New restrictions on insecticides for aphid control presents new challenges for lettuce growers. Dimethoate is soon to be unavailable and the future status of other conventional aphicides is uncertain. However, a number of new active ingredients will soon be available that offer lettuce growers valuable alternatives for aphid management in lettuce. The present dilemma and potential for implementing new chemistries into lettuce IPM programs is discussed in this report.

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This problem includes, first, the isolation and identification of microorganisms which utilized the hydrocarbon insecticide as a sole source of carbon and energy; second, a determination of the effect on plants sprayed with the hydrocarbon medium as compared with the effect on plants sprayed with the hydrocarbon medium containing a good growth of hydrocarbon-utilizers; and third, a determination of the ability of laboratory stock cultures of organisms to utilize or remain alive in the hydrocarbon medium.

Call number: LD2668 .T4 1986 J86 / Master of Science / Biology

Malaria is the leading cause of mortality and morbidity in Sudan. The annual malaria cases and deaths are estimated at 7.5 million and 35, 000 respectively. One of the possible factors that have led to this situation is the development of insecticide resistance in the main malaria vector in Sudan, Anopheles arabiensis. This study therefore, was initiated to identify the malaria vectors in Gezira and Sennar states of central Sudan, determine their susceptibility levels to the different classes of insecticides used for malaria vector control, identify mechanisms of resistance, and determine the sporozoite infection rate and the blood meal sources in these populations. The polymerase chain reaction (PCR) for species identification revealed that An. arabiensis was the only member of the An. gambiae complex present in the study area. The blood meal analysis using ELISA showed high anthropophily with 89.2% feeding on humans. The overall sporozoite infection rate was 2.3 %. WHO susceptibility tests showed complete susceptibility of An. arabiensis to bendiocarb (100% mortality) and multiple resistance to permethrin (54-78%), DDT (55-66%) and malathion (76-78%). The kdr mutation analysis revealed the presence of the West African kdr allele with the majority of specimens being heterozygous (RS). The kdr in DDT/permethrin susceptible specimens were: 15% homozygous for the kdr mutation (RR), 64.2% heterozygous (RS) and 20.8% homozygous for the susceptible allele (SS). Amongst the DDT/permethrin resistant specimens, 13% were SS, 48.7% RS and 38.3% RR. The apparent lack of correlation between kdr and resistant phenotype strongly suggests that other resistance mechanisms are playing a role.

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.

Malaria remains a serious public health challenge in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in Africa, and mostly in pregnant women and children under the age of five. Anopheles gambiae (An. gambiae) is the principal malaria vector in Africa, where vector control measures involve the use of insecticides in the forms of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The development of insecticides resistance mitigates these approaches. Glutathione (GSH) is widely distributed among all living organisms, and is associated with detoxification pathways, especially the Glutathione S-transferases (GSTs). Its direct involvement and relevance in insecticide resistance in An. gambiae has not been determined. Thus, this work examines the contribution of GSH, its biosynthetic genes (GCLM, GCLC) and their possible transcriptional regulator Nrf2 in insecticide resistance in An. gambiae sampled from agricultural setting (areas of intensive agriculture) and residential setting (domestic area). Bioinformatics analysis, W.H.O. adult susceptibility bioassays and molecular techniques were employed to investigate. Total RNA was first isolated from the adults An. gambiae mosquitoes raised from agricultural and residential field-caught larvae which had been either challenged or unchallenged with insecticides. Semi-quantitative RT-PCR using gel image densitometry was used to determine the expression levels of GCLM, GCLC genes and Nrf2. Bioinformatics’ results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) transcription factor binding sites in An. gambiae GCLC and GCLM promoters in silico. An. gambiae s.l. studied here were highly resistant to DDT and permethrin but less resistant to bendiocarb. Both knockdown resistance (kdr) mutation variants L1014S and L1014F that confers resistance to pyrethroid insecticides were identified in both An. coluzzii and An. arabiensis sampled from northern Nigeria. The L1014F was much associated with An. coluzzii. A significant positive correlation (P=0.04) between the frequency of the L1014F point mutation and resistance to DDT and permethrin was observed. However, a weak or non-significant correlation (P=0.772) between the frequency of the L1014S point mutation and resistance was also found. L1014S and L1014F mutations co-occurred in both agricultural and residential settings with high frequencies. However, the frequencies of the two mutations were greater in the agricultural settings than in the residential settings. The levels of total, reduced and oxidized GSH were significantly higher in mosquitoes from agricultural sites than those from residential sites. Increased oxidized GSH levels appears to correlate with higher DDT resistance. The expression levels of GCLM, GCLC and Nrf2 were also significantly up-regulated in adults An. gambiae raised from agricultural and residential field-caught larvae when challenged with insecticide. However, there was higher constitutive expression of GCLM, GCLC and Nrf2 in mosquitoes from agricultural setting. The increased expression levels of these genes and also GSH levels in this population suggest their roles in the response and adaptation of An. gambiae to insecticide challenges. There exists the feasibility of using GSH status in An. gambiae to monitor adaptation and resistance to insecticides.

