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Keywords:

  • Culex quinquefasciatus;
  • resistance;
  • ecology;
  • mosquitoes

ABSTRACT:

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Culex quinquefasciatus, an arboviral and filarial vector, is present in high numbers throughout sub-Saharan Africa, and insecticide-resistant populations have been reported worldwide. In order to determine the insecticide resistance status of Cx. quinquefasciatus in Macha, Zambia, adult mosquitoes reared from eggs collected from oviposition traps were tested by bioassay. High levels of resistance to DDT, pyrethroids, malathion, and deltamethrin-treated net material were detected, and molecular assays revealed that the knockdown resistance (kdr) allele was frequent in the Cx. quinquefasciatus population, with 7.0% homozygous for the kdr L1014 allele and 38.5% heterozygous (0.263 kdr frequency). The kdr frequency was significantly higher in mosquitoes that had successfully fed on human hosts, and screening archived specimens revealed that kdr was present at lower frequency prior to the introduction of ITNs, indicating that ITNs might be a selective force in this population. Additionally, metabolic detoxification enzyme activity assays showed upregulated glutathione S-transferases, α-esterases, and β-esterases. Continued monitoring and assessment of the Cx. quinquefasciatus population is necessary to determine levels of resistance.


INTRODUCTION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Culex quinquefasciatus Say, a member of the Culex pipiens group, is a medically important mosquito and major pest species with a worldwide distribution (White 1989). Culex quinquefasciatus is known to be a major vector of filariasis (White 1989), St. Louis encephalitis virus (SLEV) (Savage et al. 1993), West Nile virus (WNV) (Kwan et al. 2010), and Rift Valley Fever virus (RVFV) (Sang et al. 2010, Turell et al. 2008). It is considered to be an opportunistic feeder, and while host choice is regionally variable, it feeds on many species of birds, mammals, and occasionally reptiles and amphibians (Mackay et al. 2010, Unlu et al. 2010). In North America, the human blood index (HBI) of Cx. quinquefasciatus varies from 1% (Reisen et al. 1990) to 50% (Zinser et al. 2004). In studies conducted in Kenya, HBIs ranged from 12–88% for indoor-collected mosquitoes and 3–23% for mosquitoes collected outdoors (Beier et al. 1990, Muturi et al. 2008).

Insecticide-treated bed nets (ITNs), which are targeted to prevent mosquitoes from feeding on people indoors, and indoor residual spraying with insecticide (IRS), which prevents malaria transmission, are increasingly deployed in Africa as a means of malaria control and can have the added benefit of protecting people from filarial and arboviral diseases transmitted by culicine mosquitoes (Manga 2002). However, selective pressure from insecticides can cause resistance in vector species (Hemingway et al. 2004). Resistance can be mediated by mutations in the target site of the insecticide or its active metabolites (target-site resistance), through enzymatic modification of insecticides to product non-toxic metabolites (metabolic detoxification), or by behavioral changes or thickening of the cuticle. DDT and pyrethroids, insecticides commonly used for vector control in Zambia (Najera 2005), share a common target site, the para voltage-gated sodium channel. Knockdown resistance (kdr) mutations in this channel can therefore confer cross-resistance to both DDT and pyrethroids (Soderlund and Knipple 2003). Organophosphate and carbamate resistance can be mediated by insensitive acetylcholinesterase (ace-1), also conferred by a single nucleotide mutation (Weill et al. 2004). Metabolic detoxification is typically mediated through upregulation of endogenous detoxification enzymes, either by gene duplication or by transcriptional upregulation (Hemingway et al. 1998). Three classes of endogenous detoxification enzymes are known to affect insecticide susceptibility. Amplification of carboxylesterases, primarily through gene duplication, causes resistance to organophosphates (OPs) and carbamates (Hemingway et al. 2004). Upregulation of cytochrome P450-dependent monooxygenases by increased transcription causes resistance to pyrethroids and DDT (Hemingway et al. 2004). Upregulation of glutathione S-transferases (GSTs), usually by increased transcription rates, can cause resistance to OPs, DDT, and pyrethroids (Hemingway et al. 2004).

