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Biology of insecticide resistance in the African malaria vector Anopheles Funestus (Diptera: Culicidae)Okoye, Patricia Nkem 15 October 2008 (has links)
The emergence of pyrethroid resistant Anopheles funestus (a major African vector) in
malaria affected parts of KwaZuluNatal,
South Africa was correlated with the
malaria epidemic of 1996 2000.
This finding prompted the necessity of
incorporating insecticide resistance management strategies into formal malaria
control policy in South Africa. Resistance management strategies often rely on the
assumption of reduced fitness associated with insecticide resistance and are based on
the principle that resistance genes will tend to drift out of vector populations in the
absence of insecticide selection pressure. This study aimed to determine whether a
fitness cost is associated with pyrethroid resistance as well as to determine the
stability and mode of inheritance of the resistance genes in a pyrethroid resistant
(FUMOZR)
strain of An. funestus. It also aimed to sequence and analyze a segment
of the sodium channel gene for any kdrtype
mutation(s) that may be associated with
pyrethroid resistance. The final aim was to determine the resistance mechanisms
involved in a Ghanaian field population of An. funestus resistant to DDT and
pyrethroids.
Results obtained suggest that pyrethroid resistance in southern African An. funestus
did not incur any loss of fitness. FUMOZR
had a reproductive advantage over a
pyrethroid susceptible An. funestus strain (FANG) in terms of higher fertility,
proportion of females laying eggs and eggtoadult
survivorship, and a lower sterility
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rate. However, FUMOZR
had a slower developmental time from egg hatch to adult
emergence than FANG.
Results of crosses and backcrosses carried out between FUMOZR
and FANG were
consistent with a monofactorial and autosomal mode of inheritance in which the
resistant genes presented as incompletely dominant. The resistant gene was found to
be stable over several generations in the absence of insecticide selection pressure.
Analysis of the genomic and mRNA sequences of the IIS5 IIS6
segment of the
sodium channel gene showed a high sequence identity between FUMOZR
and
FANG suggesting that the two strains are genetically similar. The kdrtype
mutation
was absent from this region supporting previous evidence that the resistance
mechanism is primarily metabolic.
Bioassay data showed that a Ghanaian field population of An. funestus from Obuasi,
Ghana, was resistant to DDT and pyrethroids. Molecular analysis of the IIS5 IIS6
segment of the sodium channel gene showed an absence of kdrtype
mutations
previously associated with insecticide resistance. Biochemical analysis suggests that
resistance is metabolically mediated primarily by elevated levels of and esterases
with monooxygenases and GSTs playing a lesser role. The presence of an altered
acetylcholinesterase conferring carbamate resistance was also evident in the
population. These results have implications for the management of resistance in
malaria control programmes in Africa.
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Insecticide resistance and vector status of Anopheles funestus and An. gambiae populations at a sugar estate in MozambiqueKloke, Ronald Graham 12 April 2010 (has links)
MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Malaria is on the increase in Mozambique since 2001 and impacts primarily on
children < 5 years of age. Insecticide resistance in the malaria vector mosquitoes is on
the increase in Mozambique and Africa and is cause for serious concern. Maragra
sugar estate is situated in close proximity to the nKomati river floodplain in a rural
area in Mozambique and requires intense irrigation for cane growing and as a result
provides extensive breeding sites for An. funestus and other mosquitoes. In the areas
surrounding the estate there are two important vectors of malaria, Anopheles funestus
group and An. gambiae complex. There is intense malaria transmission in the areas
surrounding the sugar estate and the last entomological study on the vectors in the
Manhica area was done in 1998. It was becoming increasingly urgent to identify to
species level the vectors in this area and to monitor the insecticide resistance status of
these vectors. Due to leakage (theft) of insecticides and a change by the National
Malaria Control Programme (NMCP) to an insecticide to which the predominant
vector is resistant, an entomological survey was carried out in this area from January
2009 to March 2009 to ascertain by Polymerase chain reaction (PCR) what species of
malaria vectors were present inside and outside of the Maragra vector control area,
their population levels and their vectorial status in these two areas. Insecticide
resistance studies by insecticide exposure and the synergist piperonyl butoxide (pbo)
were carried out using the World Health Organisation (WHO) bioassay method on
collected An. funestus mosquitoes. This was done to establish this species resistance
