RESUMO
Limited evidence suggests that the cervid parasite, Babesia odocoilei, is transovarially transmitted from adult female Ixodes scapularis Say to offspring. The prevalence of B. odocoilei in unfed larval I. scapularis and whether vertical transmission is crucial to pathogen maintenance are currently unknown. To investigate these questions, 275 unfed larvae from two Wisconsin counties were tested for B. odocoilei genetic material. Sixteen of 29 pools were positive for the parasite. The maximum likelihood estimation for overall larval infection prevalence was 7.8% (95% confidence interval: 4.7-12). This vertically acquired infection appears to be sustained transstadially in nymphal ticks the following year; however, our relatively small sample and replicate size warrants additional evaluation. Our study revealed further evidence of vertical transmission, a low and consistent infection prevalence in larvae, and the potential importance of vertical transmission in B. odocoilei maintenance.
Assuntos
Babesia/fisiologia , Ixodes/parasitologia , Animais , Cervos/parasitologia , Feminino , Ixodes/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/parasitologia , Pupa/crescimento & desenvolvimento , Pupa/parasitologia , WisconsinRESUMO
Borrelia burgdorferi, the spirochete that causes Lyme disease, is endemic and widespread in Wisconsin. Research in the northeastern United States has revealed a positive association between Babesia microti, the main pathogen that causes babesiosis in humans, and Bo. burgdorferi in humans and in ticks. This study was conducted to examine associations between the disease agents in the Upper midwestern United States. Ixodes scapularis Say nymphs (N = 2,858) collected between 2015 and 2017 from nine locations in Wisconsin were tested for Babesia spp. and Borrelia spp. using real-time PCR. Two species of Babesia were detected; Ba. microti and Babesia odocoilei (a parasite of members of the family Cervidae). Prevalence of infection at the nine locations ranged from 0 to 13% for Ba. microti, 11 to 31% for Bo. burgdorferi sensu stricto, and 5.7 to 26% for Ba. odocoilei. Coinfection of nymphs with Bo. burgdorferi and Ba. odocoilei was detected in eight of the nine locations and significant positive associations were observed in two of the eight locations. The prevalence of nymphal coinfection with both and Bo. burgdorferi and Ba. microti ranged from 0.81 to 6.5%. These two pathogens were significantly positively associated in one of the five locations where both pathogens were detected. In the other four locations, the observed prevalence of coinfection was higher than expected in all but one site-year. Clinics and healthcare providers should be aware of the association between Ba. microti and Bo. burgdorferi pathogens when treating patients who report tick bites.
Assuntos
Babesia/isolamento & purificação , Borrelia burgdorferi/isolamento & purificação , Coinfecção , Ixodes , Animais , Babesiose/transmissão , Ixodes/microbiologia , Ixodes/parasitologia , Doença de Lyme/transmissão , Ninfa/microbiologia , Ninfa/parasitologia , Prevalência , Picadas de Carrapatos/microbiologia , Picadas de Carrapatos/parasitologia , WisconsinRESUMO
The blacklegged tick, Ixodes scapularis Say, is the primary vector of several tick-borne pathogens, including those causing Lyme disease and babesiosis, in the eastern United States and active collection methods for this species include dragging or wild animal sampling. Nest boxes targeting mice may be an alternative strategy for the surveillance and collection of immature I. scapularis feeding on these hosts and would be much safer for animals compared to small mammal trapping. We constructed double-walled insulated nest boxes (DWINs) with collection tubes mounted below the nesting chamber and deployed eleven in southern Wisconsin from June until September of 2020. The DWINs were occupied by Peromyscus spp. and birds (wren species, Troglodytidae family). We collected 192 ticks from collection tubes, all of which were identified as either I. scapularis (95%) or Dermacentor variabilis Say (Acari: Ixodidae) (5%). Only 12% (21/182) and 20% (2/10) of I. scapularis and D. variabilis were blood-fed, respectively. The high proportion of unfed ticks found in collection tubes may be due to grooming by hosts inside the nest boxes. Alternatively, immature ticks may have climbed trees and entered the DWIN seeking a host. Results suggest that nest boxes could be a tool for finding ticks in areas of low density or at the leading edge of invasion, when small mammal trapping or drag sampling is not feasible.
