Use of Household Insecticides against Mosquitoes in Dengue-Endemic Areas in Sri Lanka.

The use of household insecticides in the formulation of mosquito coils and vaporizers is common among the public to protect against mosquito-borne diseases. However, information regarding the use of these commonly practiced household insecticides is scarce in Sri Lanka. A questionnaire-based survey was directed to 600 households in Colombo, Gampaha, Kandy, Kalutara, Jaffna, and Galle districts in Sri Lanka. Household insecticides were used by the majority of the respondents (57.0%), and electric liquid vaporizers (46.7%) and mosquito coils (45.7%) were the commonly used forms. The active ingredients in mosquito coils were d-trans-allethrin (w/w 0.12%), metofluthrin (w/w 0.005%), and dimefluthrin (w/w 0.01%); in liquid vaporizers, they were prallethrin (w/v 1.24% and w/v 1.26%) and transfluthrin (w/v 0.9%). The majority of the household insecticide consumers were moderately satisfied with the effectiveness of the products (68.6%), and most of the participants used household insecticides daily (35.5%), especially during the evening (41.6%). Most of the consumers were unaware of the chemicals included in household insecticides (62.8%). Mosquito coils were abundantly used by rural area participants (75.8%), whereas liquid vaporizers were the most common among the urban participants (56.4%). The findings indicate that demographic and socioeconomic factors influence household insecticide practices. To our knowledge, this study reveals the use of household insecticides to control mosquitoes for the first time in Sri Lanka and highlights the importance of awareness programs and the proper utilization of these products.

Fine-scale monitoring of insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Sri Lanka and modeling the phenotypic resistance using rational approximation.

BACKGROUND: The unplanned and intensified use of insecticides to control mosquito-borne diseases has led to an upsurge of resistance to commonly used insecticides. Aedes aegypti, the main vector of dengue, chikungunya, and Zika virus, is primarily controlled through the application of adulticides (pyrethroid insecticides) and larvicides (temephos). Fine spatial-scale analysis of resistance may reveal important resistance-related patterns, and the application of mathematical models to determine the phenotypic resistance status lessens the cost and usage of resources, thus resulting in an enhanced and successful control program. METHODS: The phenotypic resistance for permethrin, deltamethrin, and malathion was monitored in the Ae. aegypti populations using the World Health Organization (WHO) adult bioassay method. Mosquitoes' resistance to permethrin and deltamethrin was evaluated for the commonly occurring base substitutions in the voltage-gated sodium channel (vgsc) gene. Rational functions were used to determine the relationship between the kdr alleles and the phenotypic resistant percentage of Ae. aegypti in Sri Lanka. RESULTS: The results of the bioassays revealed highly resistant Ae. aegypti populations for the two pyrethroid insecticides (permethrin and deltamethrin) tested. All populations were susceptible to 5% malathion insecticide. The study also revealed high frequencies of C1534 and G1016 in all the populations studied. The highest haplotype frequency was detected for the haplotype CC/VV, followed by FC/VV and CC/VG. Of the seven models obtained, this study suggests the prediction models using rational approximation considering the C allele frequencies and the total of C, G, and P allele frequencies and phenotypic resistance as the best fits for the area concerned. CONCLUSIONS: This is the first study to our knowledge to provide a model to predict phenotypic resistance using rational functions considering kdr alleles. The flexible nature of the rational functions has revealed the most suitable association among them. Thus, a general evaluation of kdr alleles prior to insecticide applications would unveil the phenotypic resistance percentage of the wild mosquito population. A site-specific strategy is recommended for monitoring resistance with a mathematical approach and management of insecticide applications for the vector population.

Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

BACKGROUND: Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. METHODOLOGY: A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. PRINCIPAL FINDINGS: Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd-0.749, π-0.00417) and Medirigiriya (Hd-0.977, π-0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. CONCLUSIONS: Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies.