Sexual conflict can occur whenever the evolutionary interests of males and females differ, and when sexually antagonistic selection acts upon traits shared between the sexes, one or both sexes can be constrained from reaching their phenotypic optima. This intralocus sexual conflict can be characterised by a tug-of-war of allelic replacement until it is resolved, but examples of well-characterised sexually antagonistic loci are rare. This thesis investigates the basis and dynamics of intralocus sexual conflict over insecticide resistance at the Cyp6g1 locus in Drosophila melanogaster, and wing colouration in Drosophila simulans. In D. melanogaster, the Cyp6g1 locus is the site of a series of insecticide resistance alleles, one of which is sexually antagonistic when back-crossed to the old isogenic lab strain Canton-S. I investigated the presence of sexual conflict over this same allele in a recently collected and genetically heterogeneous population. I found evidence of balancing selection on resistance (Ch. 2) that could not be explained by overdominance or sex-specific dominance (Ch. 3). However, balancing selection could be explained by resistance conferring increased fecundity to females (Ch. 2-4), and decreased reproductive success to males (Ch. 4). This male cost can in turn be explained by a negative genetic correlation between reproductive success and Cyp6g1 expression (Ch. 4), possibly influencing levels of reproductive investment (Ch. 2). Additionally, I explored the dynamics of the sex-specific fitness effects of resistance across three Cyp6g1 alleles back-crossed to a single genetic background. I found no evidence of sexual antagonism, but revealed that the cost of resistance increased with more derived alleles, and that all alleles were more costly to females (Ch. 5). After decades of strong selection imposed by insecticide use an unresolved sexual conflict persists at the Cyp6g1 locus despite sexual dimorphism in resistance, and it does not appear that more derived Cyp6g1 alleles are necessarily involved in mediating this conflict. Wing interference patterns (WIPs) are a newly discovered trait subject to female mate choice in Drosophila. I explored the potential for intralocus sexual conflict over WIPs by measuring WIP traits from males and females from populations of D. simulans evolved under relaxed or elevated sexual selection. In response to sexual selection male WIPs evolved to be brighter, higher contrast, and shifted to longer wavelengths of light, but there was no associated response to selection in females (Ch. 6). While WIPs did not appear to be constrained from detectably responding to selection by acute intralocus sexual conflict, male WIPs from the relaxed selection regime were similar to female WIPs, suggesting a cost to sexually selected WIPs that may be indicative of sexually antagonistic selection. IASC is pervasive and can influence a wide range of fundamental evolutionary processes including sexual selection, speciation, and extinction. The research presented in this thesis adds to a body of evidence that sexual dimorphism does not necessarily resolve IASC, and documents the first evidence that WIPs do not appear to be subject to acute IASC and can evolve in response to sexual selection.

<p>German cockroach (<i>Blattella germanica</i> L.) is an important urban pest that poses health risks. They carry pathogenic microorganisms, and the allergens present in their feces and cast skins can trigger asthma attacks. Gel bait formulations containing insecticides (indoxacarb and fipronil) have been used for German cockroach control for more than a decade. However, historical data suggests that cockroaches can develop resistance to insecticides that are repeatedly used. Therefore, we investigated the status and inheritance patterns of fipronil and indoxacarb resistance in cockroach strains collected from the state of Indiana and Illinois. In the first objective, topical dose-response bioassays were performed to determine fipronil and indoxacarb resistance levels in adult males of three field strains (D-IL, I-IN, and S-IN) and the laboratory-susceptible Orlando strain. Comparison of LD₅₀ (median lethal dose) values between the susceptible and field strains revealed that resistance to both insecticides in the D-IL and I-IN strains was <10-fold. However, fipronil and indoxacarb resistance levels in the S-IN strain were 20- and >10,000-fold, respectively. </p> <p> </p> <p>In the second objective, choice feeding bioassays were performed to test the performance of the resistant S-IN strain (adult males) on commercial fipronil and indoxacarb baits. Complete (100%) mortality of the S-IN strain was observed on fipronil baits. However, average mortality on indoxacarb baits was ~20% at 14d. In the third objective, synergist bioassays were done with PBO and DEF to investigate the mechanism of indoxacarb resistance. PBO did not significantly increase mortality in the S-IN strain at LD₅₀, but DEF did, suggesting increased hydrolase activity as a potential mechanism of indoxacarb resistance. In the last objective, reciprocal crosses were performed between the resistant S-IN strain and the susceptible Orlando strain to determine patterns of insecticide resistance. Topical bioassays and associated LD₅₀ values for the F1 generation adult males indicated that fipronil resistance was inherited as an incompletely dominant trait with sex-linkage. In contrast, indoxacarb resistance was inherited as a codominant trait and was not sex-linked. Our results indicate that resistance can evolve independently in different field strains. High-level indoxacarb resistance observed in the S-IN strain warrants additional research on the indoxacarb target-site as a possible resistance mechanism.</p>

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%.