Assuntos
Doenças das Aves/epidemiologia , Ixodes/fisiologia , Comportamento de Nidação , Peromyscus , Doenças dos Roedores/epidemiologia , Aves Canoras , Infestações por Carrapato/veterinária , Animais , Doenças das Aves/parasitologia , Comportamento Alimentar , Ixodes/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/fisiologia , Prevalência , Doenças dos Roedores/parasitologia , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/parasitologia , Wisconsin/epidemiologiaRESUMO
We sequenced and characterized two novel invertebrate-type lysozymes from the mosquito Anopheles gambiae. Alignment and phylogenetic analysis of these and a number of related insect proteins identified through bioinformatics strategies showed a high degree of conservation of this protein family throughout the Class Insecta. Expression profiles were examined for the two mosquito genes through semiquantitative and real-time PCR analysis. Lys i-1 transcripts were found in adult females in the fat body and Malpighian tubules, whereas Lys i-2 was detected only in fat bodies. Blood-feeding resulted in significantly increased transcript abundance for both genes in the midguts. Neither gene was upregulated following bacterial challenge.
Assuntos
Anopheles/enzimologia , Anopheles/genética , Muramidase/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Trato Gastrointestinal/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Dados de Sequência Molecular , Muramidase/química , Muramidase/imunologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
Phenoloxidases, including tyrosinases and laccases, are enzymes involved in the synthesis of melanin, a process that can be elicited during insect immune responses, cuticle maturation, wound healing and egg chorion development. We cloned a putative inhibitor of melanization (POI) from Anopheles gambiae on the basis of homology with a functionally characterized peptide from Musca domestica (Daquinag et al., Proc Natl Acad Sci USA 1995; 92: 2964-2968). The 335 amino acid protein predicted from the A. gambiae cDNA consists of five tandemly arranged inhibitor motifs. The A. gambiae POI gene was expressed in all mosquito stages from egg to adult. POI transcript levels were high in the fat body and were measurable but comparatively reduced in the midgut. The POI transcript level increased after wounding or Sephadex bead injection. Gene knockdown did not result in faster or more extensive bead melanization but did result in more extensive melanization of wound sites following a thoracic bead injection.
Assuntos
Anopheles/genética , Sequência de Aminoácidos , Animais , Anopheles/imunologia , Sequência de Bases , DNA Complementar , Feminino , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Insetos/química , Melaninas/imunologia , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/fisiologia , Interferência de RNA , Homologia de Sequência de AminoácidosRESUMO
Two haemolymph proteins that are processed rapidly and specifically in response to exposure to bacteria have been identified from Anopheles gambiae. Both proteins, Anopheles gambiae bacteria-responsive 1 (AgBR1) and AgBR2, are similar to chitinases but belong to a family of proteins that have lost chitinolytic activity. AgBR1 and AgBR2 are converted to smaller forms in vivo or in vitro on exposure to bacteria, and AgBR2 also can be processed on exposure to peptidoglycan alone. AgBR1 and AgBR2 do not bind to bacteria or chitin beads. The AgBR1 and AgBR2 genes are expressed in all developmental stages. In adults, AgBR1 expression is restricted to the fat body, whereas AgBR2 is expressed in many tissues.