status to the four classes of insecticides recommended by the WHO for malaria vector
control. The collections of An. arabiensis and An. merus that were identified were too
few to carry out insecticide resistance tests on these two species. Enzyme linked
v
immuno-sorbent assay (ELISA) tests were undertaken to establish the vectorial
capacity of Anopheles funestus and An. gambiae complex in this area. The
predominant malaria vector species in this area is An. funestus s.s., with the secondary
vector being An. arabiensis. An. funestus has a high vectorial capacity in this area and
found to have a Plasmodium falciparum sporozoite rate of 6.02%. This is an increase
in the sporozoite rate of 1.2% from 1998 when the last survey in this regard was
carried out. Coupled with this increase is an increase in the An. funestus populations
in this area since this time. One An. gambiae complex sample was found to be
positive but the species is not known as this particular sample did not amplify on
PCR. Anopheles funestus is highly resistant to synthetic pyrethroids and exhibits a
lower level of resistance to bendiocarb, a carbamate insecticide in use at Maragra
sugar estate. The synergist pbo mediates the resistance mechanism in both these
insecticides indicating that the metabolic resistance mechanism present in this
mosquito is strongly mediated by monooxygenase detoxification. The role of the
medical entomologist is increasingly necessary and important in the monitoring of this
resistance phenomenon in malaria vector mosquitoes, as is the role of the vector
control programme manager in implementing and managing vector control
programmes. The implication of cane sugar farming and its impact on vector
production and malaria transmission is discussed. Insecticide resistance and the
change by the NMCP to a synthetic pyrethroid to which the predominant vector of
malaria is resistant is discussed.
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Role of monooxygenases in insecticide resistant anopheles funestus(diptera: culicidae)Amenya, Dolphine Achieng' 26 February 2007 (has links)
Student Number : 0318930A -
PhD thesis -
School of Animal, Plant and Environmental Studies -
Faculty of Science / The widespread use of pyrethroid insecticides has led to the emergence of significant
insecticide resistance in various parts of the world. An unprecedented increase in the
number of annual malaria cases reported in Kwazulu Natal, South Africa in 1999 to
2000 was attributed to the re-emergence of pyrethroid-resistant Anopheles funestus
Giles. Resistance was metabolic-based with increased monooxygenase (P450)
metabolising the pyrethroid insecticides. This emphased the need to understand the
molecular mechanisms conferring pyrethroid resistance in An. funestus. The present
study aimed to firstly isolate P450 genes in An. funestus and secondly, to identify
P450 gene over-expressed in a resistant (FUMOZ-R) strain compared to a
susceptible (FANG) strain. A third aim was to construct an An. funestus cDNA
library to lay the foundation for future studies on P450 monooxygenses.
Degenerate primers based on conserved regions of three An. gambiae P450 families
were used to amplify cDNAs from An. funestus. Eleven CYP4, four CYP6 and five
CYP9 partial genes were isolated and sequenced. BLAST results revealed that An.
funestus P450s have a high sequence similarity to An. gambiae with above 75%
identity at the amino acid level. The exception was CYP9J14. The An. gambiae
P450 with the closest similarity to CYP9J14 exhibited only 55% identity suggesting
a recent duplication event in CYP9J14. Molecular phylogenetic analysis also
supported this hypothesis. Intron positions were highly conserved between the two
species.
Expression studies using blot analysis implicated CYP6P9, an ortholog of CYP6P3
in An. gambiae, as the over-expressed P450. Dot blot analysis revealed a 500-fold
expression higher in FUMOZ-R strain compared with FANG strain. Semiquantitative
PCR revealed that CYP6P9 was developmentally regulated. Expression
was not detected in eggs and was higher in larvae compared to pupae. Quantitative
real time PCR showed that CYP6P9 expression was 4.5-fold higher in 3-day old
FUMOZ-R males than females and 3.5-fold higher in the 14-day old males than 14-
day old females. Statistically, this difference was not significant suggesting that
CYP6P9 expression is not sex specific.
The An. funestus cDNA library construction in λTriplEx2 vector was successful with
a titre of 4.9 x108 pfu/ml and a transformation efficiency of 98%.
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Comparative insecticide resistance intensity studies between the pyrethroid resistant wild Zambian and the laboratory reared Mozambique Anopheles funestusSegoea, Godira January 2015 (has links)
Malaria continues to be a significant cause of morbidity and mortality in the developing world with Africa being the most affected. Malaria vector control with chemical insecticides is the primary intervention to curb transmission. However, the success of chemical based interventions is threatened by the escalating development of insecticide resistance in the major anopheline mosquito vectors.
