Insecticide resistance and Bionomics in laboratory reared and field caught Anopheles funestus Giles (Diptera: Culicidae)

Malaria is transmitted by the mature, blood feeding portion of mosquito vector populations. Malaria vector control programs based on indoor residual spraying (IRS) of insecticides are designed to target resting adult Anopheles mosquitoes before or after they have blood fed. When a female mosquito acquires a blood meal, she could also ingest harmful xenobiotics that are present in the blood. During the resting period after feeding, many processes are initiated in order to assist in the digestion and assimilation of the blood. Ultimately, this enables the mosquito to absorb those amino acids needed for the biosynthesis of yolk proteins, which are essential for subsequent egg maturation. Since the regulation of xenobiotic (including insecticides) detoxification enzyme systems is likely to be altered in response to the ingestion of blood, this study aimed to investigate the effect of a blood meal on insecticide tolerance in insecticide resistant and susceptible southern African strains of the major malaria vector Anopheles funestus. Through the use of CDC bottle bioassays it was demonstrated that blood fed An. funestus carrying a pyrethroid resistant phenotype are even more tolerant of pyrethroid intoxication than their unfed counterparts. Using another major malaria vector, An. gambiae, microarray analysis revealed that a general increase in delta class glutathione-s-transferase (GST) expression occured in response to a blood meal. One gene, GSTD3, was over-expressed in both blood fed An. gambiae and An. funestus. Although this gene could not be validated with real time quantitative PCR, it serves as a viable target for future investigations. Since the pyrethroid resistant phenotype of southern African An. funestus has been linked to the over-expression of the duplicate copy gene CYP6P9, the expression levels of both copies of this gene were investigated. CYP6P9 and its copy, CYP6P13, showed a small but significant increase in expression in response to a blood meal. The increased expression of these major effect genes in response to blood feeding may be responsible for the increase in insecticide tolerance seen in the bottle bioassays. In an effort to repeat these experiments on wild caught An. funestus, field material was collected from Karonga in northern Malawi. Specimens were morphologically identified as members of the An. funestus group. However, attempts to molecularly identify them to species level failed. Through the use of ITS2 and D3 sequence analysis, cytogenetics and cross mating studies it was possible to conclude that these wild caught specimens were a new species. They have been provisionally named An. funestus-like

Development of mucobacteriophage L5 as a marker for mutation induction in mycobacteria

Student Number : 0201444H - MSc dissertation - School of Molecular and Cell Biology - Faculty of ScienceDue to the paucity of sensitive mutation markers available for studying mycobacterial species it was decided to explore the suitability of mycobacteriophage L5 as an analogous mutation detection system to phage Lambda in E. coli. The system relies on the detection of an increased production of clear plaque mutants (CPM) arising from turbid plaques, in response to DNA damage. A number of L5 phage experimental tools were developed and optimized, including a lysogen-based CPM confirmation assay. The mutant induction system was applied to wild type M. smegmatis mc2155 and its recA mutant, dinP mutant as well as an M. smegmatis(L5) lysogen. The lysogen system proved to be insensitive with respect to mutant induction since elevated CPM frequencies could not be detected. Interestingly, the wild type M. smegmatis mc2155 system demonstrated slightly elevated CPM frequencies in response to transfection of untreated L5 on UV irradiated host cells. This result suggests that a host SOS mutagenic system is able to act on normal, undamaged DNA bases. The involvement of the SOS response in untargeted mutagenesis was confirmed by the abrogation of increased CPM frequency, in an M. smegmatis recA mutant. This data supports suggestions that RecA is responsible for the control of the SOS response. The M. smegmatis dinP mutant system showed a decrease in CPM frequency which supports evidence that this gene does have mutator polymerase activity, as is in seen E. coli dinP homologues

The effect of a single blood meal on the phenotypic expression of insecticide resistance in the major malaria vector Anopheles funestus

<p>Abstract</p> <p>Background</p> <p><it>Anopheles funestus </it>is a major malaria vector in southern Africa. Vector control relies on the use of insecticide chemicals to significantly reduce the number of malaria vectors by targeting that portion of the female population that takes blood meals and subsequently rests indoors. It has been suggested that the intake of a blood meal may assist female mosquitoes to tolerate higher doses of insecticide through vigour tolerance. It is hypothesized that during the process of blood digestion, detoxification mechanisms required for the neutralizing of harmful components in the blood meal may also confer an increased ability to tolerate insecticide intoxication through increased enzyme regulation.</p> <p>Methods</p> <p>Bottle bioassays using a range of concentrations of the pyrethroid insecticide permethrin were performed on pyrethroid susceptible and resistant laboratory strains of <it>An. funestus </it>in order to detect differences in insecticide susceptibility following a single blood meal. Based on these results, a discriminating dosage was identified (double the lowest dosage that resulted in 100% mortality of the susceptible strain). Blood-fed and unfed females drawn from the resistant strain of <it>An. funestus </it>were then assayed against this discriminating dose, and the percentage mortality for each sample was scored and compared.</p> <p>Results</p> <p>In the insecticide dose response assays neither the fully susceptible nor the resistant strain of <it>An. funestus </it>showed any significant difference in insecticide susceptibility following a blood meal, regardless of the stage of blood meal digestion. A significant increase in the level of resistance was however detected in the resistant <it>An. funestus </it>strain following a single blood meal, based on exposure to a discriminating dose of permethrin.</p> <p>Conclusion</p> <p>The fully susceptible <it>An. funestus </it>strain did not show any significant alteration in susceptibility to insecticide following a blood meal suggesting that vigour tolerance through increased body mass (and increased dilution of internalized insecticide) does not play a significant role in tolerance to insecticide intoxication. The increase in insecticide tolerance in the pyrethroid resistant strain of <it>An. funestus </it>following a blood meal suggests that insecticide detoxification mechanisms involved in insecticide resistance are stimulated by the presence of a blood meal prior to insecticide exposure, leading to enhanced expression of the resistance phenotype. This finding may be significant in terms of the methods used to control indoor resting populations of <it>An. funestus </it>if the mass killing effect of insecticide application proves increasingly inadequate against blood-feeding females already carrying the insecticide resistance phenotype.</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

