology with EcRs and distinct from other classes of nuclear receptors. Northern analysis revealed a major transcript of 9.2 kb. In fifth instar fat body, the transcript was most abundant at the end of the instar when ecdysone titres are highest. There was no obvious evidence of EcR regulation by a juvenile hormone analog. Although its role in development may be similar, the locust ecdysone receptor (LmEcR) is divergent from EcRs characterized from insects belonging to the dipteran and lepidopteran orders, presumably reflecting the more ancestral sequence in the relatively primitive locust.

We have purified a thermal hysteresis (antifreeze) protein, with up to 100 times the specific activity of fish antifreeze proteins, from the common yellow mealworm beetle, Tenebrio molitor. It is a threonine- and cysteine-rich protein, of relative molecular mass 8,400, composed largely of 12-amino-acid repeats. We estimate that a concentration of roughly 1 mg ml−1 of this protein can account for the 5.5 °C of thermal hysteresis found in Tenebrio larvae (Fig. 1).

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

We have used Drosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting. Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 degree C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenic Drosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.

Fish antifreeze proteins (AFPs) have been reported by some researchers to prolong the viability of tissues, organs, and embryos under hypothermic conditions, while others have observed no such effect or even AFP-mediated cryotoxicity. We examined the influence of Type III AFP from Atlantic wolffish on cold tolerance in a whole animal model system, transgenic Drosophila. The activity of the AFP, transgenically expressed under the transcriptional control of the female-specific yp1 and yp2 promoters and secreted into fly hemolymph, was confirmed through thermal hysteresis and differential scanning calorimetry measurements as well as through observations of ice crystal morphology. In cold exposure trials, at 0 degrees C and at -7 degrees C, transgenic adult flies of both sexes exhibited greater survival than nontransgenic controls even though the antifreeze was only produced in females. We attribute these observations to the expression of the xanthine dehydrogenase marker gene used to identify transgenics, rather than the production of AFP. Type III AFP therefore appears unable to enhance survival of adult Drosophila under hypothermic conditions.