Assuntos
Anopheles/genética , Quitinases/genética , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Anopheles/imunologia , Anopheles/microbiologia , Anticorpos/imunologia , Bactérias/imunologia , Sequência de Bases , Western Blotting , Linhagem Celular , Quitinases/sangue , Quitinases/metabolismo , Primers do DNA , DNA Complementar/genética , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Hemolinfa/química , Immunoblotting , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmodium/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/imunologia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Four state parks located in Lyme disease endemic regions of Wisconsin were surveyed for the presence of Ixodes scapularis Say during May and June of 1998 by drag sampling along hiking trails. Nymphal abundance varied between parks, with the average number of nymphs encountered in 1 h ranging from 6.2 +/- 3.8-47.1 +/- 36.3 (mean +/- SD). Questing nymphs were tested for the presence of Borrelia burgdorferi by culture in BSK medium and 7-12% was found to be infected. The average risk of encountering an infected nymph (entomologic risk index) ranged from 0.5 to 5.2 infected nymphs per hour. The highest entomological risk index was recorded from a small island park in northwestern Wisconsin during the last week in May (8.0 infected nymphs per hour). These results indicate a lower risk for human Lyme disease exposure in Wisconsin state parks in comparison with highly endemic areas of the northeastern United States.
Assuntos
Grupo Borrelia Burgdorferi/isolamento & purificação , Ixodes/microbiologia , Animais , Comportamento Apetitivo , Densidade Demográfica , Fatores de Risco , WisconsinRESUMO
Serine proteases regulate several invertebrate defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization of pathogen surfaces. These processes require the presence of serine proteases in the hemolymph where they can rapidly activate immune pathways in response to pathogen detection. Hemolymph coagulation in the horseshoe crab is controlled by several serine proteases, including two that are pathogen recognition molecules and two in the clip domain family of serine proteases. The antimicrobial peptide synthesis and melanization pathways include clip domain proteases as well as other, uncharacterized serine proteases. We have identified five serine proteases from the hemolymph of the mosquito, Anopheles gambiae. One, Sp22D, is a large protease with potential pathogen binding domains. Sp22D is expressed in three tissues that have immune functions (midgut epithelium, fat body, and hemocytes), and its transcript abundance increases after immune challenge. Sp14A, Sp14D1, and Sp14D2 are clip domain serine proteases that are similar to enzymes with presumed roles in melanization or antimicrobial peptide synthesis. They undergo changes in transcript abundance in response to infection with bacteria or malaria parasites, and they reside in a chromosomal region that has been associated with melanization of parasites. Sp18D, also a clip domain protease, is similar to a Manduca protease with a likely role in immunity, but immune challenge does not affect its mRNA abundance.
Assuntos
Anopheles/enzimologia , Anopheles/imunologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Coagulação Sanguínea , Hemolinfa , Dados de Sequência Molecular , Serina Endopeptidases/classificaçãoRESUMO
Serine proteases play critical roles in a variety of insect immune responses; however, few of the genes that code for these enzymes have been cloned. Here, we describe the molecular characterization of a serine protease gene from the mosquito Anopheles gambiae. Sp22D codes for a 1322 amino acid polypeptide with a complex domain organization. In addition to the carboxy terminal serine protease catalytic domain, Sp22D contains two putative chitin binding domains, a mucin-like domain, two low density lipoprotein receptor class A domains, and two scavenger receptor cysteine rich domains. A typical signal peptide sequence and a lack of potential transmembrane helices suggest that Sp22D is secreted. Sp22D is expressed constitutively in three immune-related cell types: adult hemocytes, fat body cells, and midgut epithelial cells. Wounding induces no changes in transcript abundance, but within 1h after injection of bacteria, Sp22D mRNA increases 1.5-fold. Based on domain organization, tissue distribution, and transcriptional up-regulation in response to immune challenge, we suggest that Sp22D has an immune function. In addition, we predict that Sp22D is secreted into the hemolymph where it may interact with pathogen surfaces and initiate an immune response.
