Symbiotic bacteria affect competence for pathogen transmission in insect vectors, including mosquitoes. However, knowledge on mosquito-microbiome-pathogen interactions remains limited, largely due to methodological reasons. The current, cost-effective practice of sample pooling used in mosquito surveillance and epidemiology prevents correlation of individual traits (i.e., microbiome profile) and infection status. Moreover, many mosquito studies employ laboratory-reared colonies that do not necessarily reflect the natural microbiome composition and variation in wild populations. As a consequence, epidemiological and microbiome studies in mosquitoes are to some extent uncoupled, and the interactions among pathogens, microbiomes, and natural mosquito populations remain poorly understood. This study focuses on the effect the pooling practice poses on mosquito microbiome profiles, and tests different approaches to find an optimized low-cost methodology for extensive sampling while allowing for accurate, individual-level microbiome studies. We tested the effect of pooling by comparing wild-caught, individually processed mosquitoes with pooled samples. With individual mosquitoes, we also tested two methodological aspects that directly affect the cost and feasibility of broad-scale molecular studies: sample preservation and tissue dissection. Pooling affected both alpha- and beta-diversity measures of the microbiome, highlighting the importance of using individual samples when possible. Both RNA and DNA yields were higher when using inexpensive reagents such as NAP (nucleic acid preservation) buffer or absolute ethanol, without freezing for short-term storage. Microbiome alpha- and beta-diversity did not show overall significant differences between the tested treatments compared to the controls (freshly extracted samples or dissected guts). However, the use of standardized protocols is highly recommended to avoid methodological bias in the data.
- Klíčová slova
- dissection, epidemiology, microbiome, mosquito, pooling, preservation, vector,
- MeSH
- Aedes mikrobiologie MeSH
- Bacteria klasifikace genetika MeSH
- Culex mikrobiologie MeSH
- komáří přenašeči mikrobiologie MeSH
- mikrobiologické techniky MeSH
- mikrobiota * MeSH
- ochrana biologická MeSH
- střevní mikroflóra MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Growth temperature range, resistance to selective antibiotics, activities of 23 enzymes, protein fingerprints and fatty acids composition of the spirochaetal strain BR91, isolated from the Culex pipiens mosquito, were tested. The spirochaetes were grown in BSK-H Complete liquid medium. The optimal in vitro growth temperature of the strain was 33 °C. Strain BR91 was sensitive to trimethoprim, nalidixic acid, 5-fluorouracil, and tolerated phosphomycin. The strain produced acid and alkaline phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and α-fucosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay revealed several major proteins in the size range of 13-16 kDa, 22-30 kDa and 37-131 kDa. Fatty acid methyl ester (FAME) analysis showed that C₁₄:₀, C₁₆:₀, C₁₈:₁ ω9c and summed feature 5 (C₁₈:₂ ω6,9c and/or C₁₈:₀ anteiso) are major fatty acids. This study highlights certain phenotypic differences between strain BR91 and the Lyme disease spirochaete Borrelia burgdorferi, and supports the hypothesis that strain BR91 represents a unique taxonomical entity in a system of spirochaetal species.
- Klíčová slova
- Culex pipiens, Enzyme activity, FAME, SDS-PAGE, Spirochaete,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny analýza MeSH
- Borrelia burgdorferi MeSH
- Culex mikrobiologie MeSH
- Culicidae MeSH
- enzymy analýza MeSH
- kultivační média chemie MeSH
- mastné kyseliny analýza MeSH
- mikrobiální testy citlivosti MeSH
- Spirochaetaceae chemie klasifikace izolace a purifikace fyziologie MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- bakteriální proteiny MeSH
- enzymy MeSH
- kultivační média MeSH
- mastné kyseliny MeSH
In this study, larvae (1,179 ex.) of mosquito genera Culex were examined for the presence of spirochaetes by Dark Field Microscopy (DFM) at the locality of Blansko (Czech Republic) in of 2004-2008. DFM spirochaete positive samples (25.4 %) were investigated by nested PCR; only 4 samples were positive for the presence of Borrelia burgdorferi sensu lato, which is 0.3 % of the total examined samples. We can conclude that only a low percentage of pathogenic borreliae are presented in mosquito larvae, while the spirochaete of undefined genera infect larvae in high amounts.
