Plasmids were generated by standard molecular biology techniques including HiFi assembly (New England BioLabs). All plasmids were prepared for microinjection using the NucleoBond Xtra Midiprep kit EF (Machery-Nagel) and confirmed by Sanger sequencing. Sequences of the plasmids designed for this manuscript and of the transgenes of the transgenic lines used are deposited into GenBank, and the accession numbers are detailed in Supplementary Table S1.

Aedes aegypti mosquitoes (Liverpool wild-type strain and transgenics derived from them) were reared at 28°C and 70% relative humidity with a 14:10 h light:dark cycle. Adults were kept in BugDorm cages with cotton wool pads soaked with 10% sucrose solution. Transgenic lines were maintained in hemizygous state by crossing transgenic males with wild-type females at a 1:2 male:female ratio. From day 5–7 post-eclosure, females were blood fed once every 5 days, three times, to collect three ovipositions. Defibrinated horse blood (TCS) was offered until most of the females were engorged using a Hemotek membrane feeding system (Hemotek Ltd.) covered with hog gut and an over-layer of parafilm. Two days after blood meal, egg cups consisting of 1 oz clear polypropylene portion pots with a strip of coffee filter paper wrapped inside and half filled with reverse osmosis water, were added to the cages to allow egg laying. The egg cups were removed 5 days later, and egg papers hatched when required. Eggs were vacuum hatched for 2 h in 50 ml solution of reverse osmosis water with LiquiFry N° 1 (Interpet) (500 ml of water +6 drops of LiquiFry). The larvae were then transferred to a new tray with reverse osmosis water and fed TetraMin ornamental Fish Flakes ad libitum. Transgenic mosquitoes were identified in larval or pupal stages based on their fluorescent marker profile using an MZ10F stereomicroscope (Leica Microsystems) equipped with suitable filters. Mosquitoes were sexed under a stereomicroscope at the pupal stage according to the shape of the genital lobe.

All mosquito procedures were reviewed and approved by the Biological Agents and Genetic Modification Safety Committee (BAGMSC) at The Pirbright Institute.

Cages with approximately 1,000 5–7 days post-eclosure wild-type females mated with 500 wild-type males were blood fed as described above in the general mosquito maintenance section. In order to collect embryos within 1 hour of oviposition for microinjection, no substrate for oviposition was provided. Five to 7 days after the blood meal, 15–20 females were transferred into a Drosophila tube with damp cotton wool covered by a layer of filter paper and kept for 20–30 min in the dark to promote oviposition. White eggs were lined up side by side in the same orientation on a piece of filter paper and then transferred onto a cover slip with Scotch Double Sided Tape 665 (3M). Eggs were covered with halocarbon oil 27 (Sigma Aldrich) to prevent desiccation before microinjection in their posterior end. Injections were performed using Sutter quartz needles (1.0 mm external diameter 0.70 mm internal diameter needles with filaments) drawn out on a Sutter P-2000 laser micropipette puller (Sutter Instrument) with the following program: Heat = 729, FIL = 4, VEL = 40, DEL = 128, PUL = 134, LINE = 1. Injections were carried out using a standard microinjection station equipped with a FemtoJet 4x microinjector (Eppendorf). The injection mix contained 500 ng/μl of the plasmid intended for insertion, 300 ng/μl of helper plasmid AePUb-hyperactive piggyBac transposase (Anderson et al., 2022) for the generation of transgenic lines or AePUb-PhiC31 (this manuscript) for PhiC31 mediated integration, 1x Injection buffer (Coates et al., 1998) and endotoxin-free water.

