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Successful integrated management of the invasive predatory sea lamprey (Petromyzon marinus) in the Laurentian Great Lakes of North America is owed largely to the long history of beneficial use of two lampricides: 3-trifluoromethyl-4-nitrophenol (TFM) and 2′,5-dichloro-4′-nitrosalicylanilide (niclosamide). Ensuring continued successful sea lamprey control necessitates consideration of possible next-generation lampricides to supplement or replace current lampricides. This review identifies fifteen hallmarks of success for current lampricides to be used as design criteria in a search for next-generation lampricides. A three-stage research approach is outlined. Targeted research using omics, computer modelling, and high-throughput technology to define molecular mechanisms and high probability molecular targets for sea lamprey selective toxic action is crucial to prioritizing chemical candidates. Targeted delivery or identifying synergists to existing or new lampricides can provide increased efficiency and reduced environmental impact. Ultimate development of next-generation lampricides will rely on traditional toxicity testing methodologies to ensure safety and regulatory compliance.

Biliary atresia (BA) is a rare neonatal disease with unknown causes. Approximately 10% of BA cases develop in utero with other congenital defects that span a large spectrum of disease variations, including degeneration of the gall bladder and bile duct as well as malformation of the liver, intestines, and kidneys. Similar developmental alterations are manifested in a unique animal model, the sea lamprey (Petromyzon marinus), in which BA occurs naturally during metamorphosis. With the likelihood of conserved developmental mechanisms underlying organogenesis and degeneration, lamprey developmental BA may be a useful model to infer mechanisms underlying human embryonic BA. We reasoned that hepatobiliary transcriptomes regulate the transition between landmark stages of BA. Therefore, we examined sea lamprey hepatobiliary transcriptomes at four stages (M0, metamorphic stage 0 or larval stage, no BA; M2, metamorphic stage 2, onset of BA; M5, metamorphic stage 5, BA, and heightened hepatocyte proliferation and reorganization; and JV, juvenile, completion of BA) using messenger RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analyses. We found gene‐expression patterns associated with the transition between these stages. In particular, transforming growth factor β (TGF‐β), hedgehog, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase‐Akt, Wnt, and mitogen‐activated protein kinase pathways were involved during biliary degeneration. Furthermore, disrupting the TGF‐β signaling pathway with antagonist or small interfering RNA treatments at the onset of BA delayed gall bladder and bile duct degeneration. Conclusion: Distinctive gene‐expression patterns are associated with the degeneration of the biliary system during developmental BA. In addition, disrupting TGF‐β signaling pathway at the onset of BA delayed biliary degeneration.

Insects face diverse biotic and abiotic stresses that can affect their survival. Many of these stressors impact cellular metabolism, often resulting in increased accumulation of reactive oxygen species (ROS). Consequently, insects will respond to these stressors by increasing antioxidant activity and increased production of heat shock proteins (HSPs). In this study, the effect of heat, cold, starvation, and parasitism by Habroacon hebetor wasps was examined in the carob moth, Ectomyelois ceratoniae, to determine which responses were common to different stresses. For all stressors, malondialdehyde levels increased, indicative of oxidative stress in the insects. The activity of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), increased with each stress, suggesting that these enzymes were serving a protective role for the insects. Heat (46°C for 100 min) and cold (-15°C for 30 min) treatments caused significant mortalities to all developmental stages, but pretreatments of moderate heat (37°C for 10 min) or cold (10°C for 10 min) induced thermotolerance and reduced the mortality rates when insects were subsequently exposed to lethal temperatures. Quantitative RT-PCR confirmed that heat and cold tolerance were associated with up-regulation of two HSPs, HSP70 and HSP90. Interestingly, HSP70 transcripts increased to a greater extent with cold treatment, while HSP90 transcripts increased more in response to high temperatures. RNA interference (RNAi)-mediated knockdown of either HSP70 or HSP90 transcripts was achieved by injecting larvae with dsRNA targeting each gene's transcripts, and resulted in a loss of acquired thermotolerance in insects subjected to the heat or cold pretreatments. These observations provide convincing evidence that both HSP70 and HSP90 are important mediators of the acquired thermotolerance. Starvation and parasitism by wasps caused differential expression of the HSP genes. In response to starvation, HSP90 transcripts increased to a greater extent than HSP70, while in contrast, HSP70 transcripts increased to a greater extent than those of HSP90 during the first 48 h of wasp parasitism. These results showed the differential induction of the two HSPs' transcripts with variable stresses. As well as, heat, cold, starvation, and parasitism induce oxidative stress, and antioxidant enzymes likely play an important role in reducing oxidative damage in E. ceratoniae.

