Biomedical subjects

S M Paskewitz

Publications and source records attributed to S M Paskewitz.

33 records · Page 2Linked to original sources

PubMed1996

Borrelia burgdorferi-infected Ixodes scapularis (Acari: Ixodidae) and Peromyscus leucopus in northeastern Wisconsin.

Populations of the blacklegged tick, Ixodes scapularis Say, are established in western and central Wisconsin in the upper midwestern United States, but appear to be expanding geographically there. Here, we report a previously unknown population in northeastern Wisconsin. Questing I. scapularis nymphs and adults were collected by flagging vegetation from a riverine site in Marinette County, Wisconsin, in spring of 1993 and 1994. Dissection and culture of tick guts in modified Barbour-Stoenner-Kelley II medium showed that some of the ticks were infected with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwald & Brenner, causative agent of Lyme disease. Fifteen of 30 white-footed mice, Peromyscus leucopus (Rafinesque), live-trapped at the site on 23-24 August 1994 were infested with immature I. scapularis, and ear-punch biopsies yielded B. burgdorferi cultures from 2 of the mice. However, none of 50 white-tailed deer, Odocoileus virginianus (Zimmermann), shot by hunters in Marinette County in November 1994 had I. scapularis on them, probably because no deer were shot at the same site where the tick population is located. These findings document existence of an established population of I. scapularis and a focus of Lyme disease in northeastern Wisconsin.

Animals↗

PubMed1996

Ixodes scapularis (Acari:Ixodidae): status and changes in prevalence and distribution in Wisconsin between 1981 and 1994 measured by deer surveillance.

A statewide survey of blacklegged ticks, Ixodes scapularis Say, on white-tailed deer was conducted in 1994 to examine the status and changes in the prevalence and geographic distribution of this tick in Wisconsin. I. scapularis adults were collected at 17 of 26 deer registration stations, including stations in eastern (Poy Sippi) and southern Wisconsin (Monroe) without previously reported populations. Nearly all of the stations where 1 or no ticks were collected were in the eastern 1/3 of the state. The prevalence of I. scapularis in 1994 was compared with prevalence estimates taken in similar surveys during 1981 and 1989. A significant increase in tick prevalence occurred at stations along the Wisconsin River Valley, which previously defined the edge of the eastern range of I. scapularis populations and in southern Wisconsin.

Animals↗

PubMed1996

A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species.

Cryptic species complexes are groups of closely related species that are difficult or impossible to distinguish by morphological traits. These complexes are known from a wide variety of arthropods and are common among the well-studied, medically-important insects. For example, many of the anopheline vectors of malaria parasites are members of cryptic species complexes. Complexes typically include both vector and non-vector species, and two or more member species are often found sympatrically. Until the late 1950, only two such Anopheles complexes were known, the A. gambiae complex from Africa and the A. maculipennis complex from Europe. Today, dozens of Anopheles cryptic species complexes are recognized, and accumulating evidence suggests that most important malaria vectors are likely to be members of such complexes. A variety of methods have been developed for identifying the species of individual specimens from these complexes, although until recently only those based on species-specific allozymes and polytene chromosome inversions were widely used. The limitations inherent in these methods have been circumvented with DNA-based procedures, which are especially useful because both sexes and all developmental stages can be identified, and DNA can be recovered from samples stored by a wide variety of simple methods. Several DNA-based identification techniques have been developed, including hybridization assays based on species-specific repeat sequences, and diagnostic PCR fragments produced either by the use of random PCR primers or by amplifying DNA with primers based on known species-specific sequences. In this review we discuss the relative marks of different methods of cryptic species identification, with emphasis on the use of ribosomal DNA as a target for species-diagnostic PCR assays.

Animals↗

PubMed1995

Effect of mosquito age and reproductive status on melanization of sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae.