Assuntos
Anopheles/genética , Proteínas de Insetos , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Anopheles/imunologia , Sítios de Ligação , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Hibridização In Situ , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/imunologia , Distribuição Tecidual , Transcrição GênicaAssuntos
Infecções Bacterianas/fisiopatologia , Culicidae/microbiologia , Animais , Contagem de Colônia Microbiana , Culicidae/imunologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Micrococcus luteus/isolamento & purificaçãoRESUMO
Haemolymph polypeptides from Plasmodium-refractory and -susceptible mosquitoes were compared by one- and two-dimensional gel electrophoresis. The refractory strain of Anopheles gambiae kills malaria parasites by a humoral melanization mechanism whereas the parasites develop normally in susceptible mosquitoes. The two strains respond in a similar manner to carboxy-methyl-Sephadex beads that have been injected into the thoracic haemocoel, i.e. beads are strongly melanized in refractory but not susceptible mosquitoes. Protein profiles were compared between strains following cold shock (naïve control), saline injection and Sephadex bead injection. Using the susceptible naïve control as the standard, eight constitutively expressed polypeptides were specific to naïve susceptible mosquitoes while twelve other spots were reduced, enhanced or specific to refractory mosquitoes. Several of the strain-specific spots probably comprise related pairs (one in each strain) which vary only in isoelectric focusing point. Nine spots were induced by sham injection or by an injection of beads or saline, but none was reproducibly different between the strains. Amino acid sequence analysis of one of the refractory strain-specific spots identified it as AgSp14D1, an A. gambiae infection-responsive serine protease that is most similar to the Drosophila gene easter and Manduca prophenoloxidase activating enzyme. This gene maps to polytene chromosome division 14, which has been implicated in the melanization phenotype by quantitative trait loci mapping.
Assuntos
Anopheles/metabolismo , Hemolinfa/química , Proteínas de Insetos/química , Melaninas , Plasmodium , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , FemininoRESUMO
We identified five new serine protease cDNAs from the hemolymph of the malaria vector, Anopheles gambiae. All five show sequence similarity to genes thought to be involved in vertebrate or invertebrate defense responses. Sp14A, Sp14D2 and Sp22D demonstrate changes in transcript abundance in response to bacteria injections. Sp14A and Sp14D2, as well as the previously characterized Sp14D1, are induced by infection with the malaria parasite, Plasmodium berghei. These three proteases, along with Sp18D, are related to a group of secreted proteases that have amino-terminal clip domains and trypsin-like substrate specificity. BLAST results and phylogenetic analyses group Sp14A, Sp14D1 and Sp14D2 with the Drosophila protease EASTER, and three prophenoloxidase activating enzymes from other insects. EASTER's substrate is SPAETZLE, a ligand involved in embryogenesis but also in activating anti-microbial peptide synthesis. Their similarity to EASTER and immune inducibility suggest that one of these proteases may activate a SPAETZLE-like ligand during anti-parasite responses in mosquitoes. Alternatively, as potential prophenoloxidase activators, Sp14A, Sp14D1 or Sp14D2 may play a role in melanotic encapsulation of Plasmodium.
Assuntos
Anopheles/genética , DNA Complementar/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Anopheles/imunologia , Anopheles/parasitologia , Clonagem Molecular , Interações Hospedeiro-Parasita , Insetos Vetores , Dados de Sequência Molecular , Plasmodium berghei/patogenicidade , Transcrição GênicaRESUMO
The rapid induction of proteins in the hemolymph of the mosquito, Anopheles gambiae, was examined after wounding or injection of immune elicitors (Escherichia coli, lipopolysaccharide, laminarin, zymosan). One-dimensional gel electrophoresis revealed at least six hemolymph polypeptides >25 kDa that consistently appeared after any breech of the cuticle. All of these polypeptides appeared in the hemolymph within 30 min and reached a maximum concentration after approx. 6 h. No proteins were specifically induced by bacteria or bacterial or fungal cell wall products, however two constitutively expressed proteins were repressed by these injections. Patterns of hemolymph proteins were further analyzed by two-dimensional electrophoresis. Seven spots were enhanced or induced 2 h after injection in four replicate experiments. An additional two spots demonstrated some variability between replicates, but were generally responsive to injection. These rapidly induced polypeptides are candidates for regulating and initiating the mosquito's responses to pathogens and wounding.
