Speciation within the Anopheles gambiae complex: high-throughput whole genome sequencing reveals evidence of a putative new cryptic taxon in 'far-west' Africa.

The two main Afrotropical malaria vectors - Anopheles coluzzii and An. gambiae - are genetically distinct and reproductively isolated across West Africa. However, populations at the western extreme of their range are assigned as "intermediate" between the two species by whole genome sequence (WGS) data, and as hybrid forms by conventional molecular diagnostics. By exploiting WGS data from 1,190 specimens collected across west Africa via the Anopheles gambiae 1000 Genomes network, we identify a novel putative taxon in the far-west (provisionally named Bissau molecular form), which did not arise by admixture but rather originated at the same time as the split between An. coluzzii and An. gambiae. Intriguingly, these populations lack insecticide resistance mechanisms commonly observed in the two main species. These findings lead to a change of perspective on malaria vector species in the far-west region with potential for epidemiological implications, and a new challenge for genetic-based mosquito control approaches.

Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations.

BACKGROUND: It is important to understand whether the potential impact of pyrethroid resistance on malaria control can be mitigated by switching between different pyrethroids or whether cross-resistance within this insecticide class precludes this approach. METHODS: Here we assess the relationships among pyrethroids in terms of their binding affinity to, and depletion by, key cytochrome P450 enzymes (hereafter P450s) that are known to confer metabolic pyrethroid resistance in Anopheles gambiae (s.l.) and An. funestus, in order to identify which pyrethroids may diverge from the others in their vulnerability to resistance. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. RESULTS: We found that the type I and II pyrethroids permethrin and deltamethrin, respectively, are closely related in terms of binding affinity to key P450s, depletion by P450s and resistance within vector populations. Bifenthrin, which lacks the common structural moiety of most pyrethroids, diverged from the other pyrethroids tested in terms of both binding affinity to key P450s and depletion by P450s, but resistance to bifenthrin has rarely been tested in vector populations and was not analysed here. Etofenprox, which also lacks the common structural moiety of most pyrethroids, diverged from the more commonly deployed pyrethroids in terms of binding affinity to key P450s and resistance in vector populations, but did not diverge from these pyrethroids in terms of depletion by the P450s. The analysis of depletion by the P450s indicated that etofenprox may be more vulnerable to metabolic resistance mechanisms in vector populations. In addition, greater resistance to etofenprox was found across Aedes aegypti populations, but greater resistance to this compound was not found in any of the malaria vector species analysed. The results for pyrethroid depletion by anopheline P450s in the laboratory were largely not repeated in the findings for resistance in malaria vector populations. CONCLUSION: Importantly, the prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin and permethrin was correlated across malaria vector populations, and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance.

Parallel evolution or purifying selection, not introgression, explains similarity in the pyrethroid detoxification linked GSTE4 of Anopheles gambiae and An. arabiensis.

Insecticide resistance is a major impediment to the control of vectors and pests of public health importance and is a strongly selected trait capable of rapid spread, sometimes even between closely related species. Elucidating the mechanisms generating insecticide resistance in mosquito vectors of disease, and understanding the spread of resistance within and between populations and species are vital for the development of robust resistance management strategies. Here, we studied the mechanisms of resistance in two sympatric members of the Anopheles gambiae species complex-the major vector of malaria in sub-Saharan Africa-to understand how resistance has developed and spread in eastern Uganda, a region with some of the highest levels of malaria. In eastern Uganda, where the mosquitoes Anopheles arabiensis and An. gambiae can be found sympatrically, low levels of hybrids (0.4 %) occur, offering a route for introgression of adaptively important variants between species. In independent microarray studies of insecticide resistance, Gste4, an insect-specific glutathione S-transferase, was among the most significantly up-regulated genes in both species. To test the hypothesis of interspecific introgression, we sequenced 2.3 kbp encompassing Gste4. Whilst this detailed sequencing ruled out introgression, we detected strong positive selection acting on Gste4. However, these sequences, followed by haplotype-specific qPCR, showed that the apparent up-regulation in An. arabiensis is a result of allelic variation across the microarray probe binding sites which artefactually elevates the gene expression signal. Thus, face-value acceptance of microarray data can be misleading and it is advisable to conduct a more detailed investigation of the causes and nature of such signal. The identification of positive selection acting on this locus led us to functionally express and characterise allelic variants of GSTE4. Although the in vitro data do not support a direct role for GSTE4 in metabolism, they do support a role for this enzyme in insecticide sequestration. Thus, the demonstration of a role for an up-regulated gene in metabolic resistance to insecticides should not be limited to simply whether it can metabolise insecticide; such a strict criterion would argue against the involvement of GSTE4 despite the weight of evidence to the contrary.

