Content area
The New World screwworm is an obligate parasitic fly and a significant economic pest of livestock in the Americas. Although eradicated from the USA using the Sterile Insect Technique (SIT), enhancing SIT efficiency remains a priority. A promising approach involves conditional female-lethal genetic strains that produce only males in the absence of tetracycline, ideally eliminating females early in development to reduce larval diet costs. However, while some strains match wild-type production levels, lower male fitness reduces the net benefit of replacing the current wild-type strain with one of these genetic-sexing strains. This study aimed to improve strain performance through female-specific expression of both the driver and effector components of the lethality system. We tested four transgenic strains using early embryo-specific promoters from the Chhalo and g6451 genes. Strains with the Chhalo promoter driving tTA expression exhibited early-stage female lethality under a modified doxycycline regimen but suffered from reduced male fitness. In contrast, one strain with the g6451 promoter produced males with excellent fitness but female lethality occurred at the late pupal stage. Despite imperfect female lethality timing, the overall fitness characteristics of this strain makes it a good candidate for future sterile or fertile male release genetic control programs.
Introduction
Cochliomyia hominivorax, commonly known as the New World screwworm, is a destructive ectoparasite that poses a significant threat to livestock industries, particularly in tropical and subtropical regions of the Americas1,2. The larvae of this fly infest the wounds of warm-blooded animals, including cattle, and can lead to severe infections, reduced productivity, and even death if left untreated. The sterile insect technique (SIT) was developed to control screwworm populations in the U.S.A3, 4–5. SIT involves the mass release of sterilized males and females over the targeted area. As matings of fertile females with sterile males produce no offspring, repeated releases of sterile males lead to the decline in the local population. Starting in the 1950s in Florida, the SIT was successfully used to progressively eradicate screwworm from the U.S.A, Mexico and Central America. Screwworm remains a major livestock pest in most of South America and the large Caribbean islands6. In 2023 screwworm cases were reported in Panama and Costa Rica and were widespread in Central America and southern Mexico in 20257,8.
The efficiency of genetic suppression is increased if only sterile males are released as the co-released sterile females compete with fertile females for matings9. Consequently, we have developed genetically engineered screwworm strains that carry conditional female lethal genetic systems10. These “male-only” strains are designed to ensure that only sterile males are released into the environment. The genetic mechanism behind these lines involves the tetracycline transactivator (tTA), a synthetic transcription activator11. The binding of tTA to its DNA binding site (tetO) is inhibited by the presence of tetracycline in the diet. This provides a simple off-switch for keeping females alive during mass rearing.
In the initial system that was developed, the tTA coding region was interrupted with the sex-specific intron from the C. hominovorax transformer (Chtra) gene12. Sex-specific RNA splicing ensures that only the female transcript encoded tTA protein13. The tTA gene was driven by a simple enhancer-promoter consisting of multiple copies of tetO and a core promoter from a Lucilia cuprina hsp70 gene14. In this autoregulatory system, binding of tTA to tetO led to overexpression of tTA, which caused lethality likely due to transcriptional squelching and/or interference with the ubiquitin protein degradation system15. Although strains performance characteristics were comparable to the production strain (J-06), females did not die until the late larval/pupal stages13.
Larval diet costs would be significantly reduced less if females died earlier in development at the embryo or first instar stages13. Consequently, we developed a two-component system where tTA was expressed early in embryogenesis using a promoter from a cellularization gene10. Similar strains have been developed for tephritid fruit flies16,17. The initial tTA driver construct, DR2, used the promoter from the L. sericata bottleneck gene18. The second component was the L. sericata hid cell death gene19 driven by tetO-hsp70 enhancer-promoter. Only female embryos died as the Lshid gene contained the Chtra intron. While the two component strains produced 100% males on diet without tetracycline and had rearing metrics (e.g. egg hatch, pupal weight) similar to J-06 on diet with tetracycline10, transgenic males were outcompeted by J-06 males for matings with J-06 females20. As the DR2 driver showed some tTA expression in pupae, we next developed two component strains with the DR6 and DR7 tTA drivers, which were more embryo-specific in L. cuprina21. However, neither DR6 nor DR7 two component strains were an improvement over the strains assembled with the DR2 driver20. Males from the DR6 and DR7 two component strains were outcompeted by J-06 males and male fly output on diet without doxycycline was significantly less than the production strain.
