Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

Spatial distribution of Culicoides species in Portugal in relation to the transmission of African horse sickness and bluetongue viruses

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midge vectors was carried out at 87 sites within a 50 x 50 km grid distributed across Portugal, using light trap collections at the time of peak midge abundance. Culicoides imicola (Kieffer) made up 66% of the 55 937 Culicoides in these summer collections. It was highly abundant in the central eastern portion of Portugal, between 37 degrees 5' N and 41 degrees 5' N, and in a band across to the Lisbon peninsula (at around 38 degrees 5' N). Of all the complexes, its distribution was most consistent with that of previous outbreaks of Culicoides-borne disease, suggesting that it may remain the major vector in Portugal. Its distribution was also broadly consistent with that predicted by a recent climate-driven model validating the use of remote sensing datasets for modelling of Culicoides distribution. Adult C. imicola were found to have overwintered at 12 of 20 sites re-surveyed in winter but it did so in very low numbers. Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) complex midges were widespread despite their low summer abundance. The observed coincidence of high abundances of C. imicola and high abundances of C. pulicaris in summer lead us to suggest that C. imicola could bring African horse sickness virus or bluetongue virus into contact with C. pulicaris and the latter complex, together with C. obsoletus, could then transmit these viruses across much wider areas of Europe. The fact that adult C. pulicaris are present in high abundances in winter may provide a mechanism by which these viruses can overwinter in these areas.     

Cytogenetics of the Anopheles gambiae complex in Sudan, with special reference to An. arabiensis: relationships with East and West African populations

The species composition of malaria vector mosquitoes belonging to the Anopheles gambiae complex (Diptera: Culicidae) from >40 localities in Sudan, representing most ecological situations, was determined by analysis of ovarian polytene chromosomes. Of 2162 females, 93% were identified as An. arabiensis Patton and 7% were An. gambiae Giles sensu stricto. No hybrids were found between the two species. Anopheles arabiensis occurred in all but two sites, whereas An. gambiae s.s. was effectively limited to the southernmost, more humid localities. For chromosomal paracentric inversions, the degree of polymorphism was low in An. gambiae s.s. (inversions 2La, 2Rb and 2Rd), higher in An. arabiensis (inversions Xe, 2Ra, b, bc, d1, s; 3Ra, d). Anopheles gambiae samples from Sudan were all apparently panmictic, i.e. they did not show restricted gene flow such as observed among West African populations (interpreted as incipient speciation). Chromosomal inversion patterns of An. gambiae in southern Sudan showed characteristics of intergrading Savanna/Forest populations similar to those observed in comparable eco-climatic situations of West Africa. Anopheles arabiensis was polymorphic for inversion systems recorded in West Africa (2Ra, 2Rb, 2Rdl, 3Ra) and for a novel 2Rs polymorphism, overlapping with inversion systems 2Rb and 2Rd1. Samples carrying the 2Rs inversion were mostly from Khashm-el-Girba area in central-eastern Sudan. In the great majority of the samples all polymorphic inversions were found to be in Hardy-Weinberg equilibrium. Sudan populations of An. arabiensis should therefore be considered as generally panmictic. Anopheles arabiensis shows more inversion polymorphism in west than in east African populations. Sudan populations have more evident similarities with those from westwards than those from eastwards of the Great Rift Valley. The possible influence of the Rift on evolution of An. arabiensis is discussed.     

Phosphoglucomutase and trehalase isoenzymes of Venezuelan simulium vectors of Onchocerca volvulus

Phosphoglucomutase (PGM) and trehalase (Tre) isoenzymes of five species of Simulium blackflies (Diptera: Simuliidae), vectors of onchocerciasis in Venezuela, were investigated by means of a portable electrophoresis field kit. Tre differed between S. incrustatum and S. oyapockense s.l. Electrophoretic variation of Tre in other members of the S. amazonicum and S. incrustatum groups merit further investigation. PGM appears to be more useful for separating populations within species complexes. Multiple populations and/or seasonal changes in population structure of S. guianense s.l., S. exiguum s.l. and S. metallicum s.l. were inferred from elecrophoretic variation of PGM.     

Further evidence that deltamethrin-impregnated collars protect domestic dogs from sandfly bites

In many foci of zoonotic visceral leishmaniasis (ZVL), domestic dogs are important reservoir hosts of the causative Leishmania parasites transmitted by phlebotomine sandflies (Diptera: Psychodidae). We tested the protective value of impregnated dog collars (20 g plastic containing deltamethrin 800 mg ai) against Phlebotomus papatasi (Scopoli) sandflies in Iran. For each assay, the dog was sedated and caged in a net with 70-100 wild-caught sandflies overnight (23.30-06.30 hours). Dogs wearing the collars were bitten by approximately 80% fewer sandflies than before collars were fitted, i.e. 51% vs. 11% of hungry female flies exposed. Sandfly mortality rates following 20 h exposure to dogs with collars (18%) or without collars (17%) were not significantly different. Effects of collars were tested when dogs had been wearing them for 8 days. A previous trial against the sandfly P. perniciosus Newstead in France, using smaller dogs, showed that effects of such collars were not fully realized until they had been worn for 2 weeks or more; they remained effective for at least 8 months and killed significant proportions of the sandflies exposed. Present results with P. papatasi, confirming that this simple device provides effective protection against sandflies, are considered sufficiently encouraging to justify a community-wide field trial of deltamethrin-impregnated dog collars against ZVL vector sandflies in Iran.     

