Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

Downstream drift of the larvae of Chironomidae (Diptera) in the River Chew,S.W. England

The drift of larval Chironomidae (Diptera) was investigated at two sites on the River Chew, using a pump filtration system with 50 µm mesh-aperture aerial nets, situated on the river bank.Chironomid larvae were found to drift in far greater numbers than previously reported, even in slow-flowing water. First and second instar larvae dominated the drift.Variation in drifting behaviour between taxa was observed, where Orthocladiinae drifted in all instars and Chironominae predominantly as first and second instars. Proportionally different rates of drift were observed between these taxa from the benthos at the two sites.It is suggested that the majority of chironomid drift represents an active dispersal and colonisation mechanism by which population redistribution and habitat selection occurs.     

Mitogenomes provide new insights into the evolutionary history of Prodiamesinae (Diptera: Chironomidae)

Evolutionary analysis of Prodiamesinae has long been impeded by lack of information, and its phylogenetic relationship with Orthocladiinae remains questionable. Here, ten complete mitochondrial genomes (mitogenomes) of Orthocladiinae sensu lato were newly sequenced, including three Prodiamesinae species and seven Orthocladiinae species. Coupled with published mitogenomes, a total of 12 mitogenomes of Orthocladiinae sensu lato were selected for a comparative mitogenomic analysis and phylogenetic reconstruction. Mitogenomes of Orthocladiinae sensu lato are conserved in structure, and all genes arrange the same gene order as the ancestral insect mitogenome. Nucleotide composition is highly biased, and the control region displayed the highest A + T content. All protein-coding genes are under purifying selection, and the ATP8 evolves at the fastest rate. In addition, the mitogenomes of Orthocladiinae sensu lato are highly conserved, and they are practically useful for phylogenetic inference, suggesting a re-classification of Orthocladiinae by sinking Prodiamesinae as a subgroup of Orthocladiinae.     

Biology of epiphytic Chironomidae (Diptera:Nematocera) in chalk streams

Submerged macrophytes are a conspicuous feature of chalk stream ecosystems, supporting large populations of diatoms and invertebrates, including larvae of the nematocerous family Chironomidae. Close temporal and spatial association is evident between chironomid larvae and diatoms, and diatoms are frequently a major component of the food of midge larvae. Larvae provided with food that is rich in diatoms survive better and grow and develop more quickly than larvae that are supplied with food that is predominantly fine organic detritus. First instar larvae selectively feed on diatoms but it is likely that selection is on the basis of particle size, rather than for diatoms per se. Three species of epiphytic larvae (2 Cricotopus spp. and Eukiefferiella ilkleyensis) transfer to a diet that is predominantly of fresh plant material (Ranunculus calcareus leaves) in the third and fourth instar. The life cycle strategies of many species of Orthocladiinae equip them to exploit temporarily favourable environmental conditions very rapidly and effectively. This fact helps to explain the coexistence of species with similar strategies of resource utilization, as part of the normal epiphytic fauna of chalk streams.     

Stable isotope measurements confirm consumption of submerged macrophytes by macroinvertebrate and fish taxa

Many macrophyte species in lowland streams exhibit signs of grazing and herbivore damage, even though herbivory by aquatic macroinvertebrates and fish is generally considered to be of little importance. In this study, we collected evidence for the hypothesis that herbivory on macrophytes by macroinvertebrates and fish is more widespread than assumed. We measured the dual stable isotope signatures (δ¹³C and δ¹⁵N) of organic matter, epiphyton, submerged macrophytes, macroinvertebrates and fish in a Belgian lowland stream. There was a clear distinction in isotopic signatures of the different basal resources, allowing the use of the SIAR mixing model. These calculations revealed the consumption of macrophyte tissue not only by the phytophagous larvae of Nymphula nitidulata Hufnagel (Lepidoptera: Crambidae), but also by Baetidae nymphs (Ephemeroptera), Orthocladiinae larvae (Diptera: Chironomidae), the crayfish Orconectus limosus Rafinesque (Decapoda: Cambaridae) and the fish Gobio gobio L. (Cypriniformes: Cyprinidae) which are classified as feeding on other resources. Although the potential share of macrophyte biomass in the diet of macroinvertebrates and fish was demonstrated to be up to 49%, this amount is only a small percentage of the total standing macrophyte biomass in a lowland stream. However, the impact of this herbivory may still be substantial because consumption may comprise a significant fraction of the daily primary production. Additionally, small-scale herbivory may still have a negative impact on macrophyte growth and survival, for example through consumption of apical meristems and the increased susceptibility to diseases and toxins if the macrophyte’s epidermis is damaged.     

Vertebrate safety of Clostridium bifermentans serovar malaysia, a new larvicidal agent for vector control.