Genetic diversity, phylogenetic and phylogeographic analysis of Anopheles culicifacies species complex using ITS2 and COI sequences.

Anopheles culicifacies is the major vector of malaria in Sri Lanka and the Indian subcontinent which is characterized as a species complex with five sibling species provisionally designated as A, B, C, D and E. The current study was carried out to understand the phylogenetic and phylogeographic relationships between the sibling species of the species complex while observing their genetic diversity and genetic differentiation. Thirty-five ITS2 and seventy-seven COI sequences of An. culicifacies species complex reported from different geographical locations of Asia and China at the NCBI public database were used for the analysis. Bayesian likelihood trees were generated for the phylogenetic analysis. The divergence of the species complex was obtained from the Bayesian phylogeographic model in BEAST. There were two clades of the sibling species of An. culicifacies species complex as A, D and B, C and E in both phylogenetic and phylogeographic analysis using ITS2 sequences. Based on the highly divergent COI sequences and the high mutation rate of the mitochondrial genome, there were four and three clades in both phylogenetic and phylogeographic analysis using COI sequences. The diversification of An. culicifacies species complex was obtained as ranging from 20.25 to 24.12 Mya and 22.37 to 26.22 Mya based on ITS2 and COI phylogeographic analysis respectively. There was a recent diversification of the sibling species A and D than the sibling species B, C and E. Low haplotype diversity was observed in the sequences reported from Sri Lanka in both ITS2 and COI analysis that can be due to bottlenecks resulting from the intense malaria control efforts. A high genetic differentiation was achieved for some populations due to the large geographical distance. The high genetic diversity based on the five sibling species implies the possibility of maintaining a relatively high effective population size despite the vector control efforts.

Impact of climatic factors on temporal variability of sand fly abundance in Sri Lanka: Longitudinal study (2018 to 2020) with two-stage hierarchical analysis.

Background: Phlebotomine sand flies serve as vectors for leishmaniasis, a major health concern, but a neglected tropical disease. The risk of vector activity is governed by climatic factors that vary in different geographic zones in the country. Thus, we aimed to quantify the effect of climatic variables on sand fly vector activity in ten sentinel sites across Sri Lanka. Methods: Mean rainfall, ambient temperature, relative humidity, wind speed, soil temperature, evaporation, sunshine hours, and vector densities were recorded at monthly intervals in each location from March 2018 to February 2020. The association between weather variables and sand fly densities was analysed using a two-staged hierarchical procedure; Distributed Lag Non-Linear (DLNM) modelling framework and the DLNM method implemented in the R package dlnm (version number 2.4.6). Results: Moderate rainfall values up to 120 mm per month and increasing RH up to 82 at lag of 0 months along with increasing soil temperature and evaporation rate at lag of 2 months were associated with statistically significant increase in the sand fly activity. These associations were heterogeneous across study settings. Whereas increasing ambient and soil temperature, sunshine hours, evaporation rate appeared to reduce the sand fly activity homogeneously at lag of 0 month in all the study settings. Conclusions: The abundance of sand fly vectors varied in relation to selected climatic variables, either in real-time or with a time lag. This information can be utilized for predicting sand fly densities and for the development of effective strategies to prevent leishmaniasis transmission in specific settings.

Genetic Variation in the Domain II, 3' Untranslated Region of Human and Mosquito Derived Dengue Virus Strains in Sri Lanka.