Malaria-refractory and -susceptible strains of the mosquito vector, Anopheles gambiae, differ in their response to negatively-charged Sephadex CM-25 beads. CM-25 beads elicit a much stronger melanization reaction in refractory mosquitoes than in susceptible mosquitoes. Light microscopic and scanning electron microscopic studies documented a progression from early stages with small spots of melanin adhering to CM-25 beads to late stages where spots had grown and coalesced to form a dark dense capsule. This reaction occurred maximally during the first 18 hr after inoculation; female mosquitoes aged 3-5 days showed 48% of beads heavily melanized by 18 hr postinoculation in the refractory strain and 0-10% in the susceptible strain. Female mosquito age and reproductive status strongly affected the ability to melanize beads. All of the beads were completely melanized in the refractory strain on the day immediately following eclosion (Day 0); thereafter these levels decreased steadily until the last time point on Day 7, when only 23% of beads were melanized in this strain. In the susceptible strain, 53% of beads were heavily melanized on Day 0 and 0-10% were melanized at all other times. At Day 1 and 2 after blood feeding, 85 and 88% of beads, respectively, were heavily melanized in refractory females in comparison with control mosquitoes of the same age which heavily melanize 23-58% of the beads. Blood feeding had little effect on the ability to melanize beads in the susceptible strain.

Aging↗

PubMed1995

Malaria: current and future prospects for control.

Malaria is the most important insect-transmitted human disease, but progress in its control has been slow, especially in Africa where approximately 90% of the infections occur. Several factors have contributed to the problem. Parasites and vectors have developed resistance to antimalarial drugs and insecticides; differences in the biology of major malaria vectors preclude the development of simple, universally applicable strategies for malaria control; and the cost of available malaria-control tools often exceeds the public health resources in the most malarious parts of the world. New tools are desperately needed. Current efforts include the testing of tools such as insecticide-impregnated bed nets that could become available in the near term, as well as long-term projects such as the development of malaria vaccines and mosquito-targeted genetic control strategies. The success or failure of any of these approaches will depend ultimately on understanding the natural patterns of malaria transmission in the field.

Animals↗

PubMed1993

Comparison of two ribosomal DNA-based methods for differentiating members of the Anopheles gambiae complex (Diptera: Culicidae).

Two DNA-based methods, the restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR), were used to identify mosquitoes of the Anopheles gambiae Giles complex collected in Kenya. Field-collected specimens of An. gambiae, An. arabiensis Patton, and An. merus Donity were tested. From a sample of 208 mosquitoes, 181 (87%) were identified by the RFLP method and 205 (99%) were identified by the PCR method. There was complete concordance between the two methods with regard to species identification. PCR assays were simpler, faster, and more reliable than RFLP assays.

Animals↗

PubMed1993

Evaluation of the polymerase chain reaction method for identifying members of the Anopheles gambiae (Diptera: Culicidae) complex in southern Africa.

Chromosomally and electrophoretically identified field samples of members of the Anopheles gambiae Giles complex from southern Africa and colony material were used to test the polymerase chain reaction (PCR) method of identification. The samples included An. arabiensis Patton, An. quadriannulatus Theobald, and An. merus Donitz. Only one of the 217 specimens examined was disputed. Twenty specimens (9%) were reamplified because of the presence of multiple bands or lack of amplification after the first assay. Specimens of An. merus from this region displayed both the diagnostic An. merus PCR band as well as the An. quadriannulatus band, possibly because of interspecific hybridization or random variation within the population. This work extends the geographical range over which the PCR technique has been verified to southern Africa.

Africa, Southern↗

PubMed1993

The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae species complex.

The primary and secondary structure of the internal transcribed spacers of ribosomal DNA (ITS1 and ITS2) and their utility for phylogenetic analysis of closely related species were examined using the Anopheles gambiae complex as a model. Restriction mapping revealed an unusual architectural feature in the ITS1 of several members of an An. gambiae cryptic species complex. Multiple spacer lengths are prevalent in An. merus and An. melas and are due to variable numbers of a repeated 250 bp sequence. Secondary structure analysis indicated that the repeat forms a helix and loop that may be involved in rDNA processing. Intra- and interspecific polymorphism within the species complex were further examined by DNA sequencing of forty-eight ITS2 clones obtained by polymerase chain reaction from individuals of the five species. Interspecies variation in the approximately 426 bp ITS2 sequence ranged between 0.4% and 1.6%; intraspecies variation ranged from 0.07% in An. arabiensis to 0.43% in An. gambiae. Intraindividual variation ranged from 0% in four individuals to a high of 0.4% in one An. quadriannulatus specimen. None of the variants were shared between species. The low level of variation supports the hypothesis that species of the complex evolved recently.