After microinjection, the eggs were washed with water and transferred onto damp filter paper in a moist chamber. Five days after injection the eggs were vacuum hatched as described for general maintenance. The collected pupae were sexed under a stereomicroscope. Each Gen₀ male was single crossed with 5 wild-type females, and the individuals were pooled in groups of 3–5 single crosses after 48 h. Gen₀ females were pooled in groups of 4–5 females and 8–10 wild-type males. The cages were fed with 10% sucrose, blood fed every 5–6 days and 5 ovipositions were collected in total. The Gen₁ egg papers were hatched as previously described, and the 3rd-4th instar larvae were screened for the presence of the transformation marker. Single positive Gen₁ individuals were crossed with wild-type counterparts and maintained as described above.

The number of transgene insertions and sex-linkage of the constructs were assessed according to standard rearing procedures by crossing transgenic males to wild-type females and quantifying the number of males and females of wild-type and transgenic phenotypes, and comparing them to the expected frequencies for an autosomal single insertion (i.e. 50% males and 50% females, 50% transgenic and 50% wild-type). Flanking PCR was also used (see protocol in the Molecular analysis section). Only lines with single insertions were used in experiments. Information regarding the insertion site of the lines used in these experiments is detailed in Supplementary Table S2.

50 females and 25/50 males of the corresponding mosquito lines (wild-type or transgenics) were pooled together according to the experiment (see Results section) and maintained as described above. For screening, 3 replicates of 300 eggs each were hatched and kept in trays with 1.5 l of reverse osmosis water and fed with TetraMin ornamental Fish Flakes ad libitum. This was done to ensure optimal developmental conditions for all the genotypes. Individuals were screened in larval or pupae stage and photographed when needed using a DFC7000 T camera (Leica Microsystems).

Chi square tests of independence were used in crosses involving the tPUb-Cre and Shu-Cre lines to study the egg to pupa survival of the double hemizygous, both single hemizygous and wild-type individuals of the different crosses. The observed values were calculated as an average of those observed in the three replicates. For individuals carrying a construct susceptible to recombination, the values of the non-recombined and recombined phenotypes were added. The expected values were calculated considering a normal segregation of the constructs and full survival of the phenotype, i.e., 25% of the average of the total number of individuals recovered per replicate.

Student’s t-tests were carried out to compare number of individuals per genotype between crosses of the Shu-Cre line.

All tests were carried out using R (ver 4.2.2) (R Core Team, 2022) in RStudio 2022.12.0 + 353, with the MASS (Venables and Ripley, 2002), rstatix (Kassambara, 2023) and dplyr (Wickham et al., 2023)

Genomic DNA was extracted from larvae or pupae of the selected lines using NucleoSpin Tissue gDNA extraction kit (Macherey-Nagel).

Flanking PCR was performed according to previously reported methods (Liu and Chen, 2007; Martins et al., 2012). Genomic DNA was digested using enzymes DpnII, MspI and NcoI (New England Biolabs) and PCRs were performed with DreamTaq (Thermo Fisher Scientific). Adaptors were built with primers 6,770 (5′-GTG​TAG​CGT​GAA​GAC​GAC​AGA​AAG​GGC​GTG​GTG​CGG​AGG​GCGGT​G-3′) and 6,776 (5′-GAT​CCA​CCG​CCC​TCC​G-3′) for DpnII, 6,770 and 6,775 (5′-CGC​ACC​GCC​CTC​CG-3′) for MspI, and 6,770 and 6,789 (5′-CAT​GCA​CCG​CCC​TCC​G-3′) for NcoI. The primary PCR used primers 6,774 (5′- GTG​TAG​CGT​GAA​GAC​GAC​AGA​A-3′) and 6,772 (5′-CAG​TGA​CAC​TTA​CCG​CAT​TGA​CAA​G-3′) to amplify the 5′ insertion site, and 6,774 and 6,773 (5′- CAG​ACC​GAT​AAA​ACA​CAT​GCG​TCA-3′) to amplify the 3′ insertion site. The nested PCR used primers 6,774 and 6,771 (5′-GGC​GAC​TGA​GAT​GTC​CTA​AAT​GCA​C-3′) to amplify the 5′ insertion site, and 6,774 and 6,787 (5′-ACG​CAT​GAT​TAT​CTT​TAA​CGT​ACG-3′) to amplify the 3′insertion site.