Genetic variations in dengue virus (DENV) play a distinct role in epidemic emergence. The DENV 3' UTR has become a recent interest in research. The objective of the study was to examine the genetic variation in the domain II, 3' UTR region of human and mosquito-derived DENV. DENV-infected human sera were orally infected to laboratory reared Aedes aegypti mosquitoes. The domain II, 3' UTR of each human- and mosquito-derived sample was amplified. The nucleotide sequence variation, phylogenetic and secondary structure analysis was carried out incorporating respective regions of so far recorded Sri Lankan and the reference genotype strains of the DENV3 and DENV1 serotypes. The human- and mosquito-derived domain II, 3' UTR were identical in nucleotide sequences within the serotypes isolated, indicating the conserved nature of the region during host switch. The sequence analysis revealed distinct variations in study isolates compared to so far recorded Sri Lankan isolates. However, despite single nucleotide variations, the maintenance of structural integrity was evident in related strains within the serotypes in the secondary structure analysis. The phylogenetic analysis revealed distinct clade segregation of the study sequences from so far reported Sri Lankan isolates and illustrated the phylogenetic relations of the study sequences to the available global isolates of respective serotypes.

Compensatory Base Changes Reveal Sexual Incompatibility among Members of the Anopheles subpictus Sensu Lato (Diptera: Culicidae) Species Complex in Sri Lanka.

The mosquito Anopheles (Cellia) subpictus sensu lato (s.l.) is a major secondary vector of malaria in Sri Lanka. The sibling species composition in this species complex in Sri Lanka remains debatable. Compensatory base changes (CBCs) in the secondary structures of internal transcribed spacer 2 (ITS2) are reliable sources to predict sexual incompatibility among closely related species. The objective of the present study was to investigate the An. subpictus s.l. populations in Sri Lanka using the CBC analysis. Mosquito DNA was amplified and sequenced for the ITS2 region. The sequences were annotated using ITS2 Database. ITS2 secondary structures were constructed and analyzed for CBCs using various bioinformatics tools. The ITS2 regions consisted of two different lengths, 575 bp and 480 bp. The two CBCs and three hemi CBCs identified in the present study suggest that there may be at least two sexually incompatible sibling species. In conclusion, it is likely that there may be only two reproductively isolated sibling species in the An. subpictus species complex in Sri Lanka. However, due to high divergence of ITS2 in these species, it is reasonable to assume that they may be undergoing a speciation event to separate as a distinct species.

Insecticide resistance and molecular characterization of knockdown resistance (kdr) in Culex quinquefasciatus mosquitoes in Sri Lanka.

Resistance to pyrethroids (PY) and organophosphate (OP) insecticides is widespread among populations of Culex quinquefasciatus, the major vector of lymphatic filariasis (LF). The present study was designed to detect the L1014F kdr (knockdown resistant) mutation among Cx. quinquefasciatus populations in the filarial belt of Sri Lanka. Mosquitoes were reared from field-caught larvae from seven localities where LF is endemic. Susceptibility status of Cx. quinquefasciatus to adulticides, 0.05% deltamethrin, 0.75% permethrin, 5% malathion, and the larvicide temephos was determined using the standard WHO susceptibility tests. A fragment of vgsc gene was amplified and sequenced to identify the responsible kdr mutations. The susceptibility test results revealed less than 90% mortalities for 0.05% deltamethrin, and 0.75% permethrin and temephos. For 5% malathion, all study sites except Maharagama revealed greater than 90% mortality. The L1014F kdr mutation was observed in all studied populations. Although the overall microfilaria rate is less than 1% in the country, there is a high risk of re-emergence of LF in Sri Lanka due to abundant Cx. quinquefasciatus mosquitoes, increased resistant status to currently used insecticides, imported LF cases, higher rates of microfilaria among neighboring countries, and the advancement of tourism.

Resistance to commonly used insecticides and underlying mechanisms of resistance in Aedes aegypti (L.) from Sri Lanka.

