Simultaneous identification of the Anopheles funestus group and Anopheles longipalpis type C by PCR-RFLP

<p>Abstract</p> <p>Background</p> <p><it>Anopheles longipalpis </it>is morphologically similar to the major African malaria vector <it>Anopheles funestus </it>at the adult stage although it is very different at the larval stage. Despite the development of the species-specific multiplex PCR assay for the <it>An. funestus </it>group, the genomic DNA of <it>Anopheles longipalpis </it>type C specimens can be amplified with the <it>Anopheles vaneedeni </it>and <it>Anopheles parensis </it>primers from this assay. The standard, species-specific <it>An. funestus </it>group PCR, results in the amplification of two fragments when <it>An. longipalpis </it>type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between <it>An. vaneedeni </it>and <it>An. parensis</it>. <it>Anopheles longipalpis </it>type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the <it>An. funestus </it>group, such as <it>An. funestus</it>, <it>An. funestus</it>-like and <it>An. parensis</it>, are included. The present study provides a multiplex assay that will identify <it>An. longipalpis </it>along with other common members of the African <it>An. funestus </it>group, including <it>Anopheles leesoni</it>.</p> <p>Methods</p> <p>A total of 70 specimens from six species (<it>An. funestus</it>, <it>An. funestus</it>-like, <it>An. parensis</it>, <it>Anopheles rivulorum</it>, <it>An. vaneedeni </it>and <it>An. leesoni</it>) in the <it>An. funestus </it>group and <it>An. longipalpis </it>type C from Malawi, Mozambique, South Africa and Zambia were used for the study. A restriction fragment length polymorphism (RFLP) assay was designed based on the DNA sequence information in the GenBank database.</p> <p>Results</p> <p>The enzyme, <it>EcoRI </it>digested only <it>An. longipalpis </it>type C and <it>An. funestus</it>-like after the species-specific <it>An. funestus </it>group PCR assay. The <it>An. longipalpis </it>and <it>An. funestus</it>-like digestion profiles were characterized by three fragments, 376 bp, 252 bp and 211 bp for <it>An. longipalpis </it>type C and two fragments, 375 bp and 15 bp for <it>An. funestus</it>-like.</p> <p>Conclusions</p> <p>An RFLP method for the group was developed that is more accurate and efficient than those used before. Hence, this assay would be useful for field-collected adult specimens to be identified routinely in malaria vector research and control studies.</p

A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance (kdr) mutations in Anopheles gambiae from the Republic of the Congo

<p>Abstract</p> <p>Background</p> <p>Knockdown resistance (<it>kdr</it>) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in <it>Anopheles gambiae </it>in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the <it>kdr </it>mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the <it>kdr </it>mutations in <it>An. gambiae </it>s.s. from the Republic of the Congo were compared with DNA sequence analysis.</p> <p>Methods</p> <p>A total of 52 pyrethroid and DDT resistant <it>An. gambiae </it>from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two <it>kdr </it>mutations (<it>kdr</it>-e and <it>kdr</it>-w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population.</p> <p>Results</p> <p>Fifty-one specimens were found to be <it>An. gambiae </it>S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the <it>kdr</it>-e and <it>kdr</it>-w resistance mutations, seven specimens (13.5%) were homozygous for the <it>kdr</it>-e mutation, and 14 specimens (26.9%) were homozygous for the <it>kdr</it>-w mutation. A single individual was genotyped as heterozygous <it>kdr</it>-e mutation (1.9%) only and another as heterozygous <it>kdr</it>-w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous <it>kdr</it>-w, six specimens (11.5%) homozygous for the <it>kdr</it>-e mutation, one specimen (1.9%) heterozygous for the <it>kdr</it>-w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous <it>kdr</it>-e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis.</p> <p>Conclusion</p> <p>Both the <it>kdr</it>-e and <it>kdr</it>-w mutations were abundant in <it>An. gambiae </it>S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both <it>kdr</it>-e and <it>kdr</it>-w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of <it>kdr </it>is still held as the gold standard assay, this paper highlights the importance of <it>kdr </it>mutation confirmation via sequencing especially in regions where <it>kdr </it>frequency has never been reported before or where both the <it>kdr</it>-e and <it>kdr</it>-w mutations are present simultaneously.</p

