Rapid and precise establishment of homozygous transgenic Anopheles gambiae lines by COPAS sorting

The goal of this work was to evaluate the possibility of performing accurate, fast and high-throughput larval screening and sorting using a flow cytometry machine, the Complex Object Parametric Analyzer and Sorter (COPAS®, Union Biometrica). To this end, the DSX::EGFP transgenic line (thereafter, DSX) [18] was used, as it comprises a combination of fluorescent markers that allow the differentiation between male and female larvae as early as the first instar stage (based on higher expression levels of an EGFP reporter gene in the male midgut), and between heterozygous and homozygous individuals (based on higher expression levels of the selectable DsRed marker in the central nervous system of homozygotes) (Figure 1). To test the efficacy and sensitivity of the COPAS machine to sort different classes of larvae, non-transgenic mosquitoes were crossed to DSX mosquitoes and the resulting F 2 larvae were analysed. Such progeny was expected to segregate into five fluorescence classes according to single gene Mendelian inheritance and sex-specific expression of the GFP marker: 12.5% of homozygous transgenic females, 12.5% of homozygous transgenic males, 25% of heterozygous females, 25% of heterozygous males, and 25% of homozygous non-transgenic males and females (for which no fluorescence-based sex separation was possible). First instar larvae were COPAS-analysed and the number and ratio of larval classes were quantified by COPAS software. First, the area corresponding to live larvae was determined using light extinction and time of flight parameters. Signals beyond this area were identified by microscopy as eggshells and debris from the larval breeding water. The selected area was next analysed by fluorescence and the five expected larval classes were identified (Figure 2). Out of 4,036 larvae screened, 493 (12.2%) showed high green (EGFP-positive) and high red (DsRed-positive) fluorescence (homozygous transgenic males), 520 (12.9%) were low green and high red (homozygous transgenic females), 1,003 (24.9%) were high green and low red (heterozygous transgenic males), 958 (23.7%) were low green and low red (heterozygous transgenic females) and 1,062 (26.3%) were negative for any fluorescence (Figure 2). These values are consistent with the expected percentages (goodness of fit χ2 =6.072, p = 0.19). About 500 larvae from each of the four transgenic populations were sorted separately (in less than 30 min) and the accuracy of sex sorting was verified by visual examination of the resulting adults. All individuals were of the predicted sex.

Figure 1 Sex-specific expression of the Dsx-GFP transgene. A mix of wild-type and heterozygous DSX first instar larvae observed under a fluorescence microscope with a 5x objective. Red fluorescence (upper panel, left) denotes larvae carrying the DsRed transgene (the upper larva shows its dorsal side, the four additional red larvae show their ventral side). Green fluorescence intensity (upper panel, right) allows distinguishing between females (less bright, arrowheads) and males (showing stronger fluorescence). Lower panels: left, transmission image; right; overlay of images on upper panels. Full size image

Figure 2 COPAS-assisted analysis of larval populations. The progeny of DSX/+ mosquitoes, containing non-transgenic as well as heterozygous and homozygous transgenic mosquitoes, was analysed by COPAS. The left diagram (Extinction vs Time of Flight) shows all detected objects. Mosquito larvae were empirically determined to be located inside the gated area (R1). The right diagram (red vs green fluorescence) decomposes the larval population into five categories: non-transgenic (+/+, R6), heterozygous females (XX; DSX/+, R5), heterozygous males (XY; DSX/+, R4), homozygous females (XX; DSX, R3), homozygous males (XY; DSX, R2), as indicated. Full size image

Next, male and female homozygous transgenic adults obtained from the sorting process were crossed and their progeny was screened (again using the COPAS instrument) to search for potential heterozygous individuals arising from inefficient sorting of homozygotes. Heterozygotes were absent, suggesting that the initial sorting of homozygous individuals by COPAS was 100% efficient, thereby permitting the establishment of a homozygous transgenic mosquito line in one generation. These results demonstrate the potential of the COPAS machine for compensating the lack of balancer chromosomes in mosquitoes for selection and maintenance of a desired transgenic genotype, thereby permitting a major gain in time and precision.

COPAS-assisted high-throughput sexing of early mosquito larvae

To test the efficiency and reliability of sorting single-sex mosquito populations, first instar homozygous DSX larvae were analysed with the COPAS machine, which detected two distinct populations of larvae displaying high and low levels of green fluorescence that were expected to correspond to male and female populations, respectively (Figure 3A).

