Biomedical subjects

M J Lehane

Publications and source records attributed to M J Lehane.

At least 19 recordsLinked to original sources

PubMed2005

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge.

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.

Amino Acid Motifs↗

PubMed2004

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco.

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 +/- 3.1 mg a.i./m2 in walls and 11.9 +/- 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 +/- 6.9 mg a.i./m2 and 34.7 +/- 10.4 mg a.i./m2 in walls and 28.8 +/- 19.2 mg a.i./m2 and 24.9 +/- 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.

Animals↗

PubMed2004

Comparative evaluation of pyrethroid insecticide formulations against Triatoma infestans (Klug): residual efficacy on four substrates.

We investigated the residual efficacy of four insecticide formulations used in Chagas disease vector control campaigns: cyfluthrin 12.5% suspension concentrace (SC), lambda-cyhalothrin 10% wettable powder (WP), deltamethrin 2.5% SC, and 2.5% WP on four types of circular blocks of wood, straw with mud, straw with mud painted with lime, and mud containing 5% of cement. Three concentrations of these insecticides were tested: the LC90 (previously determined on filter paper), the double of the LC90, and the recommended operational dose. For each bioassay test, 15 third-stage nymphs of Triatoma infestans (Klug) (Hemiptera: Reduviidae) were exposed for 120 h to each treatment at 24 h, 30, 60, 90, and 180 days post-spraying. Mortality rates, moulting history and behaviour were recorded at 24, 48, 72, and 120 h of exposure. Mortality rates were highest during the first 30 days post-spraying. Highest mortality rates (above 50%) were observed for deltamethrin 2.5% SC and lambda-cyhalothrin 10% WP on wood blocks up to three months post-spraying. Mud was the substrate on which treatments showed lowest persistence, with the other two substrates showing intermediate residual efficacy of all treatments. During the first 30 days WP formulations were not as effective as SC flowable formulations but, overall in the longer term, WP gave grater mortality rates of T. infestans nymphs exposed at up to six months post-spraying. Porous surfaces, especially mud, showed most variability presumably due to absorption of the insecticide. In contrast the less porous surfaces (i.e. wood and lime-coated mud) kept mortality rates high for longer post-treatment, irrespective of the insecticide concentration used.

Animals↗

PubMed2003

Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes.

BACKGROUND: Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. RESULTS: A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. CONCLUSIONS: The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions.

Aging↗

PubMed2003

A physiological and biochemical model for digestion in the ectoparasitic mite, Psoroptes ovis (Acari: Psoroptidae).

Mites are an important group of arthropod pests affecting crops, animals and humans. Despite this, detailed physiological studies on these organisms remain sparse due largely to their small size. Unifying models are required to draw together the diverse information from studies on different groups and species. This paper describes a model for digestion in the parasitic mite, Psoroptes ovis, the causative agent of psoroptic mange or sheep scab disease. The limited information about this species is supplemented with data from other acarines, especially house dust mites and ticks. We review the range of enzymes and allergens found in mites and consider their possible roles in digestion in mites, generally and in particular, P. ovis. Histological studies, enzyme biochemistry and molecular biology and experimental evidence suggest that P. ovis utilises a digestive system reliant upon acid peptidases functioning in a largely intracellular environment. The actions of the digestive enzymes are supplemented by the involvement of bacteria as potential direct and indirect sources of nutrition. It is possible that some extra-corporeal digestion also takes place. The interaction of bacteria and digestive enzymes on the skin surface of the sheep may be responsible for the excessive pathological reactions evident in clinical sheep scab.

Animals↗

PubMed2002

Ultrastructure of the alimentary canal of the sheep scab mite, Psoroptes ovis (Acari: Psoroptidae).

