Wolbachia from the planthopper Laodelphax striatellus establishes a robust, persistent, streptomycin-resistant infection in clonal mosquito cells.

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales: Anaplasmataceae), distorts reproduction of its arthropod hosts to facilitate invasion of naïve populations. This property makes Wolbachia an attractive "gene drive" agent with potential applications in the control of insect vector populations. Genetic manipulation of Wolbachia will require in vitro systems for its propagation, genetic modification, amplification, and introduction into target insects. Here we show that Wolbachia from the planthopper, Laodelphax striatellus, establishes a robust infection in clonal C7-10 Aedes albopictus mosquito cells. Infected cells, designated C/wStr, expressed radiolabeled proteins that were enriched in cells grown in the absence of antibiotics that inhibit Wolbachia, relative to cultures grown in medium containing tetracycline and rifampicin. Using mass spectrometry, we verified that tryptic peptides from an upregulated 24 kDa band predominantly represented proteins encoded by the Wolbachia genome, including the outer surface protein, Wsp. We further showed that resistance of Wolbachia to streptomycin is associated with a K42R mutation in Wolbachia ribosomal protein S12, and that the pattern of amino acid substitutions in ribosomal protein S12 shows distinct differences in the closely related genera, Wolbachia and Rickettsia.

Decapitation improves detection of Wolbachia pipientis (Rickettsiales: Anaplasmataceae) in Culex pipiens (Diptera: Culicidae) mosquitoes by the polymerase chain reaction.

Polymerase chain reaction (PCR) is often used to detect microorganisms, pathogens, or both, including the reproductive parasite Wolbachia pipientis (Rickettsiales: Anaplasmataceae), in mosquitoes. Natural populations of Culex pipiens L. (Diptera: Culicidae) mosquitoes are infected with one or more strains of W. pipientis, and crosses between mosquitoes harboring different Wolbachia strains provide one of the best-known examples of cytoplasmic incompatibililty (CI). When we used PCR to monitor Wolbachia in the Buckeye strain of Culex pipiens, and in a Wolbachia-cured sister colony obtained by tetracycline treatment, we noted false negative PCR reactions with DNA samples from infected mosquitoes; these results were inconsistent with direct microscopic observation of Wolbachia-like particles in gonads dissected from mosquitoes in the same population. Assays with diluted template often improved detection of positive samples, suggesting that DNA prepared from whole mosquitoes contained an inhibitor of the PCR reaction. We reconciled discrepancies between PCR and microscopy by systematic measurement of the PCR reaction in the presence of an internal standard. Mosquito decapitation before DNA extraction restored the reliability of the PCR reaction, allowing accurate determination of Wolbachia infection status in infected and tetracycline-cured mosquito populations, consistent with microscopic examination. Using PCR primers based on the Tr1 gene, we confirmed that the Wolbachia infection in the Buckeye strain of Culex pipiens belongs to the genotype designated wPip1. Finally, to explore more widely the distribution of PCR inhibitors, we demonstrated that DNA isolated from the cricket, Acheta domesticus (L.); the beetle, Tenebrio molitor L.; the honey bee, Apis mellifera L.; and the mosquito, Anopheles punctipennis Say also contained PCR inhibitors. These results underscore the importance of measuring the presence of inhibitors in PCR templates by using a known positive standard, and provide an approach that will facilitate use of PCR to monitor environmental samples of mosquitoes that harbor endosymbionts or pathogenic organisms.

Histone H1-like, lysine-rich low complexity amino acid extensions in mosquito ribosomal proteins RpL23a and RpS6 have evolved independently.