BACKGROUND: Drastic increases of dengue fever (DF) over the past few years have prompted studies on the development of resistance to insecticides in the mosquito vector, Aedes aegypti (Linnaeus). In Sri Lanka control of the vector population is essentially achieved using larvicides (temephos) and adulticides (principally pyrethroids). The present study investigates resistance to commonly used insecticides and underlying mechanisms of Ae. aegypti in selected sites in Sri Lanka. METHODS: In this study, susceptibility to three commonly used adulticides (malathion, permethrin and deltamethrin) and the larvicide temephos were tested for Ae. aegypti sampled from five localities in Sri Lanka using WHO dose diagnostics tests. In addition, we performed dose-response tests for permethrin to determine lethal concentrations (LCs) with CDC bottle bioassays. An assessment of the activity of metabolic detoxifying enzymes (multifunction oxidases (MFOs), glutathione S-transferases (GSTs) and esterases) and determination of frequency of the kdr mutations (F1534C, V1016G and S989P) were also carried out to ascertain the associated resistance mechanisms. Kdr genotype frequencies were compared with samples collected from the same sites in 2015 to determine the change of allele frequencies over the years. RESULTS: The present study revealed resistance in all Ae. aegypti populations studied, with low mortality percentages for both permethrin (10-89%) and deltamethrin (40-92%). Dose response tests revealed highest resistance ratios (RR) for permethrin and temephos from Colombo district whereas Puttalum district exhibited the lowest. High frequencies of the 1534C allele (0.052-0.802) were found in the study sites in 2017. Comparison with samples collected in 2015 revealed a substantial increase in this allele. The activity of MFOs and p-nitro phenyl-acetate esterase was significantly greater in most Sri Lankan populations in comparison to that of the New Orleans (NO) susceptible strain. In contrast, the activity of α-esterase and ß-esterase was similar or lower than that in the NO strain. CONCLUSIONS: Aedes aegypti from Sri Lanka is resistant to pyrethroid insecticides showing rapid selection for kdr mutations and varying metabolic mechanisms. Continued monitoring of vector populations is crucial to mitigate the development of resistance to commonly used insecticides and in turn, controlling the vector population.

Mitochondrial metabolic genes provide phylogeographic relationships of global collections of Aedes aegypti (Diptera: Culicidae).

Phylogeographic relationships among global collections of the mosquito Aedes aegypti were evaluated using the mitochondrial Cytochrome C Oxidase 1 (CO1) and NADH dehydrogenase subunit 4 (ND4) genes including new sequences from Sri Lanka. Phylogeographic analysis estimated that Ae. aegypti arose as a species ~614 thousand years ago (kya) in the late Pleistocene. At 545 kya an "early" East African clade arose that continued to differentiate in East Africa, and eventually gave rise to three lineages one of which is distributed throughout all tropical and subtropical regions, a second that contains Southeast Asian/Sri Lankan mosquitoes and a third that contains mostly New World mosquitoes. West African collections were not represented in this early clade. The late clade continued to differentiate throughout Africa and gave rise to a lineage that spread globally. The most recent branches of the late clade are represented by South-East Asia and India/Pakistan collections. Analysis of migration rates suggests abundant gene flow between India/Pakistan and the rest of the world with the exception of Africa.

Gene Flow Patterns Among Aedes aegypti (Diptera: Culicidae) Populations in Sri Lanka.

In Sri Lanka, dengue is the most serious arboviral disease. Recent increases in dengue cases suggest a higher infection rate and spread of the disease to new areas. The present study explores gene flow patterns of Ae. aegypti, the main vector of dengue disease, among 10 collection sites including major ports and inland cities using variations at 11 microsatellite loci. Discriminant analysis of principal components (DAPC) and k-means clustering estimated eight genetic clusters. Analysis of Molecular Variance (AMOVA) estimated equal variances among cities and among collections in Colombo, Sri Lanka. Significant evidence, although weak, was detected for isolation by distance. Analysis of gene flow rates and directions using MIGRATE-n indicated that populations throughout the island served as a source of immigrants for Colombo with abundant gene flow among major commercial cities in Sri Lanka, which appear to receive migrant mosquitoes from throughout Sri Lanka. The observed patterns probably arise through human movement of Ae. aegypti during commerce from throughout Sri Lanka into Colombo increasing the risk of spread. The patterns uncovered in this study are significant for global health as Sri Lanka is situated along a key international shipping route.