The mite Psoroptes ovis causes sheep scab disease in flocks throughout much of the world. A serious impediment to the development of novel control measures for this mite is our inability to produce in vitro colonies of the mite. Here, we describe the alimentary canal of the mite with the particular aim of determining what it feeds on, as part of a longer term goal, to develop in vitro culture techniques for P. ovis. The alimentary canal of P. ovis consists of a cuticle-lined foregut and hindgut separated by a microvilli-lined midgut. The foregut is divided into a pre-oral cavity and a muscular pharynx and oesophagus. The midgut consists of three ultrastructurally discrete areas: a stomach with bi-lobed ventriculi, a colon and a post-colon. The stomach is composed of two cell types. The most common cells (Type 1) are either cuboidal or squamous depending on the degree of gut distension and possess short microvilli, a single basally located nucleus, extensive rough endoplasmic reticulum and other components suggesting active secretion. The less common cells (Type 2), possess an extensive apical network of tubules and basally food vacuoles suggesting intracellular digestion. These cells extend into the gut lumen, become free-floating and degenerate. The colon and post-colon are composed of cells similar to Type 1 stomach cells but the post-colon epithelium possesses significantly longer microvilli. Cells from these areas have not been observed to leave the surrounding epithelium and enter the gut lumen, but it is suggested that significant absorption occurs in these two areas. Faecal pellets, often containing a significant number of bacteria, leave the digestive system through the cuticle-lined anal atrium.

Animals↗

PubMed2002

Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini.

Endodermal or midgut cells have only recently been recognized as the site of pheromone synthesis in bark beetles. Midgut cells are not only specialized for digestion, but they have also been recruited to form isoprenoid compounds that function as pheromone components in Ips pini and Dendroctonus jeffreyi. Male bark beetle midgut cells are competent to produce isoprenoid pheromones after feeding or stimulation by juvenile hormone (JH) III. Competent midgut cells share many ultrastructural features with cells that do not secrete isoprenoid pheromone, but they are distinguished from these by abundant and highly ordered arrays of smooth endoplasmic reticula. During secretion, both midgut cells that produce pheromone and cells that do not are characterized by the presence of apical extrusions (apocrine secretion) rather than the presence of vesicles that fuse with the apical membrane and undergo exocytosis (eccrine secretion). Pheromone-producing cells of the midgut do not represent a population of cells that are distinct from cells involved in digestion. All, or most, midgut cells of male I. pini and D. jeffreyi can secrete pheromones as well as digestive enzymes.

Animals↗

PubMed2002

Association of midgut defensin with a novel serine protease in the blood-sucking fly Stomoxys calcitrans.

Using ELISA we provide direct evidence that the midgut defensins of the blood-sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post-transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS-stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

Amino Acid Sequence↗

PubMed2002

Molecular cloning and sequencing of salivary gland-specific cDNAs of the blood-sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae).

Haematophagous insects produce pharmacological substances in their saliva to counteract vertebrate host haemostasis events such as coagulation, vasoconstriction and platelet aggregation. To investigate the bioactive salivary molecules of the triatomine bug Triatoma brasiliensis, we produced subtraction-enriched cDNAs of salivary-gland specific genes using suppression subtractive hybridization. Six full-length differentially expressed cDNAs (Tb113, Tb125, Tb152, Tb169, Tb180 and Tb198) were selected, cloned and sequenced. Sequence similarity searches of the databases using the putative amino acid sequence of our clones gave the following results: Tb152 - Triabin, an antithrombin induced platelet aggregation factor found in salivary gland extracts of T. pallidipennis. Tb169 - Pallidipin, an anticollagen induced platelet aggregation factor also found in T. pallidipennis salivary homogenates. Tb180 - Procalin, the major allergen of T. protracta saliva. The other three salivary-gland specific cDNAs produced no obvious homologies. Comparison of these salivary gland-specific cDNAs of with those of other triatomines combined with functional studies using recombinant proteins will allow a better understanding of the co-evolutionary process occurring between these insects and their vertebrate hosts, and may also lead to the discovery of novel antihaemostatic agents.

Amino Acid Sequence↗

PubMed2002

[Value of HPLC and fluorescent pteridines in estimating the age of Anopheles gambiae adults].