Animals↗

PubMed1992

Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae.

Two distinct site-specific retrotransposon families, named RT1 and RT2, from the sibling mosquito species Anopheles gambiae and A. arabiensis, respectively, were previously identified. Both were shown to occupy identical nucleotide positions in the 28S rRNA gene and to be flanked by identical 17-bp target site duplications. Full-length representatives of each have been isolated from a single species, A. gambiae, and the nucleotide sequences have been analyzed. Beyond insertion specificity, RT1 and RT2 share several structural and sequence features which show them to be members of the LINE-like, or non-long-terminal-repeat retrotransposon, class of reverse transcriptase-encoding mobile elements. These features include two long overlapping open reading frames (ORFs), poly(A) tails, the absence of long terminal repeats, and heterogeneous 5' truncation of most copies. The first ORF of both elements, particularly ORF1 of RT1, is glutamine rich and contains long tracts of polyglutamine reminiscent of the opa repeat. Near the carboxy ends, three cysteine-histidine motifs occur in ORF1 and one occurs in ORF2. In addition, each ORF2 contains a region of sequence similarity to reverse transcriptases and integrases. Alignments of the protein sequences from RT1 and RT2 reveal 36% identity over the length of ORF1 and 60% identity over the length of ORF2, but the elements cannot be aligned in the 5' and 3' noncoding regions. Unlike that of RT2, the 5' noncoding region of RT1 contains 3.5 copies of a 500-bp subrepeat, followed by a poly(T) tract and two imperfect 55-bp subrepeats, the second spanning the beginning of ORF1. The pattern of distribution of these elements among five siblings species in the A. gambiae complex is nonuniform. RT1 is present in laboratory and wild A. gambiae, A. arabiensis, and A. melas but has not been detected in A. quadriannulatus or A. merus. RT2 has been detected in all available members of the A. gambiae complex except A. merus. Copy number fluctuates, even among the offspring of individual wild female A. gambiae mosquitoes. These findings reflect a complex evolutionary history balancing gain and loss of copies against the coexistence of two elements competing for a conserved target site in the same species for perhaps millions of years.

Amino Acid Sequence↗

PubMed1991

A genetic study of Plasmodium susceptibility in the African malaria vector Anopheles gambiae.

We are studying the interaction between malarial parasites and their mosquito hosts by a process based on the genetic selection of lines of the mosquito vector that will not support normal parasite development. The model system we are using is the mosquito Anopheles gambiae and a number of different human and non-human primate malarial parasites. Our first effort in this general approach involved the selection of a strain of An. gambiae that was able to encapsulate the malarial parasite during or just after its penetration of the mosquito midgut. This mosquito has been found to be highly refractory to a wide variety of different Plasmodium species and at least partially refractory to all four human malarial parasites. The basis of this phenomenon appears to be the mosquito's enhanced ability to mount a normal encapsulation response against invading pathogens or parasites. The late ookinete is enclosed in a heavily melanized capsule approximately 16-24 hours after the mosquito takes an infective blood meal. Encapsulation appears to be primarily humoral; electron microscopy reveals evidence of the phenomenon as the ookinete passes through or between midgut cells, and no hemocyte involvement has been observed. Subcellular structures in newly encapsulated parasites appear normal, indicating that live rather than dead or dying parasites are encapsulated. Preliminary studies suggest that two unlinked loci contribute to the refractory mosquito phenotype. One of these loci is very closely linked to a region of the mosquito genome that includes two esterases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Use of the polymerase chain reaction to identify mosquito species of the Anopheles gambiae complex.

A nonradiometric method has been developed for distinguishing between the sibling species Anopheles gambiae Giles and An. arabiensis Patton, two important Afrotropical vectors of malaria. DNA fragments of species diagnostic length are amplified by polymerase chain reaction (PCR) from a small amount of unknown DNA and three different PCR primers. All three PCR primers are based on ribosomal DNA (rDNA) sequences. A universal plus-strand primer (A0) is derived from a conserved region at the 3' end of the 28S rDNA coding region. Two species-specific minus-strand primers (Aa0.5 and Ag1.3) are derived from sequences in the intergenic spacers. The Ag1.3 sequence is approximately 1.3 kb downstream of A0; the Aa0.5 sequence is about 0.5 kb downstream of A0. When mosquito DNA is amplified in the presence of all three primers, a 1.3 kb fragment is produced if An. gambiae DNA is used as template, and a 0.5 kb fragment is produced if An. arabiensis DNA is used. Amplification of DNA from An.gambiae/An. arabiensis hybrids produces both the 1.3 kb and the 0.5 kb fragments. Neither diagnostic fragment is produced when DNA from other species in the An. gambiae complex is used as template.

