Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions.

Although many insects successfully live in dangerous environments exposed to diverse communities of microbes, they are often exploited and killed by specialist pathogens. Studies of host-pathogen interactions (HPI) provide valuable insights into the dynamics of the highly aggressive coevolutionary arms race between entomopathogenic fungi (EPF) and their arthropod hosts. The host defenses are designed to exclude the pathogen or mitigate the damage inflicted while the pathogen responds with immune evasion and utilization of host resources. EPF neutralize their immediate surroundings on the insect integument and benefit from the physiochemical properties of the cuticle and its compounds that exclude competing microbes. EPF also exhibit adaptations aimed at minimizing trauma that can be deleterious to both host and pathogen (eg, melanization of hemolymph), form narrow penetration pegs that alleviate host dehydration and produce blastospores that lack immunogenic sugars/enzymes but facilitate rapid assimilation of hemolymph nutrients. In response, insects deploy an extensive armory of hemocytes and macromolecules, such as lectins and phenoloxidase, that repel, immobilize, and kill EPF. New evidence suggests that immune bioactives work synergistically (eg, lysozyme with antimicrobial peptides) to combat infections. Some proteins, including transferrin and apolipophorin III, also demonstrate multifunctional properties, participating in metabolism, homeostasis, and pathogen recognition. This review discusses the molecular intricacies of these HPI, highlighting the interplay between immunity, stress management, and metabolism. Increased knowledge in this area could enhance the efficacy of EPF, ensuring their future in integrated pest management programs.

Genetic variation in 12S and 16S mitochondrial rDNA genes of four geographically isolated populations of Gulf Coast ticks (Acari: Ixodidae).

Single-strand conformation polymorphism (SSCP) analysis was examined in a 303-bp region of the 16S and 12S mitochondrial rDNA genes to study haplotype frequencies among populations of Gulf Coast ticks collected from Refugio Co., TX, Payne Co., OK, and two sites in Osage Co., KS. Seven haplotypes were identified from the 16S rDNA gene fragment, whereas only two haplotypes were detected from the 12S fragment. Only the results from the 16S rDNA fragment are discussed. Haplotype diversity was greatest in Kansas (site 1), where three of the four haplotypes detected were unique to this site. All Gulf Coast tick populations shared the fourth haplotype. Two haplotypes were determined for Texas and Oklahoma populations, one of which appeared only in Texas, whereas the other was shared. Nei's haplotype diversity (h) indicated that the Texas population was relatively homogeneous (15%), whereas the remaining populations were heterogeneous (42-59%), although the Bonferroni confidence interval found no significant differences (P < 0.05). Nucleotide sequencing of the seven haplotypes and subsequent phylogenetic analysis using neighbor joining showed a monophyletic relationship among these haplotypes. One haplotype, shared by both Oklahoma and Kansas (site 2), was basal to the remaining haplotypes and formed a distinct clade. Two haplotypes, both from Kansas (site 1), formed a unique clade, whereas the remaining four haplotypes were unresolved polytomies.

Chimeric Mos1 and piggyBac transposases result in site-directed integration.

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, integration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding domain was fused to the NH(2) terminus of the Mos1 and piggyBac transposases and a target plasmid was created that contained upstream activating sequences (UAS), to which the Gal4 DBD binds with high affinity. The transpositional activity of the Gal4-Mos1 transposase was 12.7-fold higher compared to controls where the Gal4-UAS interaction was absent and 96% of the recovered transposition products were identical, with integration occurring at the same TA site. In a parallel experiment, a Gal4-piggyBac transposase resulted in an 11.6-fold increase in transpositional activity compared to controls, with 67% of the integrations occurring at a single TTAA site. This technology has the potential to minimize nonspecific integration events that may result in insertional mutagenesis and reduced fitness. Site-directed integration will be advantageous to the manipulation of genomes, study of gene function, and for the development of gene therapy techniques.

Differential gene expression during wing morph differentiation of the ectoparasitoid Melittobia digitata (Hym., Eulophidae).