Mitochondrial-COII sequence polymorphism reflects spatial genetic clustering of Anopheles culicifacies sibling species E in Sri Lanka.

BACKGROUND & OBJECTIVES: Malaria infects around 216 million people annually with estimated 445,000 deaths globally. Anopheles culicifacies is the vector of malaria in Sri Lanka, a complex of five morphologically identical sibling species of which precise identification using DNA-based methods is still under experimentation. This study was carried out in Sri Lanka to observe the utility of BCE-PCR assay based on mitochondrial Cytochrome Oxidase II (COII) developed in India, in sibling species B and E identification in Sri Lanka, to characterize nucleotide and corresponding amino acid sequences of COII region in major vector sibling species E in Sri Lanka and to analyze the spatial distribution pattern of sibling species E in Sri Lanka using microsatellite markers. METHODS: BCE-PCR was carried out for the samples to identify their sibling status. Sequencing of COII region was then carried out to investigate the genetic diversity of Sri Lankan sibling species E, sequences were aligned and compared; microsatellite genotyping was carried out and the spatial clustering pattern was analyzed. RESULTS: Identification of sibling species B and E using BCE-PCR was confusing due to the heterogeneity in the COII region of sibling species in Sri Lanka. Non-synonymous substitutions were detected in COII gene amongst sibling species E. Spatial distributed two clusters were detected in the studied population. INTERPRETATION & CONCLUSION: Existence of genetic variants among sibling species is suggested in Sri Lanka. Further, the pattern of sibling species identification in BCE-PCR was reflected in the spatial clustering of sibling E in Sri Lanka.

Bradyrhizobium and Pseudomonas strains obtained from coal-mining areas nodulate and promote the growth of Calopogonium muconoides plants used in the reclamation of degraded areas.

AIMS: The objective of this work was to isolate and characterize indigenous rhizobia from coal-mining areas able to efficiently nodulate and fix nitrogen in association with Calopogonium mucunoides (calopo). METHODS AND RESULTS: Isolation, authentication and morphological, biochemical and molecular characterization of the autochthonous rhizobia were performed and their symbiotic efficiency (SE) evaluated. Efficient rhizobial isolates suitable for the inoculation of calopo in coal-mining regions were obtained. A total of 30 isolates were obtained after nodulation authentication, of which five presented high SE with plant-growth promoting traits such as indole-3-acetic acid production, phosphate solubilization and biofilm formation. These isolates were identified as belonging to Bradyrhizobium, Pseudomonas and Rhizobium. CONCLUSIONS: Bradyrhizobium sp. A2-10 and Pseudomonas sp. A6-05 were able to promote calopo plant growth using soil obtained from coal-mining degraded areas, thus indicating their potential as inoculants aiming at land reclamation. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of Pseudomonas nodule formation in calopo. Furthermore, the results demonstrated that autochthonous rhizobia obtained from degraded soils presented high SE in calopo and possess a wide range of plant-growth promoting traits. Ultimately, they may all contribute to an increased leguminous plant growth under stress conditions. The selected rhizobia strains may be used as inoculants and present a valuable role in the development of strategies aiming to recover coal-mining degraded areas. Bacterial inoculants would greatly reduce the use of often harmful nitrogen fertilizers vastly employed in revegetation programmes of degraded areas.

Kidney Transplantation Using Gonadal Vein for Venous Anastomosis in Patients With Iliac Vein Thrombosis or Stenosis: A Series of Cases.