Geographic population structure of the African malaria vector Anopheles gambiae suggests a role for the forest-savannah biome transition as a barrier to gene flow.

The primary Afrotropical malaria mosquito vector Anopheles gambiae sensu stricto has a complex population structure. In west Africa, this species is split into two molecular forms and displays local and regional variation in chromosomal arrangements and behaviors. To investigate patterns of macrogeographic population substructure, 25 An. gambiae samples from 12 African countries were genotyped at 13 microsatellite loci. This analysis detected the presence of additional population structuring, with the M-form being subdivided into distinct west, central, and southern African genetic clusters. These clusters are coincident with the central African rainforest belt and northern and southern savannah biomes, which suggests restrictions to gene flow associated with the transition between these biomes. By contrast, geographically patterned population substructure appears much weaker within the S-form.

Insecticide resistance monitoring of field-collected Anopheles gambiae s.l. populations from Jinja, eastern Uganda, identifies high levels of pyrethroid resistance.

Insecticide resistance in the malaria vector Anopheles gambiae s.l. (Diptera: Culicidae) threatens insecticide-based control efforts, necessitating regular monitoring. We assessed resistance in field-collected An. gambiae s.l. from Jinja, Uganda using World Health Organization (WHO) bioassays. Only An. gambiae s.s. and An. arabiensis (≈70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT50 > 2 h; deltamethrin LT50 > 5 h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, while An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (≈95%), consistent with insecticide-mediated selection, but is currently at a low frequency in An. arabiensis (0.07%). Kdr 1014F was also at a low frequency in An. gambiae. These frequencies preclude adequately-powered tests for an association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s. ×An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid-impregnated bednets or indoor spraying of pyrethroids.

Evidence for a discrete evolutionary lineage within Equatorial Guinea suggests that the tsetse fly Glossina palpalis palpalis exists as a species complex.

Tsetse flies of the palpalis group are major vectors of Human African Trypanosomiasis in Africa. Accurate knowledge of species identity is essential for vector control. Here, we combine ribosomal internal transcribed spacer 1 (ITS1), mitochondrial Cytochrome Oxidase 1 (COI) and microsatellites to determine the population structure and phylogenetic relations of Glossina p. palpalis in Equatorial Guinea. CO1 sequence data suggest that G. p. palpalis in Equatorial Guinea is a distinct subspecies from previously described G. p. palpalis in West Africa and Democratic Republic of Congo. Glossina p. palpalis in Equatorial Guinea and DRC share a common ancestor which diverged from West African G. p. palpalis around 1.9 Ma. Previous ITS1 length polymorphism data suggested the possible presence of hybrids in Equatorial Guinea. However, ITS1 showed incomplete lineage sorting compared with clearly defined COI groups, and data from 12 unlinked microsatellites provided no evidence of hybridization. Microsatellite data indicated moderate but significant differentiation between the populations analysed (Rio Campo, Mbini and Kogo). Moreover, unlike previous studies of G. p. palpalis, there was no evidence for heterozygote deficiency, presence of migrants or cryptic population structure. Variance effective population size at Rio Campo was estimated at 501-731 assuming eight generations per year. This study of the population genetics of G. p. palpalis in central Africa provides the first estimate of genetic differentiation between geographically separated G. p. palpalis populations.

Concordant genetic estimators of migration reveal anthropogenically enhanced source-sink population structure in the river sculpin, Cottus gobio.

River systems are vulnerable to natural and anthropogenic habitat fragmentation and will often harbor populations deviating markedly from simplified theoretical models. We investigated fine-scale population structure in the sedentary river fish Cottus gobio using microsatellites and compared migration estimates from three FST estimators, a coalescent maximum-likelihood method and Bayesian recent migration analyses. Source-sink structure was evident via asymmetry in migration and genetic diversity with smaller upstream locations emigration biased and larger downstream subpopulations immigration biased. Patterns of isolation by distance suggested that the system was largely, but not entirely, in migration-drift equilibrium, with headwater populations harboring a signal of past colonizations and in some cases also recent population bottlenecks. Up- vs. downstream asymmetry in population structure was partly attributable to the effects of flow direction, but was enhanced by weirs prohibiting compensatory upstream migration. Estimators of migration showed strong correspondence, at least in relative terms, especially if pairwise FST was used as an indirect index of relative gene flow rather than being translated to Nm. Since true parameter values are unknown in natural systems, comparisons among estimators are important, both to determine confidence in estimates of migration and to validate the performance of different methods.