There are many reports of insecticide-resistant Cx. quinquefasciatus in Africa. Resistance to pyrethroids has been documented in Tanzania (Oxborough et al. 2010), Benin (N'Guessan et al. 2009), and Côte d'Ivoire (Asidi et al. 2004), while organophosphate and carbamate resistance, whether through upregulated esterase or insensitive acetylcholinesterase, has been reported in Benin (Corbel et al. 2007), Burkina Faso (Majori et al. 1986), and Côte d'Ivoire (Asidi et al. 2005). Additionally, DDT resistance has been recorded in Benin (Corbel et al. 2007), Burkina Faso, and Côte d'Ivoire (Magnin et al. 1988). However, no studies have been published on insecticide resistance in Cx. quinquefasciatus from Southern Africa.

As part of the malaria control scale-up, a free mass distribution of ITNs by the Zambian government provided 4,800 long lasting insecticide-treated nets (LLINs) to the Macha area in 2007. In conjunction with our ongoing investigations on the insecticide susceptibility status of Anopheles arabiensis, the primary malaria vector in the region, we tested the sympatric Cx. quinquefasciatus field population for insecticide resistance, as well as investigating the efficacy of ITNs against this species and its propensity to feed on human hosts.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Study area

This study was conducted at the Johns Hopkins Malaria Research Institute's field station in Macha, Zambia. It is located at 16.39292°S, 26.79061°E, in the southern province of Zambia, at approximately 1,100 m above sea level. The habitat around the field station, the Malaria Institute at Macha (MIAM), is Miombo woodland. There are three distinct seasons: a rainy season that lasts from December to April, a cold dry season from May to July, and a hot dry season from September to December. Culex quinquefasciatus mosquitoes were collected in the villages of Lupata and Chidakwa, which are located <10 km from Macha Hospital; in Namwalinda, located approximately 10 km from Macha Hospital; and on the Macha Hospital campus.

Mosquito collections

Culex egg rafts were collected from oviposition buckets (Service 1976) in the Macha area in January, 2010, during the rainy season. Briefly, 5 to 10 liter buckets were filled with a mixture of rainwater, baker's yeast, and hay and left outside for several days to ferment. Buckets were placed in Lupata and Chidakwa in sheltered areas outside of houses that were known to have had high numbers of Cx. quinquefasciatus in recent household mosquito collections, as well as around the Macha Hospital campus. Approximately 50 egg rafts were collected and transported to the insectary at MIAM, kept in separate containers, and reared to eclosion. After adults in each egg batch were confirmed as Cx. quinquefasciatus by morphology (Jupp 1996), they were pooled before use in insecticide susceptibility assays. Three- to five-day-old mosquitoes were used for Centers for Disease Control (CDC) bottle bioassays and ITN susceptibility assays. After the bioassays were completed, mosquitoes were killed by freezing and desiccated on silica gel for transport to the Johns Hopkins School of Public Health (JHSPH) in Baltimore.

For blood-feeding data, adult, female mosquitoes were collected by indoor CDC light trap (Beier 2002) from households in Lupata, Chidakwa, and Namwalinda throughout the rainy season in 2009 and 2010. Mosquitoes were killed by freezing, identified morphologically (Jupp 1996), and desiccated on silica gel for transport to JHSPH.

Mosquitoes for enzyme activity assays were collected by manual aspiration on the Macha Hospital campus in October, 2010, during the hot dry season. Oviposition bucket collections were attempted but yielded no egg rafts, as this method appears to work best during the rainy season. Mosquitoes were killed by freezing, identified morphologically, and maintained below 0° C during transport to JHSPH.

Archived mosquitoes, from the 2004/5 and 2005/6 rainy seasons, had been collected by pyrethrum spray catch, manual aspiration, and human landing catch (Service 1976). They were packaged in microcentrifuge tubes on silica gel and cotton and stored until used for DNA extractions and genotyped for the kdr allele.

CDC bottle bioassay

CDC bottle bioassays were used to test adult mosquitoes for insecticide susceptibility to permethrin, deltamethrin, DDT, and malathion (Sigma Aldrich, St. Louis, MO) (Brogdon 2009, Brogdon and McAllister 1998). Doses of insecticide, measured in μg/bottle, were diluted in acetone and used to coat the inside of 250 ml glass Wheaton bottles, and a control bottle was coated with acetone only. The acetone was allowed to evaporate over the course of several hours or overnight. Diagnostic dosages previously established for the Cx. quinquefasciatus S-LAB colony were used: 300 and 500 μg/bottle DDT, 30 μg/bottle permethrin (40:60 cis:trans), 20 μg/bottle deltamethrin, and 100 μg/bottle malathion (McAbee et al. 2004). After the acetone had fully evaporated, 20–25 mosquitoes were introduced to each bottle by aspiration. Every 15 min, for 3 h, the number of alive and knocked-down mosquitoes was counted in each bottle. After the 3 h timepoint, the mosquitoes were removed from the bottles and sorted into “alive” and “knocked-down” groups. Mosquito groups were kept in separate paper cups with 10% sucrose solution under insectary conditions. After 24 h, they were scored as alive or dead in order to determine delayed mortality. At least three replicates were performed for each dosage of each insecticide.