Control of sea lamprey Petromyzon marinus in the Great Lakes requires accurate assessment of the instream distribution of this pest species and the ability to distinguish it from the four lamprey species that are native to the Great Lakes (American brook lamprey Lethenteron appendix , chestnut lamprey Ichthyomyzon castaneus , northern brook lamprey Ichthyomyzon fossor , and silver lamprey Ichthyomyzon unicuspis ). We developed PCR-based environmental DNA (eDNA) assays to distinguish among the four “genetic species” of Great Lakes lampreys (silver and northern brook lampreys were genetically indistinguishable), tested them under laboratory conditions, and demonstrated in the field that both spawning and larval sea lamprey can be detected using eDNA. In the laboratory, mean detection frequency decreased with increasing flow rate, but was not significantly related to larval density or temperature over the range of relatively high densities tested. Proof-of-concept was demonstrated in the field when sea lamprey eDNA was detected during the spawning season (when large-bodied adults, their gametes, and later their carcasses were present); sampling effort involved collection of three 1–2 L water samples at each of four transects (0.4–1.0 km apart). Mean detection frequency remained high (81–97%) until spawning ceased at the end of June, but decreased thereafter, falling to 6% by mid-August. We also demonstrated that eDNA of smaller, burrowing larvae (in water collected after mid-August) was detectable in two of four streams with medium to high larval density (1–2 larvae/m 2 ), but not at low densities ( 2 ), potentially due to the exacerbating effect of high flow rates.

Lampreys diverged from the jawed vertebrate lineage approximately 500 million years ago. Lampreys undergo sex differentiation much later than most other vertebrates, and ovarian differentiation occurs several years before testicular differentiation. The genetic basis of lamprey sex differentiation is of particular interest both because of the phylogenetic importance of lampreys and because of their unusual pattern of sex differentiation. As well, differences between parasitic and non-parasitic lampreys may first become evident at ovarian differentiation. However, nothing is known about the genetic basis of ovarian differentiation in lampreys. This study examined potential differences in gene expression before, during, and after ovarian differentiation in parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzon fossor . Eight target genes (17β-hydroxysteroid dehydrogenase, germ cell-less, estrogen receptor β, insulin-like growth factor 1 receptor, daz-associated protein 1, cytochrome c oxidase subunit III, Wilms’ tumour suppressor protein 1, and dehydrocholesterol reductase 7) were examined. Northern brook lamprey displayed higher expression of cytochrome c oxidase subunit III, whereas chestnut lamprey displayed higher expression of insulin-like growth factor 1 receptor; these genes may be involved in apoptosis and oocyte growth, respectively. Presumptive male larvae had higher expression of Wilms’ tumour suppressor protein 1, which may be involved in the undifferentiated gonad and/or later testicular development. Differentiated females had higher expression of 17β hydroxysteroid dehydrogenase and daz-associated protein 1, which may be involved in female development. This study is the first to identify genes that may be involved in ovarian differentiation and fecundity in lampreys.