Assuntos
Anopheles/metabolismo , Proteínas de Insetos/biossíntese , Animais , Anopheles/imunologia , Escherichia coli/imunologia , Feminino , Glucanos , Hemolinfa/metabolismo , Lipopolissacarídeos/imunologia , Polissacarídeos/imunologia , Zimosan/imunologiaRESUMO
The nucleotide and deduced amino acid sequence of a serine protease (AgSp14D1) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 360 amino acid protein that contains two domains and has the highest sequence similarity to the Drosophila melanogaster serine protease easter and to prophenol oxidase activating enzyme (pPAE) from Manduca sexta. The catalytic domain is at the carboxy terminus and has the conserved serine, histidine and aspartic acid residues found in serine proteases as well as six cysteines common to invertebrate enzymes. The amino terminus contains critical cysteines that define a clip (=disulphide knot) domain which places this gene product in a subfamily of regulatory serine proteases that includes not only easter and pPAE but also the Drosophila proteins masquerade, stubble and snake as well as proclotting enzyme and factor B from the horseshoe crab. In situ hybridization to the polytene chromosomes detects a single band at 14D and Southern analysis with a probe from the 5' end of the gene confirms the single copy status of this gene. Northern analysis reveals changes in transcript abundance during development and following blood feeding. Interestingly, this analysis also shows an increase in transcript levels following wounding or injection of bacteria.
Assuntos
Anopheles/enzimologia , Proteínas de Insetos , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Anopheles/genética , Anopheles/imunologia , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Feminino , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
One major quantitative trait locus controls melanization of both malaria ookinetes and Sephadex CM beads in a refractory strain of the mosquito, Anopheles gambiae. Hemolymph transferred from a nonmelanizing, Plasmodium-susceptible strain (4arr) to a melanizing, Plasmodium-refractory strain (L35) caused a reduction in the melanization of CM beads. In addition, when beads were first incubated in vivo in susceptible mosquitoes and then recovered, washed, and transferred to refractory mosquitoes, a strong reduction in melanization was observed. No changes in melanization were observed when beads or hemolymph were transferred in the opposite direction or within a strain. Incubation of beads in vitro in refractory or susceptible hemolymph resulted in a reduction of melanization when these beads were subsequently transferred to refractory mosquitoes. This reduction was significantly stronger when susceptible hemolymph was used as the incubating medium. Protection from melanization was observed after 3-, 6-, and 24-h incubations of beads in susceptible mosquitoes with longer incubations resulting in greater protection. Treatment of protected beads with 1 M NaOH resulted in the loss of the protection but treatment with 1% sodium dodecyl sulfate (SDS), 1% SDS/DTT/boiling, or 1 M NaOAc (pH 8.9) did not. These results show that a melanization-preventing factor covalently binds to the surface of CM beads in susceptible mosquitoes and can subsequently prevent melanization in refractory mosquitoes.
Assuntos
Anopheles/fisiologia , Anopheles/parasitologia , Hemolinfa/fisiologia , Melaninas/metabolismo , Plasmodium/fisiologia , Animais , Cinética , Plasmodium/patogenicidade , Característica Quantitativa Herdável , Especificidade da Espécie , Fatores de TempoRESUMO
The sedge Cyperus iria, a common weed in rice, contains large amounts of the insect hormone (10R) juvenile hormone III (JH III). Given its widespread distribution in Asia and Africa, we examined the possibility that C. iria could be used as a safe, inexpensive, and readily available mosquito larvicide. Plants of varying ages were harvested and leaves tested for lethal effects on larvae of the yellow fever mosquito, Aedes aegypti. The median lethal doses (LD50s) for frozen leaves from 1- and 2-month-old plants were 267 and 427 mg/100 ml of water, respectively. Leaves from 1-month-old C. iria contained 193 micrograms JH III/g fresh weight, whereas leaves from 2-month-old plants contained 143 micrograms JH III/g fresh weight. Larval sensitivity to the plant differed with age; 4-day-old larvae displayed the greatest mortality followed in decreasing sensitivity by larvae 5, 6, 3, and 2 days old. Six Cyperus species (C. albostriatus, C. alternifolius, C. esculentus, C. iria, C. miliifolius, and C. papyrus) of similar developmental stage were assayed for JH III content. Only C. iria was found to contain significant levels of JH III.