ITN susceptibility assay

Netting cut from a deltamethrin-treated Permanet® 2.0 LLIN was used to line the inside of a one-gallon (3.79 liters) cardboard container. As a control, a second container was lined with untreated netting material. Twenty Cx. quinquefasciatus mosquitoes were gently aspirated into each container and exposed to LLIN material. After 5 min, they were gently shaken out into a cage and aspirated into labeled paper cups with 10% sucrose pads. In order to determine delayed mortality, they were then held for 24 h under insectary conditions. After 24 h, mosquitoes were categorized as dead or alive. Because pyrethroid insecticides cause mosquitoes to shed legs, which can negatively impact survival, live mosquitoes were further scored as having one to three or four to six legs. Previous data using this assay consistently showed 100% mortality in susceptible mosquitoes, and it has been used to characterize insecticide susceptibility in Anopheles arabiensis (Norris et al. unpublished data).

Enzyme activity assays

Metabolic detoxification activity was measured by microplate enzyme activity assays for glutathione S-transferases (GST), oxidases, and nonspecific α- and β-esterases, modified from the procedure developed by Brogdon (Brogdon 2009). Briefly, individual, frozen mosquito heads and thoraces were homogenized in 1,000 μl potassium phosphate buffer. Total protein concentration was determined by NanoDrop absorbance at 280 nm to correct for size differences between mosquitoes. Each homogenate was aliquoted into microplates in triplicates of 75 μl for the assays. A negative control for each assay was 75 μl of potassium phosphate buffer. For the GST assay, 75 μl reduced glutathione solution and 75 μl cDNB solution were added to each well, and the plate was read at 340 nm at 0 and 10 min. The T0 reading was subtracted from the T10 reading. For the oxidase assay, 150 μl TMBZ solution and 20 μl 3% hydrogen peroxide were added to each well, and the plate was read at 620 nm at 0 and 5 min. The T0 reading was subtracted from the T5 reading. For the esterase assays, 75 μl α- or β-naphthyl acetate was added to each well, the plate was incubated for 10 min, 75 μl dianisidinetetratotized solution was added to each well, the plate was incubated for 2 min, and read at 620 nm for α-esterase, 540 nm for β-esterase.

Data from enzyme activity assays was analyzed by averaging the triplicate absorbances, subtracting the average absorbance of the negative controls, and dividing by the total protein concentration, to give a normalized score. The upper limit for enzyme activity in the susceptible colony population was used as a cut-off for amplified enzyme in the field population.

PCR assays

Mosquito samples were split into head+thorax and abdomen for PCR assays. Each was extracted using a modified salt-extraction, with total DNA from each mosquito extraction resuspended in 50 μl dH2O (Kent et al. 2007).

Head+thorax extractions were used to genotype samples for the kdr allele, using a PCR protocol modified from Martinez-Torres and others (Martinez-Torres et al. 1999). Primer lengths were extended to increase binding efficiency (Table 1). PCR conditions are as follows: each 25 μl reaction contained 1× PCR buffer, 100 μM each dNTPs, 75 pmol CxRev primer, 75 pmol forward primer, 2.0 U Taq polymerase, and 1.5 μl DNA template. Thermocycler conditions consisted of an initial denaturation step of 95° C for 2 minutes; 50 cycles of 94° C for 30 s, 55° C for 1 min, 72° C for 45 s; followed by a final extension of 72° C for 5 min. Because both the susceptible and resistant products are the same size (∼375 bp), each forward primer was run in a separate PCR reaction, with products run alongside each other on a 2% agarose gel. In a subset of the mosquitoes (both from 2009–2010, as well as archived mosquitoes), a portion of the voltage-gated sodium channel was sequenced using previously published primers (Martinez-Torres et al. 1999) to validate the results of the PCR assay.

