Say egg rafts were collected in Harris County, TX from 2004 through 2008. Mosquitoes were reared in incubators at 27.5°C, 80–85% RH, and 14 : 10 light : dark cycle. They were aspirated into 1.5-ml microtubes 1–2 wk after they had emerged and stored in a −80°C freezer (Thermo Scientific, Waltham, MA) for future use. Fifteen mosquitoes from each collection site throughout Harris County were tested, for a total sample size of 300. In order to compare the competitive enzyme assay to the polymerase chain reaction (PCR) assay, the same mosquitoes were used for both tests. For the PCR assay and sequencing, all 6 legs were removed from the females, and the genomic DNA was extracted with the use of DNAzol® (Molecular Research Center Inc., Cincinnati, OH). The head and body were homogenized in potassium phosphate (KPO₄) buffer for the enzyme assay.

The insensitive acetylcholinesterase assay followed the procedures described by Brogdon (1988), and outlined in McAllister et al. (2012). The authors previously found that 10 min was not a sufficient amount of time to see the necessary color change (McAllister and Scott, unpublished data). The protocol was modified, and the microplate was read immediately (T₀) on a spectrophotometer with the use of a 414-nm filter, and then the microplate was stored in the refrigerator and read again after 24 h (T₂₄) (McAllister et al. 2012). The T₀ reading was subtracted from the T₂₄ reading for statistical analysis. Each mosquito sample was run in triplicate on the microplate.

Mosquito genomic DNA was extracted from the legs with the use of DNAzol. The legs from individual mosquitoes were homogenized in 25 μl of DNAzol with the use of a Kontes pellet pestle cordless motor with disposable pestles and the pestles were rinsed with 25 μl of DNAzol. The homogenate was centrifuged for 2 min at 10,621 × g to remove insoluble tissue. A volume of 48 μl of the resulting viscous supernatant was transferred to a new 1.5-ml microtube. The DNA was precipitated from the homogenate by adding 25 μl of 100% ethanol (CAS 64-17-5). The samples were mixed by inversion to ensure a homogenous solution and incubated at room temperature for 3 min. The samples were centrifuged for 4 min at 14,000 rpm. The DNA precipitate was washed twice with 500 μl of 75% ethanol, allowed to air dry for 5–15 sec, and then resuspended in 25 μl of dH₂O (CAS 7789-20-0).

The genomic DNA extracted from the legs was PCR amplified with the degenerated primers Moustdir1 5′-CCG GGN GCS ACY ATG TGG AA-3′ and Moustrev1 5′-ACG ATM ACG TTC TCY TCC GA-3′ for 30 cycles of amplification (94°C for 30 sec, 52°C for 30 sec, and 72°C for 1 min), a procedure developed by Weill et al. (2004).

The PCR fragments were digested with AluI restriction enzyme according to the manufacturer’s instructions and fractionated on a 2% agarose gel. The 194 base-pair (bp) fragment amplified by PCR on genomic DNA is cut by the Alu1 restriction enzyme only in resistant mosquitoes. Frequencies of ACE-1 alleles were calculated as described in McAllister et al. (2012).

In order to check the identity of the amplified fragments, sequences were performed directly on PCR products of all 300 mosquitoes using the Big Dye® terminator kit (Applied Biosystems, Carlsbad, CA). The sequencing procedure outlined by Labbe et al. (2007) was followed. Two specific primers: CpEx3dir 5′-CGA CTC GGA CCC ACT CGT-3′ and CpEx3rev 5′-GAC TTG CGA CAC GGT ACT GCA-3′ generated a 457-bp fragment that amplified part of exon 3 of the ACE-1 gene, including position 119.

The concordance between insensitive AChE and the presence of the G119S mutation, as detected by the PCR test and enzyme assay, was 100% (n = 300). This was confirmed by sequencing of the PCR product. The AChE activity measured biochemically in adults is therefore due to the ACE-1 mutation. Genomic DNA amplified a 194-bp fragment that is undigested by Alu1 for susceptible homozygous mosquitoes, and cut into 2 fragments (74 bp and 120 bp) for resistant homozygous individuals. Heterozygous individuals displayed a combined pattern, with 3 fragments (74 bp, 120 bp, and 194 bp).