PCR reactions for the other experiments were performed in a final volume of 100 μl using 50–150 ng of template, 5 μl of primer forward 10 μM, 5 μl of primer reverse 10 μM, 50 μl of Q5 High-Fidelity 2x Master Mix (New England Biolabs) and water.

To test for the removal of internal sequences for interaction analyses, primers 6,676 (5′-TGT​GCA​GTC​GGT​TAG​TTG​GGA​AAG​G-3′) and 5,038 (5′-GGC​CAT​TGT​GAC​TTT​GAA​GGT​GGA​GG-3′) were used. Cycling conditions included an initial denaturation step at 98° for 30 s, 35 cycles of 98° 10 s, 72° 1 min 35 s, and a final elongation at 72° for 2 min. A fragment of 3,135 bp was expected if the internal sequence was present, and of 350 bp if the sequence was absent.

To test for the removal of backbone sequences, primers 5,842 (5′-CAG​ACA​TGA​TAA​GAT​ACA​TTG​ATG-3′) and 6,792 (5′-AAT​GAC​ATC​ATC​CAC​TGA​TCG-3′) were used. Cycling conditions included an initial denaturation step at 98° for 30 s, 35 cycles of 98° 10 s, 59° 15 s and 72° 2 min 45 s, and a final elongation at 72° for 2 min. A fragment of 5,281 bp was expected if the backbone sequence was present, and of 578 bp if the sequence was absent.

To test for correct PhiC31 integrase mediated integration, primer pair 6,707 (5′-TCG​GTC​TGT​ATA​TCG​AGG​TTT​AT-3′) and 6,799 (5′-CCC​TTC​ACG​GTG​AAG​TAG​TG-3′) was used to amplify the attL region, and primer pair 6,795 (5′-GCA​CAA​GCT​GGA​GTA​CAA​CTA-3′) and 6,791 (5′-CCA​GTT​CGG​TTA​TGA​GCC​GT-3′) was used to amplify the attR region. Cycling conditions included an initial denaturation step at 98° for 30 s, 35 cycles of 98° 10 s, 63° 15 s and 72° 45 s, and a final elongation at 72° for 2 min. Fragments of 1,101 bp and 1,393 bp were expected for attL and attR, respectively.

PCR products were run in a 1% agarose gel with SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific) and photographed using a Bio-Rad Gel Doc imaging system. As clear single bands were observed in all cases, the remaining PCR product was purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and Sanger sequenced using both forward and reverse primers.

We generated transgenic mosquito lines expressing Cre recombinase either in a broad range of tissues using a truncation of the Polyubiquitin gene promoter (AAEL003888, Bartholomeeusen et al., 2018) (tPUb-Cre, Figure 1B) or the germline specific shut-down gene promoter (from OP823146, Anderson et al., 2023) (Shu-Cre, Figure 1C). The Cre coding sequence used was modified for expression in Ae. aegypti by adjusting codon usage to avoid rare codons (“codon-optimised”). We tested these lines by crossing them to different transgenic reporter lines.

To analyse these lines for their somatic effect, we generated a reporter mosquito line carrying the construct PUb-loxP-mCh-stop-loxP-AmC (Figure 2A). These individuals express the red fluorophore mCherry over the whole body; after Cre mediated recombination, the mCherry open reading frame should be removed and the cyan fluorophore AmCyan expressed instead (Figure 2A, Supplementary Figure S1).

We screened the F₁ progeny of crosses between hemizygous tPUb-Cre or Shu-Cre individuals with hemizygous counterparts from the reporter line. The three transgenic lines have their own transformation marker making it possible to identify all the genotypes of the F₁: yellow body for tPUb-Cre, blue optic nerves for Shu-Cre, and red optic nerves for the reporter (Figures 1A, B, 2A).