Carbon dioxide (CO(2)) is an important long-range chemosensory cue used by blood-feeding female mosquitoes to find their hosts. The CO(2) receptor in Drosophila melanogaster was previously determined to be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)-mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO(2) sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi-mediated knockdown of AaGr2 expression had no impact on CO(2) reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO(2) receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO(2) receptor in different species are discussed. The functional identification of the CO(2) receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.

Proposals to farm non-native and genetically modified species are often highly contentious because there is no reliable method of ensuring that they do not escape, reproduce and become environmental problems. Suggested approaches to prevent breeding outside hatcheries are unable to guarantee sterility in both sexes or cannot easily be applied to animals. We developed and tested on two contentious groups — fish and oysters — genetic constructs that render the animals functionally sterile outside of hatchery conditions, but allow normal development in captivity following brief provision of a repressor compound at a particular life history stage. “Sterile Feral” constructs consist of a stage-specific promoter, a blocker for a critical developmental gene and a repressible element. In transient assays, prototype constructs worked as expected, causing high mortality under non-repressed conditions in both fish and molluscs, and lower mortality, often equal to controls, in the presence of the repressible element (doxycycline). Sterile feral technology can potentially be applied to a wide range of species and be designed to use multiple, independent sterilizing constructs, minimizing the risk of establishing feral populations. Full tests of efficiency, however, require trials in integrated lines, currently in progress, and the usefulness of the technology could depend on the development of an improved repressible element.

Background Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. Results We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. Conclusions HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Bactrocera tryoni is a serious pest of horticulture in eastern Australia. Here we review molecular data relevant to pest status and development of a transformation system for this species. The development of transformation vectors for non-drosophilid insects has opened the door to the possibility of improving the sterile insect technique (SIT), by genetically engineering factory strains of pest insects to produce male-only broods. Transposition assays indicate that all five of the vectors currently used for transformation in non-drosophilid species have the potential to be useful as transformation vectors in B. tryoni. Evidence of cross mobilization of hobo by an endogenous Homer element emphasises the necessity to understand the endogenous transposons within a species. The sex-specific doublesex and yolk protein genes have been characterized with a view to engineering a female-specific lethal gene or modifying gene expression through RNA interference (RNAi). Data are presented which indicate the potential of RNAi to modify the sex ratio of resultant broods. An understanding of how pest status is determined and maintained is being addressed through the characterization of genes of the circadian clock that enable the fly to adapt to environmental cues. Such an understanding will be useful in the future to the effective delivery of sophisticated pest control measures.

We examined the relative efficiency of microinjection, electroporation and particle bombardment for introducing DNA into the embryos of the Kuruma prawn, Penaeus japonicus. The amount of DNA that could be delivered into one- to four-celled embryos and subsequently recovered was examined using plasmids with selectable antibiotic resistance. Microinjection proved to be the most reliable technique. The mean value for plasmid recovery from injected embryos ranged from 225 to 243 plasmids per embryo. Electroporation was effective in delivering small, but measurable amounts of DNA into embryos with values ranging from 0.5 to 0.9 plasmids per embryo. Bombardment failed to introduce any significant amount of DNA into viable embryos.

Members of the hAT transposable element family are mobile in non-host insect species and have been used as transformation vectors in some of these species. We report that the Queensland fruit fly, Bactrocera tryoni, contains at least two types of insect hAT elements called Homer and a Homer-like element (HLE). The Homer element is 3789 bp in size and contains 12-bp imperfect inverted terminal repeats. The Homer element contains a long open reading frame (ORF) that encodes a putative transposase. Three different copies of this long ORF were recovered from the B. tryoni genome and, upon transcription and translation in an in vitro system, all produced transposase. The HLE is an incomplete element since no 3' inverted terminal repeat (ITR) was found. Homer and the HLE are as related to one another as either is to the other insect hAT elements such as Hermes, hobo, hermit and hopper. The structure and distribution of these two Homer elements is described.