Histone H1-like amino acid extensions have been described at the amino terminus of Drosophila RpL22 and RpL23a, and at the carboxyl terminus of mosquito ribosomal protein RpS6. An in silico search suggested that RpL23a, but not RpL22, in Anopheles gambiae has an amino-terminal extension. Because low complexity amino acid extensions are not common on eukaryotic ribosomal proteins, and their functions are unknown, we cloned cDNAs encoding RpL23a from Aedes albopictus and Anopheles stephensi mosquito cell lines. RpL23a proteins in Aedes and Anopheles mosquitoes are rich in lysine (approximately 25%), alanine (approximately 21%), and proline (approximately 8%), have a mass of approximately 40 kDa, a pI of 11.4 to 11.5, and contain an N-terminal extension of approximately 260 amino acid residues. The N-terminal extension in mosquito RpL23a is about 100 amino acids longer than that in the Drosophila RpL23a homolog, and contains several repeated amino acid motifs. Analysis of exon-intron organization in the An. gambiae and in D. melanogaster genes suggests that a short first exon encodes a series of 11 amino acid residues conserved in RpL23a proteins from Drosophila, mosquitoes, and the moth, Bombyx mori. The histone H1-like sequence in RpL23a is encoded entirely within the second exon. The C-terminal 126 amino acid residues of the RpL23a protein, encoded by exon 3 in Drosophila, and by exons 3 and 4 in Anopheles gambiae, are well conserved, and correspond to Escherichia coli RpL23 with the addition of the eukaryotic N-terminal nuclear localization sequence. Sequence comparisons indicate that the histone H1-like extensions on mosquito RpS6 and RpL23a have evolved independently of each other, and of histone H1 proteins.

Mosquito ribosomal protein S3 lacks a critical glutamine residue associated with DNA repair activity in homologous Drosophila proteins.

In Drosophila melanogaster, ribosomal protein RpS3 has extra-ribosomal activities including apurinic/apyrimidinic lyase activity and N-glycosylase activity that participate in DNA repair. It has been suggested that these activities couple DNA repair to the translational machinery. To establish a basis for participation of RpS3 in DNA repair in mosquitoes, we cloned RpS3 cDNAs from Aedes aegypti and Aedes albopictus mosquito cell lines. The sequence data were used to reconstruct the homologous gene from the Anopheles gambiae database. Mosquito RpS3 is a single copy gene, which in Aedes albopictus, lacks introns in the amino acid coding region. Although RpS3 proteins are well-conserved among eukaryotes, a critical glutamine residue, Q59, essential to robust DNA repair activity in the Drosophila protein, is replaced by an asparagine (N) in all three mosquito RpS3 proteins. In this respect, the mosquito protein resembles human RpS3, which has relatively modest DNA repair activity. None of the insect RpS3 proteins available in the database, other than those from Drosophila, contain glutamine at position 59. However, in the Lepidoptera, N59 is consistently replaced by serine (S), and the putative interactive site at position 134 is replaced by arginine (R). These data suggest that in the case of RpS3, the Drosophila protein may be uniquely unusual in having robust DNA repair activities that are unlikely to be common to RpS3 from other insects.

Co-immunoprecipitation of putative proteins that interact with mosquito proliferating cell nuclear antigen.

We have sequenced cDNAs encoding proliferating cell nuclear antigen (PCNA) from Aedes albopictus cells and from Aedes aegypti mosquitoes. The mosquito cDNAs contained an open reading frame encoding a 260 amino acid protein with a calculated mass of 29.0 kDa and a pI of 4.46. There was a single amino acid difference between PCNA proteins from Ae. albopictus and Ae. aegypti. In An. gambiae, the PCNA homolog contained 260 residues, and the pcna gene was interrupted by a single 67 nucleotide intron in the betaC2 region of the protein. A phylogenetic comparison grouped known Dipteran PCNA sequences into two clusters, representing the Nematocera and the Cyclorrhapha. PCNA transcripts measured 1.1 kb, and were stable, as was PCNA protein. Mosquito PCNA was efficiently recognized by a commercially available mouse anti-PCNA monoclonal antibody, which coprecipitated 29 kDa and 35 kDa proteins from mosquito cells representing different growth states. These results support the feasibility of recovering mosquito cell cycle inhibitory proteins by virtue of their interaction with PCNA.

Recovery of cDNAs encoding ribosomal proteins S9 and L26 from Aedes albopictus mosquito cells and identification of their homologs in the malaria vector, Anopheles gambiae.

We used PCR-based approaches to obtain the full-length cDNA sequences encoding ribosomal protein (Rp) S9 and L26 from a mosquito (Aedes albopictus) C7-10 cell line. The deduced mosquito RpS9 protein has a mass of 22,826 Da and a pI of 11.41, while RpL26 had a mass of 17,442 Da and a pI of 11.52. Both cDNAs initiated with the 5'-polypyrimidine motif characteristic of ribosomal protein transcripts. Using the Aedes protein and nucleic acid sequences, we identified rpS9 and rpL26 as single copy genes in the Anopheles gambiae genome. In An. gambiae, the RpS9 coding region was distributed over 3 exons, spanning 2.6 kb, but the Anopheles rpL26 protein coding region lacked introns. The Aedes and Anopheles RpS9 and RpL26 proteins shared 96 and 92% identity, respectively. Despite low numbers of parsimony-informative amino acid substitutions, phylogenies based on the ribosomal protein sequences accurately group the Aedes and Anopheles proteins with high bootstrap values.