For both types of crosses, all double hemizygous F₁ individuals expressed mCherry and AmCyan over the whole body (Figures 2B–H), indicating that the removal of the loxP-mCh-stop-loxP section (leaving one loxP sequence behind) occurred in many cells. Whole body expression of Cre in the tPUb-Cre line was expected due to the nature of the promoter. In the Shu-Cre line, although expression of shut-down is germline-specific, the promoter fragment used was known to provide some somatic expression as well (Anderson et al., 2023), which was now visually corroborated.

To analyse the lines for their germline effect we generated a reporter mosquito line carrying the construct loxN-R-loxP-loxN-Y-loxP (Figure 3, Supplementary Figure S2). These mosquitoes express a red body marker from fragment R, and a yellow optic nerves marker from fragment Y. As lines tPUb-Cre and Shu-Cre have yellow body and blue optic nerves markers respectively, all the genotypes deriving from the crosses could be identified (see example in Supplementary Figure S3). The reporter line includes other sequences that are not relevant for this study (Supplementary Figure S2), and hence their effect is not addressed. The use of two overlapping pairs of incompatible lox sites—loxP and loxN—means that recombination between one pair removes one of the other pair, thus preventing recombination based on that site. Recombination therefore has two possible stable outcomes i) recombination between loxP sites deleting segment Y, retaining segment R and therefore red fluorescence; ii) recombination between loxN sites deleting segment R, retaining segment Y and therefore yellow fluorescence.

We first screened the F₁ progeny of crosses between tPUb-Cre or Shu-Cre with the reporter line. In order to address any possible sex specific effect, both reciprocal crosses were performed for line tPUb-Cre, but as line Shu-Cre is linked to the m allele of the M/m male determining locus (“m-linked”), only Shu-Cre females were used. Double hemizygous F₁ males and females were subsequently crossed to wild-type counterparts, the F₂ was screened, and their phenotypes were assessed for recombination events.

The results for line tPUb-Cre are summarised in Figure 4A and detailed in Supplementary Tables S3–S6. The results for line Shu-Cre are summarised in Figure 4B and detailed in Supplementary Tables S7, S8. The analysis of the segregation of the tPUb-Cre and loxN-R-loxP-loxN-Y-loxP constructs (adding up recombined and non-recombined versions of the latter), showed no statistically significant differences with the expected frequencies (25% each—double hemizygous, single hemizygous for each insertion and wild-type) (Chi-square test of independence: p > 0.05). This suggests no substantial lethal effect of the recombinase line or the recombined fragments (power analysis: N > 250, β > 0.9, ω < 0.3 for all crosses). Crosses with the tPUb-Cre line produced only one recombinant individual for one of the two possible phenotypes (red body from red body or yellow optic nerves). This indicated little or no expression—or function, but see below—of Cre in the germline of this strain, likely due to the PUb promoter fragment used in this line.

Crosses with the Shu-Cre line generated individuals of each of the two possible recombined phenotypes. Analysis of the observed frequencies of the segregating constructs found no evidence for lethal effect for any of the genotypes (Chi-square test of independence: p > 0.05), and expression of Cre recombinase in the germline. A linear model was constructed to compare the number of individuals per phenotype between crosses, and no differences were observed (Student’s t-test: p > 0.05, Supplementary Table S9). A power analysis indicates that there is no evidence for a difference in efficiency of the Cre recombinase between the male and female germline (β = 0.9, f = 0.2).

The efficiency of the Cre lines in the germline, calculated as the percentage of individuals carrying a recombined construct (R + Cre, Y + Cre, R and Y) was 0.16% for line tPUb-Cre and between 15.4% and 17.8% for the Shu-Cre line.

As the Shu-Cre line was found to be highly efficient at inducing recombination in the germline (15.4%–17.8% efficiency), we further analysed it using other lines containing lox sites.