Table 1.  Primers used to genotype Cx. quinquefasciatus for the kdr allele.
Primer NameSequence
CxRev (reverse primer)5′-GCA AGG CTA AGA AAA GGT TAA GAA-3′
CxSEA (susceptible kdr-e forward)5′-TGG CCA CCG TAG TGA TAG GAA ATT T-3′
CxREA (resistant kdr-e forward)5′-TGG CCA CCG TAG TGA TAG GAA ATT C-3′
CxSWA (susceptible kdr-w forward)5′-GGC CAC CGT AGT GAT AGG AAA TTT A-3′
CxRWA (resistant kdr-w forward)5′-GGC CAC CGT AGT GAT AGG AAA TTT T-3′

Mosquitoes were screened for insensitive acetylcholinesterase (ace-1) by the PCR-RFLP method of Weill et al. (Weill et al. 2004). PCR products were digested overnight for the RFLP, and run on a 2% agarose gel.

Blood meals from Cx. quinquefasciatus collected as adults were identified using a number of PCR diagnostics performed on extracted abdomens. The PCR diagnostics of Kent and Norris (Kent and Norris 2005) and Fornadel and Norris (Fornadel and Norris 2008) were used to identify mammalian blood meals and distinguish between human, cow, dog, goat, and pig blood meals. The PCR diagnostic of Ngo and Kramer (Ngo and Kramer 2003) was used to distinguish between Passeriforme, Galliforme, and Columbiforme species of birds. For samples that did not react with these PCRs, the PCR developed by Parson et al. (Parson et al. 2002) was used to amplify a universally conserved portion of the vertebrate cytb gene, which was sequenced and identified by BLAST search (Altschul et al. 1990).

RESULTS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

CDC bottle bioassays

F1 field Cx. quinquefasciatus showed some degree of resistance to all the insecticides tested at diagnostic dosages used (Figure 1). For a susceptible population, 100% knockdown occurs at 1 h, with 100% 24-h mortality. At 300 μg/bottle DDT, knockdown after 3 h ranged from 75–80%. Increasing the dosage to 500 μg/bottle did not have an appreciable effect, and caused only 80–87% knockdown after 3 h. At 30 μg/bottle permethrin, there was 89–96% knockdown at 1 h, but 100% knockdown by 3 h. At 20 μg/bottle deltamethrin, there was 90–98% knockdown at 1 h, and 97–98% knockdown at 3 h. At 100 μg/bottle malathion, there was 89–95% knockdown at 1 h, and 95–100% knockdown at 3 h.

image

Figure 1. Results of CDC bottle bioassays with F1 field Cx. quinquefasciatus, using diagnostic dosages 300 and 500 μg/bottle DDT, 30 μg/bottle permethrin, 20 μg/bottle deltamethrin, 100 μg/bottle malathion.

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After the time course experiment, mosquitoes were removed from the bottles and held in the insectary for 24 h to measure delayed mortality. For 300 μg/bottle DDT, 24–74% of mosquitoes survived both at 3 and 24 h; 7–45% of mosquitoes were knocked down at 3 h but recovered at 24; and 0–11% of mosquitoes had delayed mortality at 24 h. Overall, there was 13–69% mortality at 24 h. For 500 μg/bottle DDT, 11–39% of mosquitoes survived at both 3 and 24 h; 17–27% of mosquitoes were knocked down at 3 h but recovered at 24, and no mosquitoes had delayed mortality at 24 h. Overall, there was 37–70% mortality at 24 h.

For 30 μg/bottle permethrin, 4–20% of knocked down mosquitoes recovered at 24 h. For 20 μg/bottle deltamethrin, 2–3% of mosquitoes survived at 3 and 24 h, and 0–2% of knocked down mosquitoes recovered. For 100 μg/bottle malathion, there was 100% mortality at 24 h.

ITN susceptibility assays, kdr, and ace-1 genotyping

This assay was previously tested with S-LAB susceptible Cx. quinquefasciatus colony mosquitoes, and consistently resulted in 100% mortality. Using mixed-sex F1 field Cx. quinquefasciatus from Macha, however, resulted in only 34.2% mortality (95% CI: 26.8–41.5%). Of the remainder, 49.1% (41.3–56.8%) survived with most or all of their legs, 15.5% (9.9–21.1%) with one to three legs, and 1% (0–3.0%) were knocked down but recovered with most or all of their legs (n = 161, Figure 2).

image

Figure 2. ITN susceptibility assays with F1 field Cx. quinquefasciatus, using untreated control and deltamethrin-treated netting material.