We considered that one possibility for the reduced fitness of two component males is that tTA is expressed equally in males and females. While expression was highest in early embryos, significant expression was detected at later stages, particularly in pupae. Since high levels of tTA are toxic13, we hypothesized that the expression of tTA in males could be reducing their fitness. The aim of this study was to create and evaluate new tTA driver lines that include the Chtra intron and consequently only females should make the tTA protein. A second aim was to evaluate promoters from the Chhalo and g6451 genes that were previously identified to have high expression in very early embryos (0-1 h) but little expression at other stages22. Chhalo is the ortholog of the D. melanogaster halo gene, which is expressed in the early zygote and is essential for cellularization23. The Chhalo, g6450 and g6451 genes are closely linked in the genome, show similar developmental expression profiles and encode proteins with high similarity (about 90%)22. Early embryo-specific gene promoters would be ideal for assembly of two component male-only strains in C. hominivorax.
Results
Developmental expression and alternative splicing of tTA transcripts in DR9 and DR10 driver lines
The DR9 and DR10 driver constructs contain the predicted Chhalo and g6451 gene promoters upstream the tTAv coding region, respectively (Fig. 1A). tTAv is a variant with a nucleotide sequence that was optimized for expression in Drosophila15.The promoter fragments (1495 bp for Chhalo and 1134 bp for g6451) were synthesized based on the genome sequence and contain the entire 5’UTR and upstream DNA. The sex-specific intron from Chtra was inserted within the tTAv coding region immediately after the translation start codon for tTA, as in the tTAv overexpression constructs evaluated previously13. The vectors contain a constitutively expressed ZsGreen gene to identify transgenics24 and the 5’ and 3’ ends of the piggyBac transposon (Fig. 1A). Four DR9 and three DR10 lines were obtained by piggyBac-mediated germ-line transformation. One DR9 line could not be bred to homozygosity and was discarded. The remaining lines were initially assessed by crossing with an EF1#6 effector line10 on standard diet. If the driver line was effective, the expectation was that few female offspring would be obtained. We found that several combinations produced few, if any, females (Table S1). Based on this assessment, two autosomal DR9 lines, #3-2 and #4-1, and two autosomal DR10 lines, #4-3 and #5A were selected for further analysis.
Fig. 1 [Images not available. See PDF.]
Developmental gene expression in the DR9 and DR10 lines. (a) Schematic of the gene constructs. In the DR9 construct, tTA is driven by the promoter from the Chhalo gene, while for DR10 construct the promoter from the g6451 gene was used. The tTA coding region is interrupted by the sex specific intron from the Chtra gene in both constructs (schematized with the small triangle in yellow). ZsGreen is constitutively driving by Lchsp83. In the effector construct EF1, there are 21 copies of tetO upstream of the core promoter from the L. cuprina hsp70 gene. The promoter is driving expression of the proapoptotic gene hid from L. sericata (b-e) RT-PCR analyses of RNA isolated from 0–1 (E1H), 2–3 (E2H) and 4–6 h (E6H) embryos, wandering third instar larvae (L3) and pupae (P2 stage 48 h). (b,c) Chhalo and tTA expression in the DR9 lines. (d,e) g6451 and tTA expression in the DR10 lines. The ChGAPDH gene was used as a positive internal control. For images of the complete agarose gels refer to Figures S1 and S2 in the supplementary material.
An RT-PCR analysis was performed with RNA isolated from mixed sex pre-cellular embryos (1 h), embryos at cellularization (2–4 h), and after cellularization (5-6 h), third instar larvae and pupae. As expected, transcripts from the endogenous Chhalo gene were only detected in embryos, although there were some differences between the DR9 lines (Fig. 1 B, C, S1). In the DR9#3-2 line there was significant expression in the earliest collection (1 h) while in DR9#4-1 expression was highest in 2 h embryos, which likely reflects differences in timing of embryo collections. In both DR9 lines, tTAv transcripts were detected in all embryo samples. For the DR9#4-1 line, tTAv RNA levels were highest in the 2 h embryo sample, similar to endogenous Chhalo RNA. At later stages, tTAv was detected in larvae and pupae from DR9#3-2 but not DR9#4-1. In the DR10 lines, g6451 transcripts were detected in embryos as expected from the previous developmental RNAseq analysis22. However, unexpectedly, g6451 RNA was also detected in the larval and pupal samples, indicating the gene is not embryo-specific. Indeed, tTAv transcripts in the DR10 lines were detected at all developmental stages (Fig. 1 D, E, S2). The presence of tTAv RNA in the earliest embryo collections (1 h) suggests that either the promoters are active in precellular embryos or that there is significant maternal expression and deposition of tTAv into developing eggs. Consequently, we next performed RT-PCR analysis of RNA isolated from ovaries of adult females. tTAv RNA was not detected in ovaries from either DR9 line (Fig. S3). However, both DR10 lines showed expression in ovaries, suggesting that maternal deposition is contributing to the RNAs detected in early embryos.