Figure 3 COPAS profile of the homozygous DSX strain and sexual selection. A. About 5,000 freshly hatched larvae of the homozygous DSX line were subjected to COPAS analysis and sorting. The data generated by COPAS was treated with WinMDI software to analyse the results and to express results as artificially colored regions corresponding to males (red) and to females (green) on the fluorescence diagram (on the right). Dot colours are the same as in the time-of-flight vs extinction diagram (left). B. A total of 2,000 larvae of each sex were selected with COPAS and grown to adulthood. Careful visual examination of the cages did not reveal the presence of any adult of the wrong sex. Left panels: male mosquitoes, as identified by the hairy antennae; right panels: female mosquitoes. Bottom panels are close-ups of top panels. Full size image

After sorting, 2,000 larvae of each population were raised separately to the pupal stage and resulting pupae were placed in two different cages. Careful and repeated visual examination of the adults emerged in each cage confirmed that all adults from the larvae displaying high GFP fluorescence were males and all adults from the larvae showing low GFP fluorescence were females (Figure 3B).

In an independent experiment, groups of 150 larvae were recovered in four successive passages of the same individuals through the COPAS machine, sorting for male and for female larvae successively. These larvae were raised to adulthood and the sex of each mosquito verified. Again, visual examination confirmed the 100% accuracy of sorting at each of the four passages; none of the sorted groups contained an adult of the wrong sex. Importantly, the number of larvae from the fourth sorting that developed to adulthood (>100 individuals) was similar to the number of larvae that underwent 0, 1, 2 or 3 COPAS treatments (Table 1). These results suggest that the survival rate of COPAS-sorted larvae was not affected by the sorting procedure itself repeated up to three times.

Table 1 Efficiency of COPAS sorting and survival of sorted larvae Full size table

COPAS sorting does not impair male mating competitiveness

The next step was to examine whether COPAS treatment negatively affected fitness and reproductive capability of sorted male insects. To this end, the reproductive success of males raised in standard conditions was compared to that of males that had passed three times through the COPAS machine. Two crosses were assembled in separate cages. Each cage contained 100 non-fluorescent, wild-type virgin females, 100 non-fluorescent competitor males, and 100 DSX transgenic males that were either untreated or had been sorted three times with COPAS at the early larval stage. Freshly emerged mosquitoes of each kind were simultaneously placed in the two cages and kept together for five days. Females received a blood meal on day 5, and on day 8 they were isolated into single plastic tubes to oviposit on shallow water. Freshly hatched larvae from individual females were examined under the fluorescence microscope to score the identity of their father. No significant difference was detected in the number of progeny fathered by the COPAS-treated vs control DSX males (Table 2), suggesting that COPAS sorting does not impair male competitiveness. Note that these experiments revealed a significant proportion of females fertilized by more than one male (15 progenies, out of 80 analysed, arose from females inseminated by at least two males of different genotypes, suggesting an overall rate of multiple matings in these experiments of at least 37.5%). This result is in agreement with previous laboratory-based reports showing that multiple inseminations occur in crowded cages [23], possibly due to repeated female exposure to males in the first 24 h after mating before the mechanisms of refractoriness to further matings are fully activated.

Table 2 Effect of COPAS on male reproductive competitiveness Full size table

Three additional independent repeats of this experiment were performed with smaller mosquito numbers (45–60 mosquitoes for each group). These confirmed the absence of significant loss of fitness and mating competitiveness for males that passed through the machine compared to control males of the same genotype (data not shown). These results suggest that repeated COPAS sorting does not confer any obvious mating disadvantage to the males, at least in laboratory conditions.

Isolation of non-transgenic male-only populations from transgenic colonies

For certain types of vector control interventions (release of sterile males for non-transgenic SIT or release of selected natural disease-resistance traits), it will be desirable to obtain large populations of non-transgenic mosquito males. This would be essential in all instances where the release of transgenic insects may not be possible for regulatory reasons. A simple COPAS-based strategy was designed to obtain a large, non-transgenic, male-only population based on the inheritance of an X-linked gene in the F 1 generation. This strategy made use of a newly established transgenic mosquito line, the FKX line, carrying a GFP-expressing transgene on the X chromosome (see Methods). A total of 120 FKX males were crossed to 200 non-transgenic virgin females. In the F 1 progeny, all males inherit their fathers' Y chromosome and are non-transgenic, while all females inherit a copy of the GFP-expressing X chromosome. Taking advantage of this property, the COPAS sorter was set to select only the non-fluorescent F 1 male larvae (Figure 4A). For quality control, the sorted individuals were immediately re-analysed by the COPAS. All objects fell in the non-fluorescent region of the diagram, confirming the absence of any contaminating transgenic female larvae (Figure 4B). Visual examination of the resulting cage of adult males confirmed their purity. Therefore, the use of COPAS allows the selection of non-transgenic, male-only larvae from a cross between non-transgenic females and males from an X-linked transgenic line. The limiting step of this procedure for high-throughput applications is the separation of female-only wild type individuals for the crosses: this limitation would be overcome by the generation of Y-linked transgenic lines allowing to sort (again by the use of fluorescence-assisted sorting) large numbers of non-transgenic virgin females from their transgenic siblings.