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A subset of 50 mosquito samples were genotyped for the kdr and ace-1mutations. The ace-1 mutation was not detected in any mosquito samples. However, the L1014F (kdr-west) allele was detected in a large proportion of mosquito samples. Because this mutation causes pyrethroid resistance, all F1 Cx. quinquefasciatus from these assays were then genotyped for kdr (Table 2). 54.5% were homozygous wildtype, 38.5% were heterozygous, and 7.0% were homozygous for L1014F (0.263 kdr freqency). Individuals homozygous for the L1014F allele were more likely to survive the ITN assay, with 72.7% survival with four to six legs (95% CI: 43.3–90.3%), vs 46% survival (35.7–56.4%) with four to six legs for wildtype individuals. However, the genotypes did not significantly correlate with phenotypes (p = 0.35, Fisher's exact test). Interestingly, 46% of homozygous wildtype mosquitoes survived the ITN bioassay with most or all of their legs, indicating alternative resistance mechanisms other than the kdr mutation.

Table 2. kdr genotype vs ITN bioassay phenotype for F1 field Cx. quinquefasciatus. Numbers in bold indicate number of individuals; numbers in parentheses indicate what percentage of individuals with that genotype fell into each phenotypic category. The kdr L1014F allele appears to correlate with a survival phenotype. However, 66% of wildtype individuals also survived the assay.
phenotype# wildtype# heterozygous#kdr L1014F# PCR failure
down29 (34%)23 (38%)2 (18%)1 (18%)
up, 1–3 legs17 (20%)7 (12%)1 (9%)0 (0%)
up, 4–6 legs39 (46%)30 (50%)8 (73%)4 (80%)
total85 (100%)60 (100%)11 (100%)5 (100%)

To determine whether the kdr allele was present prior to the introduction of insecticide-treated nets in Macha in 2007, 48 archived Cx. quinquefasciatus specimens from 2004/5 and 36 specimens from 2005/6 were genotyped for kdr (Table 3). In the 2004/5 set, 47 were homozygous wildtype, one was heterozygous, and no samples were homozygous for the kdr allele (0.01 kdr frequency). In the 2005/6 set, 30 were homozygous wildtype, five were heterozygous, and one sample was homozygous for kdr (0.10 kdr frequency). 95% of the specimens from 2004/5 were collected by pyrethroid spray catch, which may have biased the results of the kdr screening. In the 2005/6 set, only 12% were collected by pyrethroid spray catch, the remainder having been collected using insecticide-free methods.

Table 3.  Genotype frequencies, number of individuals genotyped, and Hardy-Weinberg equilibrium p-values for subsets of the Cx. quinquefasciatus Macha population.
Population Subsetwildtypeheterozygotekdr L1014nkdr frequencyH-W p-value
F1 males and females, collected as egg rafts54.5%39.5%7.0%1560.2630.86
Unfed females, from indoor CDC traps50.7%41.0%8.3%2050.2880.99
Blooded females, from indoor CDC traps43.2%40.4%16.4%2920.3660.027
Human-blooded females, from indoor CDC traps39.7%42.7%17.6%1990.3890.15
Adult females from 2004/597.9%2.1%0%480.0110.94
Adult females from 2005/683.3%13.9%2.8%360.0980.21

Blood-feeding behavior

In total, blood meal hosts for 442 Cx. quinquefasciatus from indoor CDC light traps were identified. The majority, 73.8%, were human, followed by 16.3% Galliformes (chickens, turkeys, and guinea fowl), 4.1% unidentified mammal, 2.9% dog, 1.1% mixed human and Galliforme, 0.9% pig, 0.5% cow, 0.2% Passeriformes (perching birds), and 0.2% unidentified amphibian.

Of these samples, 292, as well as 205 unfed adult females from CDC light traps, were genotyped for kdr (Table 3). Unfed adults had similar allele frequencies to F1 Cx. quinquefasciatus collected as eggs. Blooded females had an increased proportion of L1014F homozygotes and fewer homozygous wildtype as compared to unfed females, and those blooded on humans had more than double the proportion of L1014 homozygotes and significantly fewer homozygous wildtype than unfed females (p= 0.0084, chi-square test). When Hardy-Weinberg values were calculated for the kdr allele, the unfed female and human-blooded female subsets were in equilibrium, while the blooded subset was not in equilibrium (Table 3).