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Physiological ecology and future distributions of two malaria vectors : Anopheles arabiensis and An. funestusLyons, Candice-Lee 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although malaria remains a major public health concern, especially in sub-Saharan Africa, little information exists on the physiological tolerances of malaria vectors. Here, I aimed to provide a comprehensive set of physiological tolerances for Anopheles arabiensis and An. funestus, by investigating thermal tolerance traits of adults, larvae and pupae; desiccation resistance of adults and development rate-temperature relationships for both vectors. Critical thermal limit (CT) and desiccation data showed significant effects of increasing adult age on reducing tolerance to temperature or dry conditions. Females of both species were more tolerant of high or low temperatures in CT experiments and were more desiccation tolerant than males in desiccation trials. Anopheles funestus was more desiccation tolerant than An. arabiensis, despite the common misconception that An. arabiensis is the more arid-adapted of the two species.
Comparisons between thermal tolerance traits of adult laboratory and wild strain progeny of both species indicated a high degree of similarity between critical thermal limits in wild and laboratory strains, suggesting that the use of laboratory populations of both mosquito strains can provide an accurate estimate of wild population responses to thermal change. Lethal temperature estimates for both vectors indicated a higher tolerance to high temperature in An. arabiensis larvae and pupae when compared with An. funestus, and a greater tolerance of high or low temperatures in adult females when compared with adult males.
Species differences between the vectors were further highlighted in development rate-temperature experiments. Under fluctuating and constant temperatures, An. arabiensis developed significantly faster than An. funestus and had higher survival to the adult stage. Under fluctuating temperatures, An. arabiensis developed faster or no different to constant temperatures, while survival under fluctuating temperatures was also comparable to constant temperature estimates. This faster development rate of this species is likely a consequence of the puddle-breeding nature of An. arabiensis and the need to develop to adulthood before evaporation of breeding sites. Anopheles funestus on the other hand, showed reduced survival and development under fluctuating temperatures when compared with constant temperatures, probably as a result of the more thermally stable breeding sites usually used by this species.
Distribution data of these species, combined with developmental parameters in a process-based distribution model, suggests that both species will show range changes in response to climate change. Areas where these species were previously only present on a seasonal basis might become more suitable for vector population establishment and persistence, while areas on the northern margins of current distributions will become less favourable, leading to an overall southerly shift in habitat suitability for both species.
Increases in temperature and changes in rainfall patterns as predicted to occur with climate change are likely to impact the distribution of both malaria vectors. Combining the physiological tolerance data collected in this thesis in a future, planned mechanistic distribution model, will provide an accurate indication of potential range shifts of these vectors and hence, provide an indication of areas that may be at increased risk of malaria. / AFRIKAANSE OPSOMMING: Alhoewel malaria „n groot publieke gesondheidskwelling bly, veral in sub-Sahara Afrika, bestaan min inligting rakende die fisiologiese toleransies van malaria vektore. Hier het ek gepoog om 'n omvattende reeks van fisiologiese toleransies te voorsien vir Anopheles arabiensis en An. funestus, deur termiese verdraagsaamheidseienskappe, uitdrogingsweerstand en ontwikkelingstempo-temperatuur verhoudings vir beide vektore te ondersoek. Kritiese termiese limiet (CT) en uitdroging data het beduidende uitwerkings getoon van toenemende ouderdom op die vermindering van verdraagsaamheid teenoor temperatuur of droë toestande. Wyfies van beide spesies was meer verdraagsaam vir hoë of lae temperature in CT eksperimente en was meer verdraagsaam teenoor uitdroging as mannetjies in die uitdrogingsproewe. Anopheles funestus was meer verdraagsaam teenoor uitdroging as An. arabiensis, ten spyte van die algemene wanopvatting dat An. arabiensis die meer ariede aangepaste van die twee spesies is.
Vergelykings tussen termiese verdraagsaamheidseienskappe van laboratorium-en wilde stamlyn nageslagte van beide spesies het 'n hoë mate van ooreenkoms tussen kritieke termiese limiete in wilde en laboratorium stamlyne aangedui, wat voorstel dat die gebruik van laboratorium bevolkings van beide muskiet stamlyne 'n akkurate skatting kan gee van wilde bevolkingsreaksies tot termiese verandering. Fatale temperatuur beramings vir beide vektore het „n hoër toleransie getoon by hoë temperature in An. arabiensis larwes en papies wanneer dit vergelyk word met An. funestus, en 'n groter verdraagsaamheid van hoë of lae temperature in wyfies, wanneer vergelyk word met mannetjies.