Assuntos
Aedes , Controle Biológico de Vetores , Poaceae , Sesquiterpenos , Animais , Folhas de PlantaRESUMO
A Plasmodium-refractory strain (L35) of the mosquito Anopheles gambiae Giles melanizes late ookinetes on the basal surface of the midgut, resulting in the death of the parasites. This strain also melanizes CM C-25 Sephadex beads, which serve as a model system for investigating the melanization response. The effects of larval nutrition, adult body size, and temperature of the adult environment on the ability of refractory females to melanize CM C-25 beads were studied. Nutritional deprivation during the larval stages significantly decreased the ability of adults to melanize beads. In addition, bead melanization decreased progressively as the environmental temperature of the adults increased from 24 to 30 degrees C. We conclude that environmental stress may affect the immune responses of An. gambiae.
Assuntos
Anopheles/fisiologia , Dextranos , Melaninas/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Anopheles/imunologia , Anopheles/parasitologia , Fenômenos Fisiológicos do Sistema Digestório , Géis , Imunidade Inata , Larva , Plasmodium/parasitologia , TemperaturaRESUMO
A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and kills many species of malaria parasites, whereas susceptible strains allow the parasites to develop normally. To study the role of surface characteristics in eliciting this immune response, 27 types of chromatography beads that differed in matrix type, charge, functional group, and functional group density were assayed for degree of melanotic encapsulation in refractory and susceptible mosquitoes. Overall, two glucan-based matrices, Sephadex (dextran) and cellulose, stimulated the strongest responses, regardless of functional group. Substituting matrix hydroxyl groups with functional groups on Sephadex and cellulose beads decreased the level of encapsulation. These results demonstrate that glucans induce melanotic encapsulation in An. gambiae. Beads with agarose, polystyrene, and acrylic matrices, and most methacrylate-based beads elicited little or no melanization; however, epoxide-methacrylate beads were encapsulated, demonstrating that glucans are not essential for eliciting a response. Comparisons between the two strains demonstrated that refractory mosquitoes melanized many bead types to a greater degree than did susceptible mosquitoes. On this basis, we propose that an important difference between the two strains is that one of the enzymes involved in the melanization pathway functions at a higher level in the refractory strain. Finally, of all beads tested, only 85% substituted CM-Sephadex beads were virtually unmelanized in susceptible mosquitoes but highly melanized in the refractory strain; thus, a specific surface microenvironment is necessary to demonstrate this effect.
RESUMO
The nucleotide and deduced amino acid sequence of a serine protease (AgSp24D) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 271 amino acid protein that contains the conserved serine, histidine and aspartic acid residues found in serine proteases, and has the highest identity to a serine protease of unknown function from Drosophila melanogaster. In situ hybridization to the polytene chromosomes detects a single band at 24D. Northern analysis reveals only low levels of transcripts in larvae and pupae, but more abundant transcription products occur in adults. Interestingly, this analysis also shows that adult males express much higher levels of AgSp24D mRNA than females. In addition, Plasmodium-refractory mosquitoes express higher levels of AgSp24D mRNA than susceptible mosquitoes although the biological significance of this remains to be examined. The thorax is the primary site for expression in the adults. The lack of a dramatic increase in AgSp24D mRNA levels following blood feeding suggests that this protease is not involved in digestive processes. Transcriptional induction does not follow cold shock, septic wounding, bacterial injection, laminarin injection or CM-Sephadex bead injection.
Assuntos
Anopheles/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Insetos Vetores/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Anopheles/fisiologia , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Dosagem de Genes , Genes de Insetos/genética , Humanos , Malária/transmissão , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Plasmodium , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.