Enzyme activity assays

GST, oxidase, α-, and β-esterase activity assays were run on 124 unfed mixed-sex Cx. quinquefasciatus collected by manual aspiration in Macha, and 48 three- to five-day-old mixed-sex S-LAB susceptible colony mosquitoes (Figure 3). Although there is the possibility that age differences between the groups affected the results of enzyme activity assays, susceptibility to insecticides appears to increase with age in resistant strains (Hodjati and Lines 1999, Rajatileka et al. 2011) and may be due to decreases in detoxification enzymes (Rajatileka et al. 2011).

image

Figure 3. Normalized detoxification enzyme activity levels for susceptible S-LAB and Macha field Cx. quinquefasciatus. Glutathione S-transferase, oxidase, α-esterase, and β-esterase activity levels are shown. Light bars indicate colony mosquitoes, dark bars indicate field samples.

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Field mosquitoes had significantly higher GST activity levels than did colony mosquitoes (p<0.0001, Mann-Whitney U test), and their distributions were significantly different (p<0.001, two-sample Kolmogorov-Smirnov test). There was no significant difference in oxidase activity between field and colony mosquitoes (p = 0.30, Mann-Whitney U test). However, the oxidase activity distribution for field mosquitoes was more skewed to the left than was the distribution for colony mosquitoes (field mosquito skewness = 0.80; colony mosquito skewness = 0.09; p=0.025, two-sample K-S test). The mean of the α- and β-esterase activity level distributions was not significantly different between colony and field mosquitoes (p = 0.22 and p = 0.098, respectively, Mann-Whitney U test). However, the α- and β-esterase distributions were far more skewed than the colony distributions (α-esterase: p=0.001; β-esterase: p<0.001, two-sample K-S test). The esterase activity levels for colony mosquitoes were normally distributed (α-esterase: skewness = 0.22, kurtosis = 2.54; β-esterase: skewness: –0.15, kurtosis: 2.70). The esterase activity levels for field mosquitoes were non-normal, significantly skewed to the right and had longer tails (α-esterase: skewness = 1.78, kurtosis = 6.27; β-esterase: skewness = 1.61, kurtosis = 5.39), such that there were many individuals with elevated α- and β-esterase activity levels.

The upper limit of enzyme activity in the susceptible colony population was used as the cut-off to discriminate individuals with amplified enzyme. Of the field population, 18% fell above this cut-off for GST activity, 10% for oxidase activity, 31% for α-esterase activity, and 26% for β-esterase activity (Figure 3). There was a significant correlation between the α- and β-esterase activity levels (R2= 0.916, linear regression, (Figure 4). There was no significant relationship between activity levels for any other enzyme classes.

image

Figure 4. Relationship between α-esterase and β-esterase activity levels in individual mosquitoes (R2= 0.916, linear regression). Dark diamonds indicate field samples; open diamonds indicate colony samples.

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DISCUSSION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

The Cx. quinquefasciatus population in Macha is highly resistant to DDT and deltamethrin-treated LLIN material, and has some degree of resistance to pyrethroids and malathion. The kdr L1014F allele is present in the population, and is a common mechanism of DDT and pyrethroid resistance. However, the lack of correlation between kdr genotype and outcome of ITN bioassays indicates that it is not the only mechanism at work.

The results of the enzyme activity assays show that this population includes individuals with amplified glutathione S-transferase, α-esterase, β-esterase, and possibly oxidase activity. Elevated GST activity has been shown to cause resistance to DDT and indirect resistance to pyrethroids (Hemingway et al. 2004) and is most likely a second mechanism causing DDT, deltamethrin, and permethrin resistance in this population. Cytochrome P450 monooxygenases, in the oxidase class, are associated with resistance to permethrin, DDT, carbamates, and organophosphates (Hemingway et al. 2004). However, this class of enzymes was not as highly upregulated as was the glutathione S-transferase class. Amplified α- and β-carboxylesterase activity has been shown to be related to pyrethroid resistance in An. gambiae (Vulule et al. 1999) and Anopheles albimanus (Brogdon and Barber 1990) but has not been shown to cause resistance to these classes of insecticides in Culex species. The linear relationship between α- and β-esterase activity suggests that a large part of the increased enzyme activity may be due to co-amplified estα21 and estβ21, a genotype found in 90% of organophosphate-resistant Cx. quinquefasciatus (Hemingway et al. 2004). Future studies will be needed to determine which, if any, of these classes are responsible for resistance to each insecticide.