The mosquito ribonucleotide reductase R2 gene: ultraviolet light induces expression of a novel R2 variant with an internal amino acid deletion.

Abstract Using RT-PCR, we examined expression of the ribonucleotide reductase R2 subunit (RNR-R2) in Aedes albopictus mosquito cells after treatment with ultraviolet light (UV). In control cells, a predominant band at 1.2 kb corresponded to the full-length cDNA. A smaller 650 bp band was unique to UV-treated cells. Sequence analysis showed that the 650 bp band encoded a protein with an internal deletion of 179 amino acids, relative to Ae. albopictus RNR-R2. The N-terminal twenty amino acids were identical between AalRNR-R2 and AalDeltaR2; downstream of the deletion, the proteins differed at only four residues. In AalDeltaR2, the internal deletion spanned five residues critical to RNR-R2 enzymatic activity, including a key tyrosine residue that generates an essential free radical. The full-length 46 kDa and truncated 25 kDa RNR-R2 proteins were shown to be expressed on Western blots, and to differ in their subcellular localization. Similarly, expression of the two proteins was differentially regulated during the cell cycle, and expression of AalDeltaR2 predominated after UV treatment. AalDeltaR2 resembled a human RNR-R2 variant called p53R2, which was induced by agents that damage DNA. As was the case with p53R2 and its antisense RNA, levels of AalDeltaR2 were diminished after treatment of mosquito cells with RNAi corresponding to p53 from Drosophila melanogaster. Examination of the AalRNR-R2 homologue in the Anopheles gambiae genome suggested that AalDeltaR2 resulted from precise splicing between Exons 1, 4 and 5, eliminating Exons 2 and 3. The likelihood that AalDeltaR2 is a non-enzymatic, functional participant in DNA metabolism is suggested by enhancement of DNA repair in an in vitro system and by the presence of a similar gene (rnr4) in yeast.

Characterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell line.

We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions -192 to -165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells.

Ultraviolet light sensitivity, unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells.

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of (3)H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m(2)), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.

Antisense expression of the 20-hydroxyecdysone receptor (EcR) in transfected mosquito cells uncovers a new EcR isoform that varies at the C-terminal end.

The insect steroid hormone 20-hydroxyecdysone initiates a cascade of regulatory events in a temporal and tissue-specific manner by first binding to a complex of an ecdysone receptor (EcR) protein and a ultraspiracle protein. Using an antisense (As) ribonucleic acid approach, we show that disruption of EcR expression in transfected C7-10 cells from the mosquito Aedes albopictus affects survival and growth. From stably transfected cells, we recovered a new isoform of A. albopictus AalEcRa, which is named AalEcRb. The deduced amino acid sequence of AalEcRb was almost identical to that of AalEcRa, with the exception of a seven amino acid sequence near the C-terminus. Using polymerase chain reaction followed by restriction enzyme analysis, we found that AalEcRa is the predominant species expressed by wild-type C7-10 cells, while cells transfected with As-EcR expressed both isoforms at approximately equal levels.

Two-dimensional electrophoretic analysis of Aedes aegypti mosquito fat body proteins during a gonotropic cycle.

We have used two-dimensional polyacrylamide gel electrophoresis to examine changes in the relative abundance and diversity of non-secreted proteins in Aedes aegypti fat body preparations during a reproductive cycle. Electrophoretic profiles were evaluated at four time points after eclosion, and at five time points after a blood meal. In contrast to the dramatic changes in abundance of specific secreted proteins such as vitellogenin, our results show that the complement of proteins internal to the fat body remains relatively constant during the mosquito reproductive cycle. Of the approximately 5-10% of proteins that do change in abundance, the majority undergo a dramatic decrease within 24 hours after eclosion.

Increased ribonucleotide reductase activity in hydroxyurea-resistant mosquito cells.