Melittobia digitata is an ectoparasitoid of solitary bees and wasps that displays a trade-off between reproduction and dispersion through the development of two wing morphs (long and short wing morphs (LWM and SWM)). The morph differentiation of this species is an exceptional adaptation to maximize host exploitation and habitat colonization, and an understanding of the mechanisms underlying this developmental process will shed light on how nutrients or environmental elicitors alter regulatory pathways leading to physiological and metabolic changes resulting in such drastic developmental rearrangements. Here we describe the differential gene expression between SWM and LWM larvae of M. digitata in order to unravel the molecular mechanisms controlling the morph differentiation in this minute parasitoid and pinpoint the pathways involved in the regulation of this developmental process. The suppression subtractive hybridization (SSH) methodology was used to isolate differentially expressed genes using mRNA populations collected soon after morph development commitment. Dot blot analysis of 384 clones from a forward SSH library identified approximately 200 differentially expressed clones, including those transcripts present in very low abundance. Further DNA sequence analysis of a sub-population of 42 clones revealed 31 putatively unique transcripts, from which 5 were further analyzed by Northern blot analysis and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The complete cDNA of one of these transcripts, a putative metalloprotease, was fully sequenced and is described. The role of the putative differentially expressed genes during the wing morph differentiation of M. digitata is discussed.

Analyses of cis -acting elements that affect the transposition of Mos1 mariner transposons in vivo.

The left (5') inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3') ITR. The effects on the transposition frequency resulting from the use of two 3' ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3' ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3' ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3' ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.

Predictive validity of a screen for partner violence against women.

BACKGROUND: While public health leaders recommend screening for partner violence, the predictive value of this practice is unknown. The purpose of this study was to test the ability of a brief three-question violence screen to predict violence against women in the ensuing months. METHODS: We conducted a prospective cohort study of adult women participating in the Colorado Behavioral Risk Factor Surveillance System (BRFSS), a population-based, random-digit-dialing telephone survey. During 8 monthly cohorts, 695 women participated in the BRFSS; 409 women participated in follow-up telephone interviews approximately 4 months later. Violent events during the follow-up period, measured using a modified 28-item Conflict Tactics Scale, were compared between women who initially screened positive and those who screened negative. RESULTS: Among BRFSS respondents, 8.4% (95% confidence interval [CI]=6.3%-10.5%) had an initial positive screen. During the follow-up period, women who screened positive were 46.5 times (5.4-405) more likely to experience severe physical violence, 11.7 times (5.0- 27.3) more likely to experience physical violence, 3.6 (2.4-5.2) times more likely to experience verbal aggression, and 2.5 times (1.2-5.1) more likely to experience sexual coercion. In a multivariate model, separation from one's spouse and a positive screen were significant independent predictors of physical violence. CONCLUSIONS: A brief violence screen identifies a subset of women at high risk for verbal, physical, and sexual partner abuse over the following 4 months. Women with a positive screen who are separated from their spouse are at highest risk.

Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti.

Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.

Structure of hermes integrations in the germline of the yellow fever mosquito, Aedes aegypti.

The Hermes transposable element is derived from the house fly, Musca domestica, and can incorporate into the germline of the yellow fever mosquito, Aedes aegypti. Preliminary Southern analyses indicated that Hermes integrated along with the marker gene into the mosquito genomic DNA. Here we show that Hermes integrations are accompanied by the integration of the donor plasmid as well. In addition, breaks in the donor plasmid DNAs do not occur precisely, or at the end of the terminal inverted repeats, and are accompanied by small deletions in the plasmids. Furthermore, integrations do not cause the typical 8-bp duplications of the target site DNA. No integrations are observed in the absence of a source of Hermes transposase. The Hermes transposase clearly did not catalyse precise cut-and-paste transposition in these transformed lines. It may have integrated the transposon through general recombination or through a partial replicative transposition mechanism. The imprecision of Hermes integration may result from interactions of the transposase with an endogenous hAT-like element in the mosquito genome.

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system.

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.