RNA interference has already proven itself to be a highly versatile molecular biology tool for understanding gene function in a limited number of insect species, but its widespread use in other species will be dependent on the development of easier methods of double-stranded RNA (dsRNA) delivery. This study demonstrates that RNA interference can be induced in the mosquito Aedes aegypti L. (Diptera: Culicidae) simply by soaking larvae in a solution of dsRNA for two hours. The mRNA transcripts for β-tubulin, chitin synthase-1 and -2, and heat shock protein 83 were reduced between 30 and 50% three days post-dsRNA treatment. The dsRNA was mixed with a visible dye to identify those individuals that fed on the dsRNA, and based on an absence of RNA interference in those individuals that contained no dye within their guts, the primary route of entry of dsRNA is likely through the gut epithelium. RNA interference was systemic in the insects, inducing measurable knock down of gene expression in tissues beyond the gut. Silencing of the β-tubulin and chitin synthase-1 genes resulted in reduced growth and/or mortality of the larvae, demonstrating the utility of dsRNA as a potential mosquito larvicide. Silencing of chitin synthase-2 did not induce mortality in the larvae, and silencing of heat shock protein 83 only induced mortality in the insects if they were subsequently subjected to a heat stress. Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae were also soaked in dsRNA designed to specifically target either their own β-tubulin gene, or that of A. aegypti, and significant mortality was only seen in larvae treated with dsRNA targeting their own gene, which suggests that dsRNA pesticides could be designed to be species-limited.

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.

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.

Carbon dioxide (CO(2)) is an important long-range chemosensory cue used by blood-feeding female mosquitoes to find their hosts. The CO(2) receptor in Drosophila melanogaster was previously determined to be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)-mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO(2) sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi-mediated knockdown of AaGr2 expression had no impact on CO(2) reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO(2) receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO(2) receptor in different species are discussed. The functional identification of the CO(2) receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.

International audience; Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments. (C) 2010 Elsevier Ltd. All rights reserved.

A serious shortcoming of many insecticides is that they can kill non-target species. To address this issue, we harnessed the sequence specificity of RNA interference (RNAi) to design orally-delivered double-stranded (ds) RNAs that selectively killed target species. Fruit flies (Drosophila melanogaster), flour beetles (Tribolium castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta) were selectively killed when fed species-specific dsRNA targeting vATPase transcripts. We also demonstrate that even closely related species can be selectively killed by feeding on dsRNAs that target the more variable regions of genes, such as the 3' untranslated regions (UTRs): four species of the genus Drosophila were selectively killed by feeding on short (40 nt) dsRNAs that targeted the 3' UTR of the gamma-tubulin gene. For the aphid nymphs and beetle and moth larvae, dsRNA could simply be dissolved into their diets, but to induce RNAi in the drosophilid species, the dsRNAs needed to be encapsulated in liposomes to help facilitate uptake of the dsRNA. This is the first demonstration of RNAi following ingestion of dsRNA in all of the species tested, and the method offers promise of both higher throughput RNAi screens and the development of a new generation of species-specific insecticides.

With twelve Drosophila genomes now sequenced, there is a growing need to develop higher-throughput methods for identifying the functions of the many newly identified genes. Genetic transformation and RNA interference are two technologies that have been used extensively to facilitate gene-function studies in Drosophila melanogaster, to introduce genes or block the expression of endogenous genes, respectively. Both of these technologies typically require the delivery of nucleic acids into developing insect embryos, and virtually all studies to date have relied on microinjection as the DNA delivery method of choice. In this study, we describe the use of biolistics as a higher-throughput method of nucleic acid delivery. By bombarding dechorionated D. melanogaster embryos with 1 microm gold beads coated with P-element or piggyBac transformation vectors, we observed transformation frequencies (3-4%) that are comparable to those achieved using microinjection methods, but in only a fraction of the time required for the DNA delivery. Biolistic delivery of double-stranded RNA (dsRNA) specific to a beta-glucuronidase (gus) transgene resulted in a significant (71%) reduction in gus transcripts in embryos and the RNA interference (RNAi) persisted through two successive larval molts, albeit at reduced levels. DsRNAs specific to four essential genes were delivered to embryos and resulted in arrested development and phenotypes that closely match that of null mutations. These results suggest that biolistic delivery of dsRNA into embryos could be adapted for high throughput RNAi screens of early Drosophila developmental genes.