Hydroxyurea-resistant Aedes albopictus mosquito cells were selected by incremental exposure of unmutagenized cells to hydroxyurea concentrations ranging from 0.1 to 8 mM. Clonal populations that had become 40-fold more resistant to hydroxyurea than wild-type cells varied in morphology, and their growth rate decreased to a;45 h doubling time, relative to an 18 h doubling time in unselected cells. At this level of resistance, the cells remained diploid, with a modal chromosome number of 6. When labelled with (35)S[methionine/cysteine], clone HU1062, which grew in the presence of 8 mM hydroxyurea, overproduced a labeled protein with the approximate size of the 45,000 dalton M2 subunit of ribonucleotide reductase. Consistent with this observation, ribonucleotide reductase activity in HU-1062 cells was approximately 10-fold higher than in wild-type control cells. This is the first example of an hydroxyurea-resistant insect cell line. [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:31-41 (1997).]

Exploration of mosquito immunity using cells in culture.

The propagation of immune-responsive cells in vitro has provided the basis for substantial contributions to our understanding of many aspects of the mammalian immune response. In contrast, the potential for exploring the innate immune response of insects using cultured cells is only beginning to be developed, particularly with various mosquito cell lines from the genera Aedes and Anopheles. Immune-reactive mosquito cell lines express various defensive factors, including transferrin, lysozyme, cecropin, defensin, and prophenoloxidase activities. In this review, we discuss insect immunity in the context of key concepts that have emerged in the study of the mammalian immune system, with emphasis on the properties of the cells that participate in the immune response. The nature of established cell lines and their contributions to our understanding of immune functions in humans and insects is described, with emphasis on our own work with the C7-10 and Aag-2 mosquito cell lines from Aedes albopictus and Aedes aegypti, respectively. Finally, we offer some speculation on further advances in insect immunology that may be facilitated by work with cells in culture.

Immune activation upregulates lysozyme gene expression in Aedes aegypti mosquito cell culture.

After stimulation with heat-killed bacteria, cultured cells from the mosquito Aedes aegypti (Aag-2 cells) secreted an induced protein with a mass of approximately 16 kDa that cross-reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria-treated cells, we used polymerase chain reaction-based approaches to obtain the complete lysozyme cDNA from Aag-2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is processed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anopheles gambiae and Anopheles darlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induction of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag-2 cells with heat-killed bacteria, followed by maximal expression 12-36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag-2 cells.

Differential regulation of ribosomal protein gene expression in Aedes aegypti mosquitoes before and after the blood meal.

In fat body of the mosquito, Aedes aegypti, a cycle of ribosome accumulation and degradation accompanies synthesis of the yolk protein precursor, vitellogenin. Here we compare the transcription and translation of ribosomal proteins rpS6, rpL8 and rpL34, relative to rRNA and vitellogenin genes in Aedes aegypti fat body after eclosion, and in response to a blood meal. Analysis using Northern blots and reverse-transcription polymerase chain reactions (RT-PCR) showed that the rpS6, rpL8 and rpL34 genes are coordinately regulated with respect to one another, and that ribosomal protein gene expression in this system was predominantly regulated by transcription during the 3-4 days between adult eclosion and blood feeding. After a blood meal, ribosomal protein mRNA levels remained similar to those in unfed females during the first 18 h, then declined to minimum levels by 48 h after the blood meal. These data indicate that transcription of ribosomal protein genes is low in vitellogenic mosquitoes, relative to previtellogenic females. Experiments with the dissected fat body, however, showed that levels of acetic acid-soluble proteins increased by approximately threefold between 12 and 24 h after the blood meal. Taken together, these observations suggest that the active translation of ribosomal proteins from stable mRNA accompanies ribosome biosynthesis after the blood meal. Thus, in the fat body of adult female mosquitoes, the expression of ribosomal protein genes is regulated at the level of transcription before the blood meal, while translational control is the predominant regulatory mechanism after the blood meal.

The implementation of clinical guidelines in a managed care setting: implications for children with special health care needs.

OVERVIEW: The project tracked the development and implementation of a pediatric asthma guideline to determine its usefulness as a quality assurance mechanism for children with special health care needs. METHODS: Interviews were conducted with clinic staff to gather descriptive information about guideline implementation at owned clinics within a large HMO. RESULTS: Providers developed multiple implementation strategies emphasizing patient/family education. Service coordination within the health plan was well established, while coordination of services beyond the health plan was less clearly related to guideline implementation. CONCLUSION: Guideline implementation appeared to be a highly variable process. Clinical guidelines alone may not be sufficient tools of quality assurance for children with chronic or complex conditions.