Animals↗

PubMed1989

Site-specific ribosomal DNA insertion elements in Anopheles gambiae and A. arabiensis: nucleotide sequence of gene-element boundaries.

The nucleotide sequence of the junctions between the 28S ribosomal gene and site-specific insertion elements from two sibling mosquito species, Anopheles gambiae and A. arabiensis, is reported. In both species, elements insert at the same point within the 28S gene, but this site is 634 basepairs (bp) 3' of the R1 (Type I) insertion site in Drosophila melanogaster. The two mosquito elements each have poly A tails and a polyadenylation signal, but the extreme 3' and 5' ends show no other similarity to each other or to any other insertion element. In both mosquito species, identical target site duplications of 17 bp are generated. The sequence TNTCCCTNT found in this duplication is also found in the 14 bp target site duplications that flank R1 elements in D. melanogaster. Another sequence in this duplication, GGGATAACT, is very similar to the sequence GGGAGTAACT found in the 24 base sequence required by the Bombyx mori R2 endonuclease.

Animals↗

PubMed1989

Ultrastructural localization of phenoloxidase in the midgut of refractory Anopheles gambiae and association of the enzyme with encapsulated Plasmodium cynomolgi.

A melanogenic enzyme, phenoloxidase, was localized ultrastructurally in the midgut epithelia of 2 strains of Anopheles gambiae, a refractory strain that melanotically encapsulates Plasmodium cynomolgi ookinetes on the midgut, and a susceptible strain that does not. Midguts were incubated with either dopa or dopamine, and the resultant electron-dense product of phenoloxidase activity was localized on the basal lamina (BL) and cellular basal membrane labyrinth (BML) in uninfected mosquitoes of both strains. In infected refractory mosquitoes, the reaction products still were observed on the BL and BML but were especially dense in the BML of midgut cells near encapsulated ookinetes and in the capsule itself. In infected susceptible mosquitoes, phenoloxidase localization was reduced or absent in the BL and BML and was not observed near parasites. Phenylthiourea (PTU) inhibited the phenoloxidase reaction, indicating that the reaction product deposited in the absence of PTU resulted from enzyme activity and not autooxidation of the substrates. It is concluded that higher levels of phenoloxidase in the refractory strain following a blood meal may contribute to the ability to encapsulate ookinetes.

Animals↗

PubMed1988

Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae.

Using transmission electron microscopy, we investigated the encapsulation of the simian malaria parasite, Plasmodium cynomolgi, in a refractory strain of the mosquito, Anopheles gambiae. After the ookinete penetrates the mosquito midgut epithelium and lodges between the basal membrane and the basal lamina, an electron-dense, melanin-like substance begins to coalesce around the parasite. Completely encapsulated parasites were found as early as 16 hr after the blood meal. Granules of the melanin-like substance often appeared to condense onto the parasite from the fluid in the extracellular spaces of the basal membrane labyrinth. Melanin granules also appeared to condense from the hemolymph onto the basal lamina underlying the parasite. In addition, groups of tubules, vesicles, and membranous whorls often were found in midgut cells that were located next to or were enclosing parasites. These structures were unusually electron-dense, and may have been associated with melanization. Hemocytes rarely were observed near completed capsules and neither hemocytes nor their remnants were components of the capsules. During later stages of encapsulation, parasites appeared abnormal and often were infiltrated with melanin. Although late-stage capsules were usually located basally, completed capsules enclosed by membranes were occasionally observed near the apical border of the midgut. Other capsules associated with cellular debris, were found in the lumen of the midgut from 1 to 6 days after the blood meal.

Animals↗

PubMedDate not supplied

Induction of mosquito hemolymph proteins in response to immune challenge and wounding.

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.

Animals↗