The Queensland fruit fly, Bactrocera tryoni, contains multiple members of the hAT family of transposable elements.

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.

Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly.

The mosquito Aedes aegypti is the world's most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germ-line transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G0 adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn+-carrying transposon. The cn+ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.

Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti.

The mariner transposable element is capable of interplasmid transposition in the embryonic soma of the yellow fever mosquito, Aedes aegypti. To determine if this demonstrated mobility could be utilized to genetically transform the mosquito, a modified mariner element marked with a wild-type allele of the Drosophila melanogaster cinnabar gene was microinjected into embryos of a kynurenine hydroxylase-deficient, white-eyed recipient strain. Three of 69 fertile male founders resulting from the microinjected embryos produced families with colored-eyed progeny individuals, a transformation rate of 4%. The transgene-mediated complementation of eye color was observed to segregate in a Mendelian manner, although one insertion segregates with the recessive allele (female-determining) of the sex-determining locus, and a separate insertion is homozygous lethal. Molecular analysis of selected transformed families demonstrated that a single complete copy of the construct had integrated independently in each case and that it had done so in a transposase-mediated manner. The availability of a mariner transformation system greatly enhances our ability to study and manipulate this important vector species.

The white gene from the yellow fever mosquito, Aedes aegypti.

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti. Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae, but not in the vinegar fly, Drosophila melanogaster. The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.

The Caring Efficacy Scale: nurses' self-reports of caring in practice settings.

The Caring Efficacy Scale (CES) assesses belief in one's ability to express a caring orientation and to develop caring relationships with clients or patients. This article describes the development of this Likert-type self-report scale to assess nurses' caring efficacy. It also discusses a preliminary reliability and validity study assessing the caring efficacy of novice and experienced nurses from three nursing programs: baccalaureate, nursing doctorate, and master's. The CES was found to have high internal consistency, and it possessed significant positive relationships with a measure of clinical competence.

Utilizing narrative inquiry to evaluate a nursing doctorate program professional residency.

Because the University of Colorado (CU) School of Nursing Nursing Doctorate (ND) Program initiated an innovative nursing educational reform, emerging program evaluation challenges were addressed to ensure successful implementation and program quality. The study's purpose was to evaluate the effectiveness of the ND professional clinical residency (fourth and final year) from the students' perspectives. Therefore, this evaluation was exploratory and inductive to focus on the primary questions: "How does one become an ND nurse during the residency?" (process) and "What is an ND nurse?" (outcome). Additionally, an explanation of how interactive processes affected residency experiences was addressed. The narrative inquiry framework made available a special access to the human experiences of time, order, and change during the residency process. Ten students in the first CU ND Program residency participated. Narrative data for qualitative analysis were obtained from students' monthly written vignettes and verbal sharing of their clinical experiences during conferences. Vignette formats directed students to describe significant residency experiences and share reflections on the events. One finding suggested that students' formative progression through the residency occurred in four phases similar to cognitive development theories. Additional findings confirmed students' growth toward and summative attainment of ND outcome behaviors, including holistic clinical proficiency, client advocacy, and promotion of professional growth for colleagues.

Interplasmid transposition of the mariner transposable element in non-drosophilid insects.

Plasmid-based transposition assays were performed in developing embryos of the Australian sheep blowfly Lucilia cuprina and the Queensland fruit fly Bactrocera tryoni, using the mariner transposable element from Drosophila mauritiana. Transposition products were recovered that were identical in structure to those recovered from D. melanogaster. Only sequences delimited by the mariner terminal repeats were transposed and all insertions occurred at TA residues, and duplicated these. These are the hallmarks of mariner transpositions observed in the chromosomes of D. melanogaster and D. mauritiana, indicating that the plasmid-based assays are accurate indicators of mariner transposition activity. The recovery of precise transposition products from L. cuprina and B. tryoni demonstrates that mariner should be capable of producing germline transformants in these species. The results obtained from these assays suggests that they will be extremely useful in determining if mariner can transpose in other non-drosophilid insects and for investigating factors that might affect mariner transposition frequency.