Field study site selection, species abundance and monthly distribution of anopheline mosquitoes in the northern Kruger National Park, South Africa

BACKGROUND: Knowledge of the ecology and behaviour of a target species is a prerequisite for the successful development of any vector control strategy. Before the implementation of any strategy it is essential to have comprehensive information on the bionomics of species in the targeted area. The aims of this study were to conduct regular entomological surveillance and to determine the relative abundance of anopheline species in the northern Kruger National Park. In addition to this, the impact of weather conditions on an Anopheles arabiensis population were evaluated and a range of mosquito collection methods were assessed. METHODS: A survey of Anopheles species was made between July 2010 and December 2012. Mosquitoes were collected from five sites in the northern Kruger National Park, using carbon dioxide-baited traps, human landing and larval collections. Specimens were identified morphologically and polymerase chain reaction assays were subsequently used where appropriate. RESULTS: A total of 3,311 specimens belonging to nine different taxa was collected. Species collected were: Anopheles arabiensis (n = 1,352), Anopheles quadriannulatus (n = 870), Anopheles coustani (n = 395), Anopheles merus (n = 349), Anopheles pretoriensis (n = 35), Anopheles maculipalpis (n = 28), Anopheles rivulorum (n = 19), Anopheles squamosus (n = 3) and Anopheles rufipes (n = 2). Members of the Anopheles gambiae species complex were the most abundant and widely distributed, occurring across all collection sites. The highest number of mosquitoes was collected using CO2 baited net traps (58.2%) followed by human landing catches (24.8%). Larval collections (17%) provided an additional method to increase sample size. Mosquito sampling productivity was influenced by prevailing weather conditions and overall population densities fluctuated with seasons. CONCLUSION: Several anopheline species occur in the northern Kruger National Park and their densities fluctuate between seasons. Species abundance and relative proportions within the An. gambiae complex varied between collection methods. There is a perennial presence of an isolated population of An. arabiensis at the Malahlapanga site which declined in density during the dry winter months, making this site suitable for a small pilot study site for Sterile Insect Technique as a malaria vector control strategy.The National Research Foundation and by the International Atomic Energy Agency (Contract no SAF16780 (under the G34002) and CRPG34002).http://www.malariajournal.com/am201

Identification and characterization of microRNAs expressed in the African malaria vector Anopheles funestus life stages using high throughput sequencing

Background: Over the past several years, thousands of microRNAs (miRNAs) have been identified in the genomes of various insects through cloning and sequencing or even by computational prediction. However, the number of miRNAs identified in anopheline species is low and little is known about their role. The mosquito Anopheles funestus is one of the dominant malaria vectors in Africa, which infects and kills millions of people every year. Therefore, small RNA molecules isolated from the four life stages (eggs, larvae, pupae and unfed adult females) of An. funestus were sequenced using next generation sequencing technology. Results: High throughput sequencing of four replicates in combination with computational analysis identified 107 mature miRNA sequences expressed in the An. funestus mosquito. These include 20 novel miRNAs without sequence identity in any organism and eight miRNAs not previously reported in the Anopheles genus but are known in non-anopheles mosquitoes. Finally, the changes in the expression of miRNAs during the mosquito development were determined and the analysis showed that many miRNAs have stage-specific expression, and are co-transcribed and co-regulated during development. Conclusions: This study presents the first direct experimental evidence of miRNAs in An. funestus and the first profiling study of miRNA associated with the maturation in this mosquito. Overall, the results indicate that miRNAs play important roles during the growth and development. Silencing such molecules in a specific life stage could decrease the vector population and therefore interrupt malaria transmission.IS

Antibodies to neutralising epitopes synergistically block the interaction of the receptor‐binding domain of SARS‐CoV‐2 to ACE 2

OBJECTIVES: A major COVID‐19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor‐binding domain (RBD) and angiotensin‐converting enzyme 2 (ACE2). These vaccines will also induce T‐cell responses. However, concerns were raised that aberrant vaccine‐induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. METHODS: We procured a series of overlapping 20‐amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID‐19 convalescent patients. Identified epitopes were conjugated to diphtheria‐toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. RESULTS: Seven putative vaccine epitopes were identified. Memory B‐cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. CONCLUSION: COVID‐19 convalescent patients have SARS‐CoV‐2‐specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope‐specific antibodies synergistically block RBD–ACE2 interaction