A group of transposons, named maT, with characteristics intermediate between mariner and Tc1 transposons, is described. Two defective genomic copies of MdmaT from the housefly Musca domestica, with 85% identity, were found flanking and imbedded in the MdalphaE7 esterase gene involved in organophosphate insecticide resistance. Two cDNA clones, with 99% identity to each other and 72%-89% identity to the genomic copies were also obtained, but both represented truncated versions of the putative open reading frame. A third incomplete genomic copy of MdmaT was also identified upstream of the putative M. domestica period gene. The MdmaT sequences showed high identity to the transposable element Bmmar1 from the silk-worm moth, Bombyx mori, and to previously unidentified sequences in the genome of Caenorhabditis elegans. A total of 16 copies of full-length maT sequences were identified in the C elegans genome, representing three variants of the transposon, with 34%-100% identity amongst them. Twelve of the copies, named CemaT1, were virtually identical, with eight of them encoding a putative full length, intact transposase. Secondary structure predictions and phylogenetic analyses confirm that maT elements belong to the mariner-Tc1 superfamily of transposons, but their intermediate sequence and predicted structural characteristics suggest that they belong to a unique clade, distinct from either mariner-like or Tc1-like elements.

Malathion resistance in a strain of Culex tarsalis mosquitoes is due primarily to the activity of a malathion carboxylesterase (MCE). The resistant strain was 150 times more resistant to malathion than the susceptible strain and was weakly resistant to malaoxon and carbaryl, but not to any other insecticide tested. The phenotype could be reversed with the carboxylesterase inhibitor triphenylphosphate, but no synergism was observed with either the phosphatase or polysubstrate monooxygenase inhibitors, NaF and piperonyl butoxide. MCE is expressed throughout development and is most concentrated in the gut tissues of the larvae. Subcellular fractionation indicated that MCE was localized primarily in the mitochondria of resistant insects and the cytoplasm of susceptible insects. The enzyme was purified to homogeneity from both strains, and has a molecular weight of 59,000. However, chromatofocusing indicated that resistant insects have two MCEs with pIs of 6.8 and 6.2, while susceptible insects possessed only one MCE with a pI of 6.8. The MCE unique to the resistant strain hydrolysed malathion 18 times faster than the MCE common to both strains, suggesting that malathion resistance in C. tarsalis is due to the presence of a qualitatively different esterase in the resistant strain.

Resistance to the organophosphorus insecticide malathion in genetically related strains of the Australian sheep blowflyLucilia curprina was examined. Separate lines of blowflies were established by homozygosis of the fourth chromosome of the parental RM strain. Both the RM and the derived resistant (der-R) strains are approximately 100 times more resistant to malathion than the related susceptible der-S strain, resistance being correlated with a 45- to 50-fold increase in a malathion carboxylesterase (MCE) activity. MCE has a pH optimum ranging between 6.6 and 8.0 and is strongly inhibited by the carboxylesterase inhibitors triphenyl phosphate, paraoxon, and diiospropylfluorophosphate. Subcellular fractionation revealed that MCE was localized predominantly to the cytosol and mitochondria in both resistant and susceptible blowflies. A single MCE was purified to homogeneity from RM blowflies. It has a pI of 5.5, is a monomer of 60.5 kDa, and hydrolyzes malathion with aV max of 755 nmol/min/mg protein and aK m of 11.0 µM. L. cuprina have thus evolved a remarkable MCE which is faster and more efficient at hydrolyzing a specific insecticide than any other insect esterase yet described.

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.