Antagonists and the purinergic nerve hypothesis: 2, 2'-pyridylisatogen tosylate (PIT), an allosteric modulator of P2Y receptors. A retrospective on a quarter century of progress.

2,2'-Pyridylisatogen tosylate (PIT) is a selective antagonist of P2Y responses in smooth muscle and does not antagonise the effects of adenosine. Responses to purinergic nerve stimulation are resistant to PIT. PIT is an allosteric modulator of responses to ATP in recombinant P2Y(1) receptors expressed in Xenopus oocytes with potentiation of ATP at low concentrations (0.1-10 microM) and antagonism at higher ones (>10 microM). A radioligand binding profile showed that PIT did not interact with any other receptors, with the exception of low affinity for the adenosine A(1) receptor (pK(i), 5.3). The compound recognises purine sites and then may cause irreversible binding to sulfhydryl groups following prolonged incubation or high concentrations. PIT is a potent spin trapper.

Water temperature influences the shoaling decisions of guppies, Poecilia reticulata, under predation threat.

In previous experiments, we found antipredator behaviours in female Trinidadian guppies to be influenced by water temperature. To distinguish between hypotheses suggested to explain these observations, we examined the effect of an increase in water temperature on the choice of shoal size of individual female guppies, in the absence and presence of predation threat from a confined cichlid. At 22 degrees C, guppies showed no significant preference for either of two shoal sizes of female guppies, although other responses indicated a reaction to the threat. At 26 degrees C, females chose to shoal with the smaller group when the predator was absent, but, once threatened, switched to a strong preference for the larger group. Variation in preference between individuals was quite high in all treatments, and was somewhat lower for guppies under threat in warmer water, although not significantly so. Our evidence indicates that guppies in warmer water behave as if they are at greater risk of predation, but only when under threat. Temperature-dependent increases in predation risk from ectothermic piscivores could result from less efficient escape responses and from greater vulnerability because of increased foraging activity. We suggest that selection favouring guppies displaying an optimal balance between foraging and antipredator behaviours should act more strongly at higher temperature. Copyright 1999 The Association for the Study of Animal Behaviour.

Effects of temperature on anti-predator behaviour in the guppy, Poecilia reticulata.

Warmer environmental temperatures are likely to increase the frequency of predator-prey interactions in ectothermic animals, and therefore might be expected to influence anti-predator behaviour. In a first experiment, groups of recently fed guppies, placed in a novel environment, schooled significantly more closely at 26 degrees than at 22 degreesC. Changes in two of three measures of schooling tendency over time indicated that aggregation increased during the trial periods, probably as a result of increased familiarity within the experimental groups. In a second experiment, pairs of female guppies were tested at 22 degrees and 26 degreesC, with and without predation threat from a confined cichlid. From multifactor analysis of 18 behaviour types, temperature was shown to affect behavioural time budgets profoundly, particularly in the presence of the predator. At the higher temperature, a shift occurred from inactive anti-predator behaviours and minimal foraging activity towards active predator inspection-related behaviours and a much higher level of feeding. Guppies in the warmer water might have been physically able to school more closely as a result of faster swimming ability, or might have used temperature as a cue indicating higher potential predation risk, and aggregated accordingly. The use of temperature as a source of information about the biotic environment is discussed. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

An in vitro study of the coronal leakage of two root canal sealers using an obligate anaerobe microbial marker.

The aim of this in vitro study was to investigate the coronal leakage of obligate anaerobes into root canals obturated with lateral condensation of cold gutta-percha with two root canal sealers. Sixty extracted human teeth with straight, single root canals were prepared using the modified double-flared technique with balanced force under copious irrigation until the master apical file was size 40. The teeth were divided randomly into experimental groups (40 teeth) and control groups (20 teeth). In the experimental groups, 20 teeth were obturated with lateral condensation of cold gutta-percha and AH26 sealer and 20 teeth were obturated with the same technique using TubliSeal EWT sealer. In the control groups, 10 teeth were obturated with the same technique either with AH26 or TubliSeal EWT sealer. These teeth were completely sealed to serve as negative controls. The remaining 10 teeth were not obturated and served as positive controls. The root surface of each tooth was sealed with nail varnish except the apical 2 mm. The coronal part of each root canal was sealed with the cut end of polypropylene tube and placed in a glass bottle containing sterile Fastidious Anaerobe Broth (FAB). Aliquots of 0.5 mL of FAB were injected into the polypropylene tube and the model system was centrifuged at 168 g. An inoculum of Fusobacterium nucleatum in FAB was placed in each coronal chamber at 7-day intervals and daily observations were made for bacterial growth in the apical reservoir for 12 weeks. All positive control teeth showed bacterial leakage within a week, while the negative control teeth remained uncontaminated throughout the test period. All the experimental teeth exhibited leakage of bacterial metabolites within 12 weeks, ranging from 1 to 12 weeks. The mean time for complete leakage in the AH26 and the TubliSeal EWT groups was 8.4 and 8.2 weeks respectively. There was no statistically significant difference (P > 0.05) in leakage between the AH26 and the TubliSeal EWT groups.