The safety of bacterial cells of Clostridium bifermentans serovar malaysia, which is highly toxic to mosquito larvae, was tested on mice, guinea pigs, rabbits, and goldfish. Inoculations of at least 1 x 10(8) cells per animal by routes recommended by World Health Organization (subcutaneous, percutaneous, inhalation, force-feeding, intraperitoneal, intravenous) and tests of subacute toxicity, anaphylactic shock, persistence in heart blood, and virulence by successive passages, were performed on mice, guinea pigs, or both. Growth was monitored for 1 mo before necropsy. Ocular irritation and skin scarification were tested with rabbits. C. bifermentans serovar malaysia did not induce any mortality or abnormal reactions in mammals at a dose 1,000 times higher than the level established by W.H.O. for the demonstration of safety. Bacterial cells are not toxic to goldfish at a dose 1,000 times higher than the LD50 for the target-mosquito larvae. We conclude that C. bifermentans serovar malaysia bacterial cells are safe for laboratory mammals and goldfish.     

Chironomids (Diptera,Chironomidae) fauna of reservoirs in Bashkortostan

An annotated list of chironomid species from small reservoirs located in the Republic of Bashkortostan in the basins of Kama, Ural, and Ob rivers is given. The list includes 54 valid species. In addition, data on the abundance of larvae in the studied waters are presented. A total of 60 species of 31 genera from 3 subfamilies, Tanypodinae (5), Orthocladiinae (18), and Chironominae (37), are recorded. The largest number of species is registered in the Sakmara Reservoir. Palearctic species dominate.     

Chironomid pupal exuviae and larvae of two storage reservoirs in The Netherlands

For three years chironomid larvae were collected bimonthly with a grab sampler in two deep storage reservoirs in The Netherlands. Chironomid pupal exuviae were collected monthly, from April to November, with a handnet. Net samples yielded more taxa than grab samples. The taxa found in net samples originated from more habitats, than taxa found in grab samples. The relative contribution of Orthocladiinae was much larger in net samples. Based on the results obtained in the two reservoirs, some advantages and disadvantages of both sampling methods are discussed.     

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinating B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.     

Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae ( Dicentrarchus labrax ) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila , Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.     

Diel drift of Chironomidae larvae in a pristine Idaho mountain stream

Simultaneous hourly net collections in a meadow and canyon reach of a mountain stream determined diel and spatial abundances of drifting Chironomidae larvae. Sixty-one taxa were identified to the lowest practical level, 52 in the meadow and 41 in the canyon. Orthocladiinae was the most abundant subfamily with 32 taxa and a 24 h mean density of 294 individuals 100 m⁻³ (meadow) and 26 taxa and a mean of 648 individuals 100 m⁻³ (canyon). Chironominae was the second most abundant subfamily. Nonchironomid invertebrates at both sites and total Chironomidae larvae (meadow) were predominantly night-drifting. Parakiefferiella and Psectrocladius were day-drifting (meadow) whereas 8 other chironomid taxa (meadow) and 2 taxa (canyon) were night-drifting. All others were aperiodic or too rare to test periodicity, Stempellinella cf brevis Edwards exhibited catastrophic drift in the canyon only. The different drift patterns between sites is attributed to greater loss of streambed habitat in the canyon compared to the meadow as streamflow decreased. Consequent crowding of chironomid larvae in the canyon caused catastrophic drift or interfered with drift periodicty. This study adds to knowledge of Chironomidae drift and shows influences on drift of hydrologic and geomorphic conditions.     

A new genus of trans-Tasman midge: Anzacladius gen. n. (Diptera: Chironomidae: Orthocladiinae)

Based on larvae, pupae and adults of Australian and New Zealand Orthocladiinae (Chironomidae) midges, a genus new to science, Anzacladius , is described. Two species, A. numbat Cranston sp. n. and A. kangaroo Cranston sp. n., are described from temperate Australia (both western and south-eastern). A. kiwi Cranston sp. n. is described from both North and South Islands, New Zealand: association of the pharate pupa with its putative larva used sequence similarity of the CO1 ( cytochrome oxidase 1 ) gene. Pupal exuviae, the major stage for species recognition, show the genus occurs in running waters, especially in Australian acidic and sandy-bedded creeks, and Anzacladius species are found also in perched lakes of Queensland's Cooloolla region and Fraser Island. Previous morphological phylogenetic studies (under the code 'SO3') indicate a relationship to austral genera Botryocladius Cranston & Edward, Naonella Boothroyd and Echinocladius Cranston.     

Longitudinal zonation and food of larval chironomids (Insecta: Diptera) along the course of a river in temperate Canada

The chironomid larvae of the Rouge River, Ontario, Canada, showed a longitudinal zonation which may have been influenced by water temperature and substrate composition. Species of Orthocladiinae predominated in the cooler headwaters while species of Chironomini dominated the comparatively warmer water near the estuary. In the middle section the chironomid assemblage was of a more mixed nature.
Differences occurred in both food availability and feeding between sites along the river. Within genera, there were differences in the proportions of major food items between the larval guts and the available food material sampled from the same area. In most cases (e.g. Cryptochironomus, Polypedilum and Orthocladius ) larvae selected detritus over diatoms. Some genera (e.g. Microtendipes ) changed the proportions of food items ingested seasonally, while others (e.g. Psectrocladius ) exhibited a more stable diet throughout the year. Second instar larvae appeared to seasonally adjust their diet most often (e.g. Cricotopus. Tanytarsus and Eukiefferiella ), however these changes were not just in the types of food eaten, but also in the proportions of food types consumed. Larvae of all genera continued feeding throughout the cold water conditions (<5°C) of winter. Incidence of predation was very low, for example <10% in the Tanypodinae. In the laboratory, species of Chironomini ingested and assimulated, to varying degress, isolated populations of diatoms, detritus and bacteria.     