Diversion of Anopheles gambiae from children to other hosts following exposure to permethrin-treated bednets

Permethrin-treated bednets reduce mortality and morbidity from malaria in Gambian children. However, it is not certain how this effect is achieved, as neither mosquito numbers nor the human blood index of indoor-resting female Anopheles gambiae Giles sensu lato (Diptera: Culicidae) mosquitoes have been reduced when treated bednets were introduced into a community. One possibility is that insecticide-treated bednets divert mosquitoes from children to adults. To investigate this hypothesis, a cross-over trial with insecticide-treated bednets was undertaken in two small Gambian villages. To differentiate mosquitoes that had fed on children from those that had fed on adults, all children in the study villages were immunized with rabies vaccine before the trial. Using the detection of rabies antibody in a bloodmeal as an indicator that a mosquito had bitten a child, it was found that the percentage of blood-fed mosquitoes caught indoors that had bitten a child fell significantly from 30.8% to 9.2% and from 28.0% to 6.9% in each village after insecticide-treated bednets were introduced. To investigate the possibility that some diversion to animals had occurred, a PCR analysis for human beta-globin DNA was undertaken on selected samples. The results of this investigation were confusing, as some rabies-antibody positive bloodmeals were negative for human DNA. This may have been due to cross-reacting antibodies in animal sera and/or DNA degradation by digestion in the mosquito. Although good evidence for diversion of mosquitoes away from children was obtained, it remains uncertain whether diversion was mainly to adult humans, to animals or to both.     

Insecticide resistance in Brazilian Triatoma infestans and Venezuelan Rhodnius prolixus

Resistance levels to insecticides used in control of Chagas Disease vectors were assessed in two species of bugs (Hemiptera: Reduviidae): Triatoma infestans (Klug) from Brazil and Rhodnius prolixus Stål from Venezuela. The resistance ratios, compared to susceptible laboratory strains, were determined by topical application bioassays. The T. infestans PA strain exhibited resistance ratios of 7× to deltamethrin, 3.6× to β‐cyfluthrin and 3.3× to cypermethrin, but was susceptible to β‐cypermethrin and lambda‐cyhalothrin. Rhodnius prolixus CA strain showed resistance to all the pyrethroids evaluated, the resistance ratios ranging between 4.5× to lambda‐cyhalothrin and 12.4× to cypermethrin. Deltamethrin resistance in both strains was decreased by piperonyl butoxide, suggesting oxidative metabolism as cause of resistance.     

Seasonal Population Dynamics of Ixodes Ticks and Tick-Borne Encephalitis Virus

Seasonality of the epidemic and epizootic processes of tick-borne encephalitis (TBE) depend on the period of activity of ixodid ticks Ixodes persulcatus Schulze and I. ricinus Linnaeus, which are the main reservoirs and vectors of TBE virus, and also on the process of their activation. The period of activity is the period during which the ticks occur in the active state. Activation is the transition into this state of ticks that moulted from the preceding stage and completed post-moulting development. For I. persulcatus, the first adult ticks generally emerge between April 10 and May 9. Under a variety of natural conditions, activation of adult I. persulcatus after wintering lasts for 45–86 days and this period may be even longer in certain areas of the Far East. The period during which one-half of the entire tick population becomes activated (AT₅₀) comprises no more than 10–20 days. In adult I. ricinus ticks the activation period may last even longer than in I. persulcatus. The data on duration of the period of activity and on activation of larval and nymphal stages of both tick species were considered. Ticks exhausting their nutrient reserves and failing to find a host die quickly. The period during which 50% of the entire tick population die under natural conditions is designated LT₅₀. The main types of I. persulcatus and I. ricinus seasonal activity within their species ranges were reviewed. Data on the relationship between TBE virus reproduction in a natural focus and physiological age, pattern of activation, and seasonal changes in age structure of the tick population were analyzed. Seasonal changes in the prevalence of infection among active unfed adult ticks in a natural population are determined by virus content in individual ticks at the moment of their activation and also by the duration of subsequent virus persistence (the rate of virus loss) in ticks. Apparently, the opportunity and frequency of horizontal TBE virus transmission under natural conditions, change during the season of tick activity.     

Species and endosymbiont diversity of Bemisia tabaci (Homoptera: Aleyrodidae) on vegetable crops in Senegal

Bemisia tabaci‐transmitted geminiviruses are one of the major threats on cassava and vegetable crops in Africa. However, to date, few studies are available on the diversity of B. tabaci and their associated endosymbionts in Africa. More than 28 species have been described in the complex of B. tabaci cryptic species; among them, 2 are invasive pests worldwide: MED and MEAM1. In order to assess the species diversity of B. tabaci in vegetable crops in Senegal, several samplings in different localities, hosts and seasons were collected and analyzed with nuclear (microsatellite) and mitochondrial (COI) markers. The bacterial endosymbiont community was also studied for each sample. Two species were detected: MED Q1 and MEAM1 B. Patterns of MED Q1 (dominance on most of the samples and sites, highest nuclear and mitochondrial diversity and broader secondary endosymbiont community: Hamiltonella, Cardinium, Wolbachia and Rickettsia), point toward a predominant resident begomovirus vector group for MED Q1 on market gardening crops. Furthermore, the lower prevalence of the second species MEAM1 B, its lower nuclear and mitochondrial diversity and a narrower secondary endosymbiont community (Hamiltonella/Rickettsia), indicate that this genetic group is exotic and results from a recent invasion in this area.