BACKGROUND: Kidney transplantation is the treatment of choice for patients with end-stage renal disease. The standard surgery uses the recipient's iliac vessels for vascular anastomosis. Thrombosis and/or stenosis of the iliac vein, which are possible complications of multiple vascular access points for dialysis, can be detected intraoperatively, constituting a surgical challenge. An infrequently reported option is the use of the gonadal vein. OBJECTIVES: This study aims to evaluate the outcomes of venous anastomosis in the gonadal vein in patients with iliac vein thrombosis and/or stenosis submitted to kidney transplantation. METHODS: We reviewed the records of five adult recipients with iliac vein thrombosis and/or stenosis detected intraoperatively during emergency kidney transplantation with deceased donor due to vascular access failure from February 2013 to December 2014. Antithrombotic prophylaxis was not performed. We evaluated the postoperative complications, length of stay, early graft echo-Doppler, and renal function during the first year postoperatively. RESULTS: Delayed graft function occurred in three cases. Two patients developed postoperative infection requiring antibiotics. One patient required reoperation due to post-renal biopsy complications. The mean length of stay was 31.2 days and the mean serum creatinine levels at discharge, at 6 months, and at 12 months postoperatively were 1.42 mg/dL, 0.86 mg/dL, and 0.82 mg/dL, respectively. All patients had normal ultrasonography. There were no losses of graft or deaths during follow-up. CONCLUSION: Venous anastomosis using the gonadal vein in kidney transplantation for patients with iliac vein thrombosis and/or stenosis showed good clinical and surgical results, showing this method to be a viable alternative to venous drainage in these complex patients.

Effect of Adhesive Restoration and Bleaching Technique on the Concentration of Hydrogen Peroxide In the Pulp Chamber.

This study aimed to quantify the concentration of hydrogen peroxide into the pulp chamber in the presence or absence of adhesive enamel restorations and to analyze the resin-dentin interface of bleached groups. Bovine incisors (120) were randomly divided into three groups according to enamel treatment (n=40 each): (1) enamel without restoration (control); (2) enamel cavities (3 mm diameter × 1.5 mm depth) restored with a silorane-based (SB) system; or (3) enamel cavities (3 mm diameter × 1.5 mm depth) restored with a dimethacrylate-based (DB) system. Restorations were thermocycled, and all groups were submitted to one application of 35% hydrogen peroxide (HP) agent for 45 minutes and subjected to four light activation methods (n=10 each): without light, light-emitting diode (LED), LED/diode laser, or halogen light. Acetate buffer solution was placed into the pulp chamber before bleaching, and this solution was collected to spectrophotometrically determine the concentration of HP that reached the pulp chamber after bleaching. Rhodamine B was added to the HP agent and applied on additional enamel samples of each group for 24 hours. Samples were sectioned mesiodistally, and the bleaching agent was traced using confocal microscopy. According to two-way analysis of variance and Tukey test (α=0.05), the HP concentration in the pulp chamber of the control group was significantly lower than that of the SB group (p<0.05), regardless of light activation. No differences were observed between DB and SB groups and between control and DB groups, except for the DB halogen light activated group, which exhibited higher HP intrapulpal concentration (p<0.05). Confocal microscopy exhibited HP diffusion through the interface of the SB and DB restored groups as well as enamel prisms in the control group. The SB restorative system increased the HP diffusion into the pulp chamber, but HP was able to diffuse even in the absence of enamel restorations.

Oviposition and vertical dispersal of Aedes mosquitoes in multiple storey buildings in Colombo district, Sri Lanka.