Cryptic diversity within the major trypanosomiasis vector Glossina fuscipes revealed by molecular markers

Background: The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units. Principal Findings: The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f.fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled fromEthiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations. Conclusion/Significance: We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme

Insecticide resistance in malaria vector mosquitoes at four localities in Ghana, West Africa

<p>Abstract</p> <p>Background</p> <p>Malaria vector control programmes that rely on insecticide-based interventions such as indoor house spraying with residual insecticides or insecticide treated bed nets, need to base their decision-making process on sound baseline data. More and more commercial entities in Africa, such as mining companies, are realising the value to staff productivity of controlling malaria transmission in their areas of operation.</p> <p>This paper presents baseline entomological data obtained during surveys conducted for four mining operations in Ghana, West Africa.</p> <p>Results</p> <p>The vast majority of the samples were identified as <it>Anopheles gambiae </it>S form with only a few M form specimens being identified from Tarkwa. <it>Plasmodium falciparum </it>infection rates ranged from 4.5 to 8.6% in <it>An. gambiae </it>and 1.81 to 8.06% in <it>An. funestus</it>. High survival rates on standard WHO bioassay tests were recorded for all insecticide classes except the organophosphates that showed reasonable mortality at all locations (i.e. > 90%). The West African <it>kdr </it>mutation was detected and showed high frequencies in all populations.</p> <p>Conclusions</p> <p>The data highlight the complexity of the situation prevailing in southern Ghana and the challenges facing the malaria vector control programmes in this region. Vector control programmes in Ghana need to carefully consider the resistance profiles of the local mosquito populations in order to base their resistance management strategies on sound scientific data.</p

OCT for non-destructive examination of the internal biological structures of mosquito specimen

The Study of mosquitoes and their behavioral analysis are of crucial importance to control the alarmingly increasing mosquito-borne diseases. Conventional imaging techniques use either dissection, exogenous contrast agents. Non-destructive imaging techniques, like x-ray and microcomputed tomography uses ionizing radiations. Hence, a non-destructive and real-time imaging technique which can obtain high resolution images to study the anatomical features of mosquito specimen can greatly aid researchers for mosquito studies. In this study, the three-dimensional imaging capabilities of optical coherence tomography (OCT) for structural analysis of Anopheles sinensis mosquitoes has been demonstrated. The anatomical features of An. sinensis head, thorax, and abdomen regions along with internal morphological structures like foregut, midgut, and hindgut were studied using OCT imaging. Two-dimensional (2D) and three-dimensional (3D) OCT images along with histology images were helpful for the anatomical analysis of the mosquito specimens. From the concurred results and by exhibiting this as an initial study, the applicability of OCT in future entomological researches related to mosquitoes and changes in its anatomical structure is demonstrated

Modulation of Skin Collagen Metabolism in Aged and Photoaged Human Skin In Vivo

To the best of our knowledge, no study has been conducted to date to directly compare the collagen metabolism of photoaged and naturally aged human skin. In this study, we compared collagen synthesis, matrix metalloproteinase-1 levels, and gelatinase activity of sun-exposed and sun-protected skin of both young and old subjects. Using northern blot analysis, immunohistochemical stain, and Western blot analysis, we demonstrated that the levels of procollagen type I mRNA and protein in photoaged and naturally aged human skin in vivo are significantly lower than those of young skin. Furthermore, we demonstrated, by northern blot analysis, that the procollagen α1(I) mRNA expression of photoaged skin is much greater than that of sun-protected skin in the same individual. In situ hybridization and immunohistochemical stain were used to show that the expression of type I procollagen mRNA and protein in the fibroblasts of photoaged skin is greater than for naturally aged skin. In addition, it was found, by Western blot analysis using protein extracted from the dermal tissues, that the level of procollagen type I protein in photoaged skin is lower than that of naturally aged skin. The level of matrix metalloproteinase-1 protein and the activity of matrix metalloproteinase-2 were higher in the dermis of photoaged skin than in naturally aged skin. Our results suggest that the natural aging process decreases collagen synthesis and increases the expression of matrix metalloproteinases, whereas photoaging results in an increase of collagen synthesis and greater matrix metalloproteinase expression in human skin in vivo. Thus, the balance between collagen synthesis and degradation leading to collagen deficiency is different in photoaged and naturally aged skin