For the competitive enzyme assay, absorbance values were designated that signified susceptible homozygous, resistant homozygous, and heterozygous individuals by comparing the results to the restriction enzyme and the sequenced product. An absorbance reading of 0–0.44 nm indicated a susceptible homozygous individual, >2.45 nm a resistant homozygous individual, and a reading from 0.45–2.44 nm specified a heterozygous individual.

As seen in Table 1, of the 300 mosquitoes tested, 13 (4%) were resistant homozygous, 80 (27%) were susceptible homozygous, and 207 (69%) were heterozygous. Figure 1 shows absorbance (nm) levels detected in the tested mosquitoes. There was a wide range, 1.9 nm, of absorbance values for the heterozygous mosquitoes with the homozygous mosquito values spanning a range of 0.4 nm.

Competitive biochemical assays help detect specific resistance mechanisms in individual insects and can be used to estimate the frequency of resistance genes in populations. Propoxur is used in this assay to inhibit the activity of the sensitive (i.e., susceptible) AChE, allowing the detection of the altered enzyme when it is present. In resistant mosquitoes, the insecticide fails to inhibit AChE. The number of alleles of insensitive AChE is greater as the yellow color darkens; however, it can be difficult to determine exact cut-off points with the naked eye.

The costs, time, and skill set needed for each test vary significantly. For the enzyme assay, the cost per sample is $0.07 (assuming a 96-well plate); this includes the materials (acetone, ATCH, DTNB, propoxur, and KPO₄ buffer) and the consumables (microtiter plates, 1.5-ml tubes, and pestles). The initial equipment (spectrophotometer and pH meter) costs can range from $5,000 to $30,000. It is important to note that a spectrophotometer is not necessarily needed, as it is possible, using the naked eye, to distinguish roughly between resistant homozygous, heterozygous, and susceptible homozygous individuals by their discrete absorbance classes.

For the molecular assay, the cost per sample is $1.66 (assuming a 96-well plate), which includes the materials (Taq, DNAzol, ladder, primers, dNTPs, agarose, EtBR, ethanol, etc.) and the consumables (PCR plates, cap strips, 1.5-ml tubes, and pestles). The initial equipment (centrifuge, thermal cycler, gel system, transilluminator, and hot water bath) costs are $40,000+.

The enzyme assay (30 mosquitoes per plate) requires 45 min, which includes making the stock chemicals; homogenizing the mosquitoes; loading the plates with homogenate, ATCH, and DNTB; and running the plates on the spectrophotometer. The plates must also be incubated for 24 h in the refrigerator in order to see the necessary color change. In total, the enzyme assay requires 45 min to complete, and a 24-h incubation period. For the molecular assay (96 mosquitoes total), one has to extract the DNA (2 h), run the PCR program (2 h), incubate the PCR product with the restriction enzyme (2–4 h), and run a gel (30–45 min), which totals 6½ to 7¾ h.

Training to conduct both assays is straightforward, but with the molecular assay it is advised that the newly trained individual be evaluated to assure proper technique. All samples are run in triplicate in the enzyme assay, so outlying data points due to pipetting errors or a large piece of homogenate can be disregarded. If an error arises when running the gel or sequencing the PCR product, it is necessary to start the assay over minus the extraction step. Also of importance is that the enzyme assay is not species specific, but can be run on any species of mosquito with no changes in chemicals or procedures. However, it is advised that the absorbance values indicating resistant/susceptible homozygous and heterozygous individuals on each species and on an individual-laboratory basis be determined.

The restriction enzyme assay was devised to detect the ACE-1 mutation in Anopheles gambiae Giles and Cx. pipiens, 2 mosquitoes belonging to different genera. The primers have also been shown to work on An. albimanus Wiedemann and Cx. quinquefasciatus. This indicates that it probably has a broad applicability within the Culicidae family (Weil et al. 2004). The assay has also been adapted to work on An. funestus Giles, An. arabiensis Patton, and An. quadriannulatus (Theobald) with the use of a different set of primers (Djegbe et al. 2011, Yewhalaw et al. 2011). Primers must be developed for each species of interest if not already available.

In conclusion, both assays detected ACE-1 genotypes with 100% agreement. The enzyme assay requires less time by personnel, costs less, employs a simpler technique than the PCR assay, and is not species specific. Either assay would be an invaluable option for any laboratory, and would greatly aid resistance management decisions.