Spesies verskille tussen die vektore is verder uitgelig in die ontwikkelingstempo-temperatuur eksperimente. Onder wisselende en konstante temperature ontwikkel An. arabiensis aansienlik vinniger as An. funestus en het hoër oorlewing tot die volwasse stadium getoon. Onder wisselende temperature ontwikkel An. arabiensis vinniger of met geen verskil van konstante temperature nie, terwyl oorlewing onder wisselende temperature ook vergelykbaar was met konstante temperatuur beramings. Die vinniger tempo van hierdie spesie is waarskynlik 'n gevolg van die poel-broeiende aard van An. arabiensis en die behoefte om tot volwassenheid te ontwikkel voor die verdamping van broeiplekke. Anopheles funestus aan die ander kant, het verminderde oorlewing en ontwikkeling onder wisselende temperature gewys wanneer dit vergelyk word met konstante temperature, waarskynlik as gevolg van die meer termies stabiele broeiplekke wat gewoonlik gebruik word deur hierdie spesie.
Verspreidingsdata van hierdie spesies, gekombineer met ontwikkelings-parameters in 'n proses-gebaseerde verspreidingsmodel, dui daarop dat beide spesies reeks veranderinge sal wys in reaksie tot klimaatsverandering. Gebiede waar hierdie spesies voorheen slegs teenwoordig was op 'n seisoenale basis, mag dalk meer geskik word vir vektor bevolkingsvestiging en volharding, terwyl areas op die noordelike grense van die huidige verspreidings minder gunstig sal word, wat sal lei tot algehele suidelike verskuiwing in die habitat geskiktheid vir beide spesies.
Toenames in temperatuur en veranderinge in reënvalpatrone, soos voorspel word om voor te kom met verandering van die klimaat, sal waarskynlik die verspreiding van malaria vektore beïnvloed. Deur die fisiologiese toleransie data, versamel in hierdie tesis, te kombineer met 'n toekoms, beplande meganistiese verspreidingsmodel, sal dit 'n akkurate aanduiding gee van die potensiële verspreidingsverskuiwings van hierdie vektore en dus 'n aanduiding gee van gebiede wat onder verhoogde risiko van malaria sal wees.
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Identification and characterization of microRNAs and their putative target genes in Anopheles funestus s.sAli, Mushal Allam Mohamed Alhaj January 2013 (has links)
Philosophiae Doctor - PhD / The discovery of microRNAs (miRNAs) is one of the most exciting scientific
breakthroughs in the last decade. miRNAs are short RNA molecules that do not encode proteins but instead, regulate gene expression. Over the past several years, thousands of miRNAs have been identified in various insect genomes through cloning and sequencing, and even by computational prediction. However, information concerning possible roles of miRNAs in mosquitoes is limited. Within this context, we report here the first systematic analysis of these tiny RNAs and their target mRNAs in one of the principal African malaria vectors, Anopheles funestus s.s. Firstly, to extend the known repertoire of miRNAs expressed in this insect, the small RNAs from the four developmental stages (egg, larvae, pupae
and the adult females), were sequenced using next generation sequencing
technology. A total of 98 miRNAs were identified, which included 65 known Anopheles miRNAs, 25 miRNAs conserved in other insects and 8 novel miRNAs that had not been reported in any species. We further characterized new variants for miR-2 and miR-927 and stem-loop precursors for miR-286 and miR-2944. The analysis showed that many miRNAs have stage-specific expression, and co-transcribed and co-regulated during development. Secondly, for a better understanding of the molecular details of the miRNAs function, we identified the target genes for the Anopheles miRNAs using a novel approach that identifies overlap genes among three target prediction tools followed by filtering genes based on functional enrichment of GO terms and KEGG pathways. We found that most of the miRNAs are metabolic regulators. Moreover, the results suggest implication
of some miRNAs not only in the development but also in insect-parasite interaction.
Finally, we developed the InsecTar database (http://insectar.sanbi.ac.za) for miRNA targets in the three mosquito species; Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus, which incorporates prediction and the functional analysis of these target genes. The proposed database will undoubtedly assist to explore the roles of these regulatory molecules in insects. This type of analysis is a key step towards improving our understanding of the complexity and regulationmode of miRNAs in mosquitoes. Moreover, this study opens the door for exploration of miRNA in regulation of critical physiological functions specific to vector arthropods which may lead to novel approaches to combat mosquito-borne infectious diseases.
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