- MeSH
- Aedes mikrobiologie MeSH
- Borrelia burgdorferi izolace a purifikace MeSH
- časové faktory MeSH
- Culex mikrobiologie MeSH
- larva mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the presented work, six Borrelia burgdorferi sensu lato isolates were recovered from Ixodes ricinus ticks and one strain of spirochete with uncertain taxonomic position isolated from Culex pipiens mosquito collected in the Valtice area (South Moravia, Czech Republic), as well as representative type strains, were further characterized by ribotyping and whole-cell protein fingerprinting. The ribotype patterns obtained with HindII and EcoRV ribotype patterns showed good correlations with the recently proposed genotypes and grouped each genospecies in a well-separated cluster. The whole-cell protein profiles of genospecies were more heterogeneous than the ribotype patterns. The non-identified spirochetal strain was clearly separated from the Borrelia isolates in both methods thus proving their good capacity to characterize Borrelia burgdorferi sensu lato strains.
- MeSH
- bakteriální proteiny chemie MeSH
- Borrelia burgdorferi komplex klasifikace genetika izolace a purifikace MeSH
- Culex mikrobiologie MeSH
- fylogeneze * MeSH
- klíště mikrobiologie MeSH
- lymeská nemoc epidemiologie MeSH
- ribotypizace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Názvy látek
- bakteriální proteiny MeSH
A total of 662 samples (winter period: 469; summer period: 193 specimens) of female mosquitoes of the genus Culex, Aedes and Anopheles were collected during the period March 2000-April 2001 from the locality of Vysoke Myto (Eastern Bohemia, Czech Republic). They were examined by dark field microscopy for the presence of spirochetes. The motile spirochetes were observed in 4.2 % of all species of investigated mosquitoes. One spirochetal strain out of the 8 isolation attempts (BRZ14) was obtained (cultivation rate was 12.5 %) and the spirochetal strain was then successfully cultivated and identified using PCR for the presence of Borrelia burgdorferi s.l., and subsequently with the RFLP as genomospecies Borrelia afzelii. This strain was derived from overwintering Culex (Culex) pipiens biotype molestus female mosquitoe. This is apparently one of the sporadic cases of the occurrence of pathogenic borreliae in haematophagous arthropods, other than Ixodes ricinus complex ticks.
- MeSH
- arachnida jako vektory mikrobiologie MeSH
- Borrelia burgdorferi komplex izolace a purifikace MeSH
- Culex mikrobiologie MeSH
- DNA bakterií chemie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- lymeská nemoc mikrobiologie MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- roční období MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- DNA bakterií MeSH
After first finding Borrelia in the midgut of imago mosquitoes, we concentrated on the presence of Borrelia in mosquito development stages--larvae of the third or fourth instar. In the summer season in the years of 2000-2001 a total of 439 Culex (Culex) pipiens pipiens larvae were collected from a barrel of rainwater in the Obrany holiday area of Brno city (East Moravia, Czech Republic). The larvae midgut was observed under dark-field microscopy. Ten DFM positive samples (2.28%) were further analysed using the single-tube nested PCR method for the presence of flagellum DNA sequence specific for Borrelia burgdorferi sensu lato, of which 5 were positive. Borrelian positivity of Culex (C.) pipiens pipiens larvae was 1.14%. One spirochete isolated strain in BSK-H medium was obtained. PCR detection for borrelian DNA of the isolated strain was negative. From these results we can conclude that a low percentage Borreliae can be also found in mosquito larvae and are likely to survive into imago stage.
- MeSH
- Borrelia burgdorferi genetika izolace a purifikace MeSH
- Culex mikrobiologie MeSH
- DNA bakterií genetika MeSH
- elektroforéza v agarovém gelu MeSH
- hmyz - vektory mikrobiologie MeSH
- larva MeSH
- lymeská nemoc epidemiologie mikrobiologie MeSH
- polymerázová řetězová reakce MeSH
- roční období MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Názvy látek
- DNA bakterií MeSH
- MeSH
- Aedes mikrobiologie MeSH
- Culex mikrobiologie MeSH
- flagella ultrastruktura MeSH
- lidé MeSH
- lymeská nemoc mikrobiologie MeSH
- Spirochaetales izolace a purifikace ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
During the years 1995-1996, a total of 1,743 overwintering Culex pipiens biotype molestus female mosquitoes were tested for the presence of spirochetes in several localities in South Moravia, Czech Republic.The spirochetes were observed in 5% of the mosquitoes investigated. One of the five isolated strains of spirochetes (BR-84) was identified as Borrelia afzelii. The potential role of mosquitoes in the ecology and epidemiology of Lyme disease (LD) borreliae should be further investigated.