BACKGROUND & OBJECTIVES: The Colombo City in Sri Lanka is experiencing tremendous development and construction of multiple storey buildings and high rise apartments. The change in housing types and microhabitats might have altered the flight and breeding behaviour of Aedes mosquito population. This study was carried out to determine the vertical dispersal and abundance of Aedes mosquitoes in multiple storey buildings in the Colombo district, with respect to abiotic factors such as rainfall, humidity and wind speed. Hence, this study is of paramount importance, particularly for planning and implementation of control measures against Aedes mosquitoes. METHODS: An ovitrap based study was carried out at four selected multiple storey buildings in four residential areas located in Colombo, Sri Lanka, from August to December 2013. Results were analyzed using four indices; ovitrap index, mean number of larvae, mean number of eggs and mean number of larvae per ovipaddle. RESULTS: The results implied that Aedes mosquitoes could be found in different elevations from ground floor to the highest floor (130 ft). There was a significant difference between height and ovitrap index (p<0.05), and height and mean number of larvae per recovered ovipaddle (p<0.05). The highest index value for mean number of eggs was observed as 3.492 ± 0.655 at the 6th floor (60 ft high from ground level). At the same height (60 ft height) other indices (ovitrap index, mean number of larvae and mean number of larvae per ovipaddle) also displayed higher values, i.e. 13.19 ± 2.98%, 1.366 ± 0.527, and 2.070 ± 0.421%, respectively. Abiotic factors such as wind speed, coastal nature, etc. displayed a significant effect to the vertical dispersal of Aedes mosquitoes (p<0.05). INTERPRETATION & CONCLUSION: The study suggested that Aedes mosquitoes are able to breed at any level of the buildings and not restricted by their height. The indices (mean number of larvae, mean number of eggs) representing the vertical dispersal with respect to abundance seemed to be statistically non-significant (p>0.05) with height which indicates high abundance of Aedes mosquitoes at higher floors. Abiotic factors also seemed to cause significant effect to the vertical dispersal of Aedes mosquitoes in high rise buildings.

Microcystin-LR-induced cytotoxicity and apoptosis in human embryonic kidney and human kidney adenocarcinoma cell lines.

Microcystin-LR (MC-LR) is a potent hepatotoxin, and increasing evidence suggests that it might also induce kidney injury. The aim of the present work was to evaluate the cytotoxicity and possible apoptotic effects of MC-LR on a human embryonic kidney cell line (HEK-293) and human kidney adenocarcinoma cell line (ACHN). Cells were exposed for 24 h to pure MC-LR (1.0-200 µM) and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulphorhodamine B (SRB) cell viability assays. Cell viability in both cell lines was significantly decreased after treatment with MC-LR at 50 µM for 24 h (P<0.001). Moreover, MC-LR-treated ACHN and HEK-293 cells exhibited a marked dose-dependent loss of confluence as judged by phase-contrast microscopy. Similarly, fluorescence microscopic observations following acridine orange-ethidium bromide (AO/EB) staining confirmed that both cell types were undergoing apoptosis after treatment with MC-LR for 24 h. Expression of three apoptosis-related genes, Bax, Survivin and p53, was analysed by quantitative reverse transcriptase PCR analysis. Both Bax and p53 functioned as promoters of MC-LR-mediated apoptosis in ACHN and HEK-293 cells. The Survivin gene acted as a suppressor of apoptosis at lower MC-LR concentration (1 µM) and the gene was upregulated at higher MC-LR concentration (10 µM) (P<0.001). Significant increases of caspase 3 (P<0.0001) and caspase 9 (P<0.0001) activity were detected in both cell lines after exposure to MC-LR for 24 h, indicating the MC-LR induces cytotoxicity and a marked apoptosis in both ACHN and HEK-293 kidney cell lines.

Foraging behaviour of the Scale-throated Hermit Phaethornis eurynome Lesson, 1832 (Aves, Trochilidae) in Vriesea incurvata Gaudich (Bromeliaceae).

In this study we tested for density-dependent relationships between visitation rates of the Scale-throated Hermit (Phaethornis eurynome) and the plant density and flower number of the bromeliad Vriesea incurvata, by comparing plots with varying densities of this bromeliad. Eight 100 m2 plots were established at least 200 m from each other; four plots contained 10-15 individuals of V. incurvata each, whereas the other four contained 4-5 individuals each. The visitors, number of visits, behaviour (nectar thief or potential pollinator) and the height of foraging were recorded during focal observations on the plants. The number of visits of P. eurynome varied according to the local density of V. incurvata, showing that the heterogeneous distribution of this bromeliad species may promote adjustments in the pollinator populations, through resource variation at a local scale.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates.

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.