Fluorescent pteridines are photosensitive pigments of mosquito cuticle. Their quantity decreases with time during the adult life of mosquitoes. In order to test the feasibility of the reversed-phase HPLC (High Pressure Liquid Chromatography) technique, to measure pteridine fluorescence and to estimate the calendar age, reared Anopheles gambiae of known age are used. Head and thorax were studied on mosquitoes aged 0, 5, 10, 20 days after emergence. There was significant difference between male and female of 0 and 5 days of age; the difference was not significant at 10 days of age. The level of fluorescence between a female's head and thorax non separated does not differ from the sum of fluorescence level of separated head and thorax. Pteridin fluorescent of female's head and thorax does not correlate. It decreases significantly with chronological age between 0 and 5 days either for head + thorax or for head alone. Conversely, this fluorescence quantity is weak and maintained constant beyond 5 days. The use of thorax alone of the female mosquito allows the differentiation of mosquito aged less than 5 days, between 5 and 10 days and aged more than 10 days. Reversed-phase HPLC technique, at least in the way we have demonstrated, does not appear sensitive enough to estimate the age of the species An. gambiae reared in an insectarium. It seems that this technique, relatively complex to manage, does not bring a substantial advantage compared to the method of Detinova, which allows the separation of nulliparous and parous females (i.e. < or = 3 days versus > 3 days).

Age Factors↗

PubMed2001

Tsetse immune responses and trypanosome transmission: implications for the development of tsetse-based strategies to reduce trypanosomiasis.

Tsetse flies are the medically and agriculturally important vectors of African trypanosomes. Information on the molecular and biochemical nature of the tsetse/trypanosome interaction is lacking. Here we describe three antimicrobial peptide genes, attacin, defensin, and diptericin, from tsetse fat body tissue obtained by subtractive cloning after immune stimulation with Escherichia coli and trypanosomes. Differential regulation of these genes shows the tsetse immune system can discriminate not only between molecular signals specific for bacteria and trypanosome infections but also between different life stages of trypanosomes. The presence of trypanosomes either in the hemolymph or in the gut early in the infection process does not induce transcription of attacin and defensin significantly. After parasite establishment in the gut, however, both antimicrobial genes are expressed at high levels in the fat body, apparently not affecting the viability of parasites in the midgut. Unlike other insect immune systems, the antimicrobial peptide gene diptericin is constitutively expressed in both fat body and gut tissue of normal and immune stimulated flies, possibly reflecting tsetse immune responses to the multiple Gram-negative symbionts it naturally harbors. When flies were immune stimulated with bacteria before receiving a trypanosome containing bloodmeal, their ability to establish infections was severely blocked, indicating that up-regulation of some immune responsive genes early in infection can act to block parasite transmission. The results are discussed in relation to transgenic approaches proposed for modulating vector competence in tsetse.

Amino Acid Sequence↗

PubMed2001

Microfloral diversity of cultured and wild strains of Psoroptes ovis infesting sheep.

PCR amplification of 16S rDNA was used to determine the diversity of bacteria associated with 3 strains of sheep scab mite, Psoroptes ovis. Eight species of bacteria were identified by phylogenetic analysis of the PCR product sequences. Seven of these species are previously unreported in association with sheep scab mites. Five species were matched to Serratia marcesens, Proponibacteium acnes, Phyllobacterium rubiacearum, Pantoea agglomerans, Curacaobacter baltica, whereas the remaining 3 sequences matched unclassified sequences belonging to the gamma proteobacteria, pseudomonads and streptococci. Bacterial diversity of the in vivo cultured strain was very low and did not match the diversity of the 2 wild collected isolates. The diversity of the bacteria in relation to the disease of sheep scab and the possible importance of these bacteria in the diet of the mites are discussed.

Animals↗

PubMed2001

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans.

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up-regulated in response to a bloodmeal and further up-regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down-regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up-regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel-like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non-functional.

Animals↗

PubMed2000

Grouping of trypanosome species in mixed infections in Glossina pallidipes.

Trypanosomes in the dissection-positive proboscis of Glossina pallidipes were identified by PCR using species-specific primers. Of the 3741 flies dissected 643 were proboscis positive. PCR was performed on 406 dissection-positive probosces giving positive identifications in 352 (86.7%) and infection rates of 14.8% for congolense-type infections, 2.8% for vivax-type infections and 1.4% for the unidentified group. Of the 352 PCR identified infections 225 were single, 111 were double, 13 were triple infections and there were 3 quadruple infections. Statistical analysis suggests that mixed infections group into 3 largely separate divisions among the tsetse population (i) Trypanosoma congolense savannah and T. congolense Kenya coast, (ii) T. simiae, T. congolense Tsavo and T. godfreyi and (iii) T. vivax. We conclude that either differing feeding patterns among members of the fly population or the ability of the trypanosomes in each of the infection categories to significantly influence the maturation of trypanosomes in the other categories are the most likely causes of the groupings noted. Chi-squared analysis of dissection and PCR methods of trypanosome identification revealed profound differences (chi 2 = 19.1; D.F. = 1; P > 0.05). If confirmed in other studies these findings have serious implications for our understanding of trypanosome epidemiology in tsetse flies, much of which is founded on data from dissection-based trypanosome identifications.