Larval Chironomidae (Diptera) of the Big Thicket streams

Eighty-seven species of chironomid larvae were collected from streams in the Big Thicket area of southeast Texas. Samples of leaf debris, wood debris, and sand were taken from the streams and the larvae occupying the substrates were analyzed. Four subfamilies were represented including Tanypodinae (12 species), Diamesinae (2 species), Orthocladiinae (30 species), and Chironominae (43 species). Dominant species included Lopescladius sp., Polypedilum ? convictum, Polypedilum ? halterale, Cladotanytarsus sp. group A, Rheotanytarsus sp., and Tanytarsus sp. 2. A brief account of the habitat and the distribution of each species encountered during this study is given.     

Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.     

Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan

Of 809 soil samples collected from the seven islands of the Ryukyus, Japan, 107 samples (13.2%) contained Bacillus thuringiensis. The frequency of B. thuringiensis among the B. cereus group was 1.1% (235/21842) on the average. The B. thuringiensis soil populations of the Ryukyus consisted of more than 22 H serogroups. The predominant H serotype was the H5ac/21 (serovar canadensis/colmeri), followed by the H3ad (serovar sumiyoshiensis) and H16 (serovar indiana). Geographically, most widely distributed H serogroups were the H16 and H10ac (serovar londrina); the former was recovered from five islands and the latter from three islands. Parasporal inclusions of the isolates were morphologically heterogeneous, roughly grouped into four categories: bipyramidal/cuboidal, spherical/ovoid, irregularly-pointed, and irregular-shaped. About 53% of the isolates formed spherical to ovoid parasporal inclusions. None of the isolates exhibited larvicidal activity against the silkworm, Bombyx mori. Only four isolates belonging to four different serotypes killed larvae of the mosquito, Aedes aegypti. These mosquito-specific isolates all produced spherical parasporal inclusions.     

The role of RamA on the development of ciprofloxacin resistance in Salmonella enterica serovar Typhimurium

Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.     

Development of a real-time PCR assay for detection of gyrA mutations associated with reduced susceptibility to ciprofloxacin in Salmonella enterica serovar typhi and paratyphi A

A real-time PCR assay with the cycling probe method was used to detect mutations at codons 83 and 87 in the DNA gyrase A subunit encoded by gyrA in Salmonella enterica serovar Typhi and Paratyphi A clinical isolates. The susceptibility estimated from the results of the gyrA mutation assay was consistent with that identified by the culture method using an E-test. This assay allows rapid screening of S. enterica serovar Typhi and Paratyphi A with reduced susceptibility to ciprofloxacin.     

Drift dynamics of Chironomidae larvae: I. Preliminary results and discussion of importance of mesh size and level of taxonomic identification in resolving Chironomidae diel drift patterns

Diel drift samples utilizing nets with mesh size less than 200 microns were taken in Linesville Creek, Pennsylvania, an eastern deciduous forest stream, and Inlet Run, Wyoming, an alpine snow melt stream. Identification of drifting Chironomidae larvae to lowest level taxonomic categories indicated 51 species or species group categories representing 51.95% of the total insect drift in Linesville Creek and 18 species or species group categories representing 70.47% of the total insect drift in Inlet Run. Orthocladiinae were the predominant larvae in the drift in Linesville Creek, with 19 species comprising 43.84% of the Chironomidae drift. In decreasing abundance were Chironomini (12 species, 40.36% of Chironomidae drift), Tanytarsini (10 species, 8.89%), and Tanypodinae (10 species, 6.91%). By contrast, Diamesinae were the predominant larvae in the drift in Inlet Run, (5 species, 71.43%) followed by Orthocladiinae (10 species, 27.25%), Tanytarsini (2 species, 1.20%), and Podonominae (1 species, 0.12%). Comparison of drift composition with substrate samples and/ or emergence data indicated a close relationship between relative abundance in drift and relative abundance in the benthos. Behavioral drift patterns with nocturnal peaks were seen for 3 species or species groups in Linesville Creek. Four species with diurnal drift peaks were present in Inlet Run. Analysis of the size distribution of drifting larvae indicates that a mesh size as small as 200 microns is required to resolve diel drift patterns. It is postulated that random factors greatly influence the apparent diel drift pattern of Chironomidae when nets with mesh size in excess of 400 microns are employed in drift studies. Conflicting literature reports of behavioral drift for Chironomidae may be due to differing species composition of drifting larvae and net mesh size related artifacts.