Evidence for expression of EcR and USP components of the 20-hydroxyecdysone receptor by a mosquito cell line.

The reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine whether the C7-10 cell line from the mosquito, Aedes albopictus, expresses transcripts encoding 20-hydroxyecdysone receptor (EcR) and ultraspiracle (USP) isoforms known to constitute a functional 20-hydroxyecdysone receptor. Here we describe recovery and analysis of products with high similarity to the EcR and to the USP isoform "a" that have been reported from the related mosquito, Aedes aegypti. The C7-10 EcR was 97% identical to Aedes aegypti EcR in amino acid sequence. Key features of the nuclear/steroid hormone receptor superfamily, including the zinc fingers, proximal (P)-box, and distal (D)-box were well conserved. However, the C7-10 EcR contained 5 additional amino acids in the C-terminal domain F, which required introduction of gaps to maximize alignment. The 5'-untranslated regions of the two mosquito EcRs were 98% identical, but the function of this region remains unknown. The C7-10 USP was 95% identical in amino acid sequence to the longer Aedes aegypti isoform "a." Although only the C7-10 EcR was detected on Northern blots using total RNA from the cell line, transcripts for both EcR and USP were detected using the RT-PCR procedure. These transcripts appeared to be expressed constitutively and expression levels were not affected by treatment of cells with 20-hydroxyecdysone. Arch.

A successful collaboration between a private nonprofit health plan and a state health department.

This article describes the experience of a private, nonprofit health plan in establishing a collaborative relationship with a state health department. Through a federal grant project, efforts toward assuring quality care for children with special health care needs in managed care settings provided unique opportunities to form partnerships between multiple health plans, community groups, and other stakeholders. Collaborative activities included (1) formation of a pediatric asthma task force and a performance measurement and quality assurance committee; (2) planning and execution of a statewide conference; (3) development of a teaching manual for incorporating asthma education into elementary classroom curricula; and (4) publication of a parent resource manual for health plan members. Key ingredients and influencing factors for successful public-private partnerships are discussed.

The ribosomal protein L34 gene from the mosquito, Aedes albopictus: exon-intron organization, copy number, and potential regulatory elements.

We describe the structural analysis of genomic DNA encoding ribosomal protein (rp) L34 from the mosquito, Aedes albopictus. Comparison of genomic DNA sequences encompassing approximately 8 kb with the rpL34 cDNA sequence showed that the gene contains three exons and two introns, encoding a primary transcript with a deduced size of 6196 nucleotides from the transcription start site to the polyadenylation site. Exon 1, which is not translated, measures only 45 bp, and is separated from Exon 2 by a 359 bp intron. Exon 2 measures 78 bp, and contains the AUG translation initiation codon 14 nucleotides downstream of its 5'-end. Downstream of Exon 2 is a 5270 bp intron, followed by the remainder of the coding sequence in Exon 3, which measures 444 bp including the polyadenylation signal. We used a novel PCR-based procedure to obtain 1.7 kb of DNA upstream of the rpL34 gene. Like the previously described Ae. albopictus rpL8 gene and various mammalian rp genes, the DNA immediately upstream of the rpL34 gene lacks the TATA box, and the rpL34 transcription initiation site is embedded in a characteristic polypyrimidine tract. The 5'-flanking DNA contained a number of cis-acting elements that potentially interact with transcription factors characterized by basic domains, zinc-coordinating DNA binding domains, helix-turn-helix motifs, and beta scaffold factors with minor groove contacts. Particularly striking was the conservation of an AP-4 binding site within 100 nucleotides upstream of the transcription initiation site in both Aal-rpL34 and Aal-rpL8 genes. Comparison of Southern hybridization signals using probes from the 5' and 3'-ends of the 5.3 kb second intron and the cDNA suggested that the Ae. albopictus rpL34 gene most likely occurs as a single expressed copy per haploid genome with restriction enzyme polymorphisms in the upstream flanking DNA and the likely presence of one or more pseudogenes.

Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedes aegypti and a defensin D from Aedes albopictus.

An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7-10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12-36 h after induction.