Animals↗

PubMed2000

Transformation of Stomoxys calcitrans with a Hermes gene vector.

The ability of the Hermes transposable element to function as a germ line transformation vector was tested in the stable fly, Stomoxys calcitrans. Plasmid-based transposable element mobility assays indicated moderate mobility of Hermes in this species. Germline transformants were created using a Hermes element containing the enhanced green fluorescent protein (EGFP) under the regulatory control of the promoter from Actin5C gene of Drosophila melanogaster. Approximately 4% of the fifty-five adults that developed from the 1903 G(0) embryos injected with the vector produced transgenic progeny. In the four transgenic lines established, the EGFP expression pattern was distinctly nonuniform and levels of expression were low. Promoters other than the one from the Actin5C gene of D. melanogaster should be considered for widespread, constitutive expression. All transgenic lines contained multiple (2-4) integrated Hermes elements. Hermes integration events occurred through a canonical cut-and-paste mechanism.

Animals↗

PubMed1999

Pteridine fluorescence for age determination of Anopheles mosquitoes.

The age structure of mosquito populations is of great relevance to understanding the dynamics of disease transmission and in monitoring the success of control operations. Unfortunately, the ovarian dissection methods currently available for determining the age of adult mosquitoes are technically difficult, slow and may be of limited value, because the proportion of diagnostic ovarioles in the ovary declines with age. By means of reversed-phase HPLC this study investigated the malaria vectors Anopheles gambiae and An. stephensi to see if changes in fluorescent pteridine pigments, which have been used in other insects to determine the age of field-caught individuals, may be useful for age determination in mosquitoes. Whole body fluorescence was inversely proportional to age (P < 0.001, r2 > 91%) up to 30 days postemergence, with the regression values: y = 40580-706x for An. gambiae, and y = 52896-681x for An. stephensi. In both species the main pteridines were 6-biopterin, pterin-6-carboxylic acid and an unidentified fluorescent compound. An. gambiae had only 50-70% as much fluorescence as An. stephensi, and fluorescent compounds were relatively more concentrated in the head than in the thorax (ratios 1:0.8 An. gambiae; 1:0.5 An. stephensi). The results of this laboratory study are encouraging. It seems feasible that this simpler and faster technique of fluorescence quantification could yield results of equivalent accuracy to the interpretation of ovarian dissection. A double-blind field trial comparing the accuracy of this technique to marked, released and recaptured mosquitoes is required to test the usefulness of the pteridine method in the field.

Age Factors↗

PubMed1999

Identification of bacterial species associated with the sheep scab mite (Psoroptes ovis) by using amplified genes coding for 16S rRNA.

This was the first molecular study of the bacterial flora of the sheep scab mite (Psoroptes ovis). A sequence analysis of genes coding for 16S rRNA revealed that Serratia marcescens and bacteria closely related to Staphylococcus intermedius or Staphylococcus chromogens and Alloiococcus otitidis were present. These bacteria were associated with skin lesions, dermatitis, and otitis media caused by P. ovis.

Animals↗

PubMed1998

Factors influencing the prevalence of trypanosome infection of Glossina pallidipes on the Ruvu flood plain of Eastern Tanzania.

We report the pattern of infection of Glossina pallidipes with Trypanosoma vivax and T. congolense at a site in the Coast region of eastern Tanzania, studied between November 1993 and December 1994. Of the 2315 flies dissected 114 (4.9%) were T. congolense positive, 77 (3.3%) were T. vivax positive and 2 (0.1%) were T. brucei positive. Fly age was determined by the pteridine fluorescence method. Prevalence of infection was most strongly affected by month and the linear effect of age with the interaction of month and age having an effect for T. congolense-type infections. Sex and sex by month also have some predictive capacity when data for T. congolense and T. vivax-type infections are combined. In contrast to other similar studies our results suggest that the infection rate is non-linearly related to age of the tsetse fly, with older flies having progressively more chance of infection. The potential biological factors underpinning these interactions are discussed.

Aging↗