Potentiation by 2,2'-pyridylisatogen tosylate of ATP-responses at a recombinant P2Y1 purinoceptor.

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.

Current knowledge of health effects from volatile organic chemicals in the environment. Report of the international meeting organised by Indoor Air International in London on 27-28 October 1993.

The conference attracted 60 papers and 150 delegates from 22 countries. The various sessions covered air quality management and legislation, industrial control and product reformulation, vehicle emissions and fuel composition, evaluation technologies, and health effects and air quality case studies. Delegates were presented with the 606 page book of the proceedings at registration (1). Thus those who attended were briefed right up to the present situation about volatile organic compounds (VOCs) in the environment.

Intra- and inter-individual differences in salivary sucrose clearance over time.

Salivary clearance of sugars influences acid production of dental plaque. The aim of the present study was to follow the clearance of sucrose over time. Ten subjects participated in five or six experiments each during a period of 20 months. The subjects rinsed with a 20% sucrose solution. Before the rinse and after 2, 5, 10 and 30 min a saliva sample was collected. The salivary sucrose concentration was determined by an enzymatic technique. As a measure of the clearance of sucrose in saliva, the area under the concentration versus time curve (AUC) was used. Analysis of variance revealed no differences with time in the intra-individual AUC. However, the differences in AUC among individuals were highly significant (p < 0.001). The results show that the clearance pattern of sucrose may be an individual property which is constant over long time periods.

Comparison of the plaque microflora from natural and appliance-borne enamel surfaces.

Human enamel sections and slabs, mounted on a mandibular removable appliance, were worn by 5 adult subjects for a 1-week period. Plaque was allowed to accumulate on the in situ test sites and on the adjacent natural dentition. At the end of the experimental period, the plaque microflora associated with (1) the enamel sections, (2) the enamel slabs, and (3) the acrylic base of the appliance test site was compared with that obtained from lingual and interproximal areas of the lower molar teeth. In addition, the acid anion and pH profiles of plaque obtained from both the exogenous and natural tooth surfaces were also determined. Although some quantitative differences were found between the proportions of isolates obtained from the different enamel surfaces, qualitatively the microflora was very similar, and no significant differences were found in the plaque lactate/acetate ratios or pH measurements following a sucrose mouthrinse. Thus, human tooth specimens mounted on the intra-oral device produced a plaque ecosystem similar to that present on the adjacent natural dentition, suggesting that the model is suitable for studies on early plaque development and the microbiology of enamel demineralization.

The effect of salivary clearance of sucrose and fluoride on human dental plaque acidogenicity.

Ten subjects rinsed with a 20% (0.58 M) sucrose solution with or without 0.2% NaF (905 parts/10(6) F-) added in two separate experiments. Saliva and plaque were collected before rinsing and after 2, 5, 10 and 30 min. Sucrose and fluoride concentrations in saliva and acid anion and fluoride concentrations in plaque were analysed. There was a statistically significant and positive correlation between the concentration of sucrose in the saliva 2 min after the rinse and the subsequent concentrations of lactate in plaque at 10 and 30 min after the rinse with sucrose alone but not in the presence of fluoride. Salivary fluoride concentrations during 2-30 min after the sucrose rinse were significantly correlated with plaque fluoride concentrations during the same time. The addition of fluoride to the sucrose rinse significantly inhibited lactate production.

Growth and acid production of Candida species in human saliva supplemented with glucose.

Growth characteristic and acid production of oral isolates of Candida albicans and Candida glabrata in glucose supplemented and glucose-free, pooled, human whole saliva were examined. Both Candida species exhibited sigmoidal growth curves in batch cultures of mixed saliva, supplemented with glucose. The growth of Candida in saliva was accompanied by a rapid decline in pH from 7.5 to 3.2 over 48 h and the major acidic components initiating and sustaining this pH drop were pyruvates and acetates. These acidic metabolites may play an important role in the pathogenesis of oral Candida infections.