Fasting Plasma Glucose Cutoff Value for the Prediction of Future Diabetes Development: A Study of Middle-Aged Koreans in a Health Promotion Center

We determined optimal fasting plasma glucose (FPG) cutoff values predictive of future diabetes development in a group of middle-aged Koreans who visited a health promotion center. The medical records of 2,964 subjects, who attended the Health Promotion Center in 1998 and 2003, were examined. Subjects were classified into four groups according to their baseline FPG values (Group 1:FPG <5.0 mM/L; Group 2: 5.0≤FPG <5.6 mM/L; Group 3: 5.6≤FPG <6.1 mM/L; Group 4: 6.1≤FPG <7.0 mM/L). No significant difference was observed between Group 1 and Group 2 in terms of diabetes incidence. However, incidence in Group 3 was significantly higher than that in Group 1 [hazards ratio 4.88 (1.65-14.41), p=0.004] and the hazards ratio in Group 4 for diabetes was 36.91 (13.11-103.61), p<0.001, versus Group 1. Receiver operator characteristics curve analysis showed that an FPG of 5.97 mM/L represents the lower limit and gives the best combination of sensitivity and specificity. Our data shows that the risk of future diabetes development started to increase below an FPG of 6.1 mM/L and suggests the importance of efforts to modify diabetes development risk factors at lower impaired fasting glucose levels

The Associations of Total and Differential White Blood Cell Counts with Obesity, Hypertension, Dyslipidemia and Glucose Intolerance in a Korean Population

Although many studies have reported an association between total white blood cell count and metabolic syndrome, relatively few reports are available on the association between differential white blood cell counts and metabolic syndrome. The medical records of 15,654 subjects (age, median 46, range 14-90 yr; 8,380 men and 7,274 women) who visited the Center for Health Promotion were investigated. It was found that as total white blood cell (WBC) and differential WBC counts increased the frequencies of diabetes, hypertension, obesity, dyslipidemia, and metabolic syndrome also increased. Moreover, these significant relationships persisted after adjusting for age, gender, smoking, alcohol intake, educational background, and household income. The odds ratios (95% CI) for metabolic syndrome was 2.64 (2.30-3.04) in the highest quartile of total WBC count, with corresponding figures of 2.14 (1.88-2.44) for neutrophils, 2.32 (2.03-2.64) for lymphocytes, 1.56 (1.37-1.78) for monocytes, 1.36 (1.20-1.54) for basophils, and 1.82 (1.59-2.08) for eosinophils versus the lowest quartiles of the appropriate total and differential counts, respectively, after adjusting for the variables mentioned above. These independent associations were also observed by subgroup analyses according to the smoking status. Our data suggest that even within normal ranges, total WBC count and the differential WBC counts are associated with the presence of metabolic syndrome

Protection of nigral dopaminergic neurons by AAV1 transduction with Rheb(S16H) against neurotoxic inflammation in vivo

We recently reported that adeno-associated virus serotype 1 (AAV1) transduction of murine nigral dopaminergic (DA) neurons with constitutively active ras homolog enriched in brain with a mutation of serine to histidine at position 16 [Rheb(S16H)] induced the production of neurotrophic factors, resulting in neuroprotective effects on the nigrostriatal DA system in animal models of Parkinson&apos;s disease (PD). To further investigate whether AAV1-Rheb(S16H) transduction has neuroprotective potential against neurotoxic inflammation, which is known to be a potential event related to PD pathogenesis, we examined the effects of Rheb(S16H) expression in nigral DA neurons under a neurotoxic inflammatory environment induced by the endogenous microglial activator prothrombin kringle-2 (pKr-2). Our observations showed that Rheb(S16H) transduction played a role in the neuroprotection of the nigrostriatal DA system against pKr-2-induced neurotoxic inflammation, even though there were similar levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1 beta), in the AAV1-Rheb(S16H)-treated substantia nigra (SN) compared to the SN treated with pKr-2 alone; the neuroprotective effects may be mediated by the activation of neurotrophic signaling pathways following Rheb(S16H) transduction of nigral DA neurons. We conclude that AAV1-Rheb(S16H) transduction of neuronal populations to activate the production of neurotrophic factors and intracellular neurotrophic signaling pathways may offer promise for protecting adult neurons from extracellular neurotoxic inflammation.1