Culex quinquefasciatus is widely accepted to be an opportunistic feeder. While host choice is regionally variable, it feeds on many species of birds, mammals, and occasionally reptiles and amphibians (Mackay et al. 2010, Unlu et al. 2010). Previous studies have shown varying degrees of anthropophilicity, with the percentage of human blood meals varying from 1% (Reisen et al. 1990) to 50% (Zinser et al. 2004). Nearly three-quarters of blooded mosquitoes collected in this study had fed on humans. Although this figure is biased because mosquitoes were only collected indoors, it shows that Cx. quinquefasciatus in this area often bite humans. Individuals that had fed on human hosts were more likely to be homozygous for the kdr allele. This suggests that, given the high ITN coverage in Macha, individuals homozygous for the kdr gene are more successful at obtaining human blood meals. Where ITN use is high, kdr may provide a survival advantage in peridomestic environments, or in urban environments where humans are the most widely available host species.

The large population of Cx. quinquefasciatus present in the hot dry season, coupled with human feeding and resistance to ITNs, provides a high potential for arboviral or filarial disease transmission in the Macha region. Additionally, Cx. quinquefasciatus’ variable host choice could bridge transmission of zoonotic viruses such as West Nile Virus, which is reservoired in birds, to humans. Historically, diagnosis of arboviral diseases in humans have been neglected in Macha, due to the overwhelming burden of malaria as the major cause of febrile illness. Wucheraria bancrofti transmission is not documented in the immediate Macha area, but is still endemic to other areas of Zambia (WHO 2009).

Preliminary bioassay experiments on Cx. quinquefasciatus showed insecticide resistance as early as January, 2008 (Meera Venkatesan, unpublished data), although insecticides were not used for vector control in Macha prior to the start of mass LLIN distribution in 2007. However, there has been light agricultural pesticide use, especially cattle dipping. The kdr genotyping results from 2004/5 and 2005/6 reveal that the kdr allele was present prior to the introduction of ITNs in 2007, albeit at a lower frequency. The kdr frequency in 2004/5 was 0.01, although that number is most likely biased by the pyrethrum spray catch collection method. The 2005/6 frequency, 0.15, is likely a more representative figure for the pre-ITN kdr period. The presence of kdr prior to the introduction of ITNs, coupled with bioassay data from 2008, suggests that low levels of resistance to DDT, pyrethroids, and malathion evolved in response to insecticide pressure from agriculture. There is currently no data on population structure or migration in Cx. quinquefasciatus in Macha, so it is unclear whether resistance genotypes developed locally, in response to light agricultural pesticide use, or whether it emerged in nearby areas with more intense agricultural pesticide use and a history of insecticide-based vector control. Regardless of where the mutation originally occurred, the introduction of pyrethroid-treated ITNs in 2007 may have posed a selective pressure for kdr, even though Cx. quinquefasciatus is not limited to feeding on humans. Post-ITN, the kdr frequency leapt from 0.15 to 0.42. Unfortunately, archived mosquitoes from 2006/7, the year of the ITN roll-out, are not available.

An additional concern is the effect of pyrethroid-resistant Cx. quinquefasciatus on human behavior. Culex quinquefasciatus is the dominant mosquito species in Macha during the rainy season, making up 32% of indoor CDC collections in January and February of 2010, as opposed to only 9% for the malaria vector An. arabiensis. During the hot dry season, the majority of mosquitoes collected are Cx. quinquefasciatus. If villagers in Macha and surrounding areas notice that LLINs are ineffective against these mosquitoes, it may cause them to devalue LLINs and stop using them consistently, even if they are effective against the anophelines that transmit malaria. Finally, Culex quinquefasciatus is a medically important disease vector and a major pest mosquito in Zambia. Emerging insecticide resistance in this species may hinder vector control efforts, especially in areas where filarial and arboviral disease transmission by culicine mosquitoes is still ongoing.

Acknowledgments

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

The authors thank Meera Venketesan for preliminary work that led to this investigation and Shadreck Habbanti, Limonty Simubali, Musapa Mulenga, and the Malaria Institute at Macha field team for invaluable help with field work and mosquito rearing.

REFERENCES CITED

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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