- MeSH
- Borrelia klasifikace izolace a purifikace MeSH
- Culex mikrobiologie MeSH
- incidence MeSH
- infekce bakteriemi rodu Borrelia mikrobiologie přenos MeSH
- infekce přenášené vektorem klasifikace MeSH
- pulzní gelová elektroforéza MeSH
- rizikové faktory MeSH
- techniky typizace bakterií MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Laboratory and field comparisons were made with two wettable powder formulations of Bacillus thuringiensis serotype H-14 (B. t. H-14) prepared in Czechoslovakia ("Moskitur") and the USSR ("Baktokulicid"). Expressed in the international Aedes aegypti toxic units (TU X mg-1) the potency of these two test formulations was greater than that of the Institute Pasteur Standard IPS-78 (= 1,000 TU X mg-1), i.e. Moskitur had a potency of about 1,500 TU X mg-1 and the Soviet Baktokulicid 2,000 TU X mg-1. The Baktokulicid and Moskitur LC 90 values for laboratory-reared Aedes aegypti larvae were, respectively, 0.11 and 0.16 mg X l-1. The range of LC 90 values for the Czechoslovak wild-caught mosquito species of the genera Aedes and Culex was 0.14-0.31 mg X liter-1 with Moskitur, 0.11-0.41 mg X l-1 with Baktokulicid, and 0.16-0.48 mg X l-1 with IPS-78. The susceptibility of laboratory Anopheles stephensi larvae was close to that of Aedes aegypti, larvae of An. messeae required many times as much Baktokulicid (1.6 mg X l-1) and Moskitur (more than 6.4 mg X l-1) for 90% mortality as did other mosquito species. The aim of outdoor assays was to establish the minimum Moskitur and Baktokulicid rates giving a 100% control of mosquito larvae. For Ae. cantans breeding habitat in flood plain forest areas these rates ranged between 0.1-0.5 mg X l-1 (0.2-1.0 kg X ha-1), for Ae. vexans control on artificially irrigated meadows between 0.8-2.0 mg X l-1 (1.2-3.0 kg X ha-1). Consistently with laboratory bioassays, Baktokulicid gave 100% control of An. messeae 4th instar larvae at a dose as high as 3.2 mg X l-1, Moskitur gave 23.1% kill at 6.4 mg X l-1. The effect of Moskitur and Baktokulicid formulations was immediate, larvae that hatched 7-14 days posttreatment survived. The efficacy of B. t. H-14 outdoor treatments tended to markedly decrease with the larval densities exceeding 100 larvae per 1 dm2. Species of nontarget aquatic organisms, including the Diptera Chaoborus crystallinus, Mychlonyx sp. and Dixidae, were not noticeably affected by treatments with B. t. H-14 formulations used.
- MeSH
- Aedes mikrobiologie MeSH
- Anopheles mikrobiologie MeSH
- Bacillus thuringiensis * MeSH
- biologická kontrola škůdců * MeSH
- Culex mikrobiologie MeSH
- Culicidae * MeSH
- larva MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Geografické názvy
- Československo MeSH
- SSSR MeSH
254 pools of 4,115 mosquito larvae belonging to nine species were examined by isolation experiments. The larvae were collected in breeding places in an inundated forest--a natural focus of Tahyna virus, in April, June and July 1974 and 1975. Tahyna virus was isolated from one pool of 10 Culiseta annulata larvae collected in July 1974. Ecological questions concerning this finding are discussed.
- MeSH
- Aedes embryologie MeSH
- Anopheles mikrobiologie MeSH
- Culex mikrobiologie MeSH
- Culicidae embryologie MeSH
- larva mikrobiologie MeSH
- viry encefalitidy izolace a purifikace MeSH
- viry kalifornské encefalitidy izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Československo MeSH