If the tTAv transcripts are correctly spliced, only female embryos in the DR9 and DR10 lines will produce a transcript that codes for tTA protein. To determine if tTAv transcripts were alternatively spliced, we next performed RT-PCR analysis using a forward primer from within the 5’UTR and a reverse primer in the tTAv coding sequence. RNA was isolated from mixed sex embryos from the DR9#3-2B and DR10#5A lines. Amplification products were size separated, cloned and the nucleotide sequences determined of cloned fragments. Three RT-PCR products were obtained from DR9#3-2B embryo RNA (Fig. 2). The smallest corresponded to precise excision of the Chtra intron, as would be expected in females. The largest product retained the male exon. The intermediate product showed excision of a small intron from within the male exon. Although we had not previously detected additional splicing of Chtra transcripts in males12, two male transcripts were found in L. cuprina with one showing similar splicing of a small intron from the male exon25. Two predominant RT-PCR products were obtained from DR10#5A RNA (Fig. 3). The smaller product showed precise excision of the Chtra intron and the larger retention of the male exon. Both products showed evidence of splicing in the g6451 promoter fragment, indicating the presence of an unannotated intron in the 5’UTR of the g6451 gene.
Fig. 2 [Images not available. See PDF.]
Sex specific splicing of tTAv transcripts in the DR9#3-2B line. (a) Excision of the Chtra intron in females is expected to produce a 639 bp RT-PCR product. (b) Retention of the male exon should produce a 933 bp product in males. (c) DNA fragments obtained after amplification of RNA from mixed sex embryos at 2 h. Green, blue and yellow arrowheads indicate the 639 and 933 bp fragments, respectively. The full gel image is shown. Alignments of DNA sequences of cloned RT-PCR products supported these assignments (a,b).
Fig. 3 [Images not available. See PDF.]
Sex specific splicing of tTAv transcripts in the DR10#5A line. Alignment of transcript sequences identified an intron in the 5’UTR of the g6451 gene promoter fragment that was not annotated in the assembly22 (a) Excision of the Chtra intron in females is expected to produce a 526 bp RT-PCR product. (b) Retention of the male exon should produce a 820 bp product in males. (c) DNA fragments obtained after amplification of RNA from mixed sex embryos. Green and yellow arrowheads indicate the 526 and 820 bp fragments, respectively. The full gel image is shown. Alignments of DNA sequences of cloned RT-PCR products supported these assignments (a,b).
Embryo and female lethality of the homozygous double-component of DR9 and DR10
The DR9 and DR10 lines were crossed with the EF1#6 line, which carries a constitutively expressed DsRed marker gene10. Lines that were double homozygous (DH) for driver and effector were obtained by selecting for larvae with bright red and green fluorescence. The DH strains were maintained on diet containing 25 μg/mL doxycycline rather than tetracycline as it is more cost effective and has higher bioavailability26. To determine if the DH strains produced only males on diet without doxycycline, we initially followed the same antibiotic feeding regimen as previously with DH strains maintained on tetracycline10. With this regimen, doxycycline was omitted from the paternal adult diet and larval diet of offspring. The control cages were maintained continuously on diet with doxycycline. The number of embryos that hatched (around 8 h) and survived to the first larval stage (24 h), second larval stage (48 h) and third larval stage (72 h) were counted (Table S2). On diet with doxycycline, the DH strains were generally comparable to the parental J-06 strain with some exceptions such as DR10#4-3xEF1#6 at hatching and first instar (Fig. 4). On diet without doxycycline, the expectation was that DH strain females would die in the embryo or L1 stages, which would have reduced overall survival. However, there was no significant decrease in survival on diet without doxycycline compared to diet with doxycycline at the early developmental stages (0, 24, 48 h), with the exception of DR9#3-2BxEF1#16 at 24 h. At the late larval stage (72 h), DH strains DR9#3-2BxEF1#6 and DR10#5AxEF1#6 showed a significant decrease in survival. There does not appear to be a significant decrease in the number of embryos that hatched on diet without doxycycline. We recently obtained similar results with DH strains assembled using DR6 and DR7 driver lines20. Apparently, there is enough maternal transfer of doxycycline taken up during larval development to suppress the female lethal system. Thus, we next performed a variation in the feeding protocol by removing doxycycline earlier, the parental third larval stage (Δ-Dox in L3). In the standard rearing protocol for C. hominivorax, the third larval feeding is the largest with more diet provided than at earlier stages20,27. The DR10#5AxEF1#6 strain could not be assessed under the modified feeding regimen as adults were not produced when doxycycline was omitted from the L3 diet. Without doxycycline, both DR9 DH strains showed a reduction of about half the number the embryos that hatched, suggesting that females were dying before first instar larvae (Fig. 4B, Table S3). In contrast, for the DR10#4-3xEF1#6 strain there was no decrease in survival at any stage without doxycycline in the diet. For mass rearing, productivity on diet with doxycycline is important. The survival of the DR9#3-2BxEF1#6 strain on doxycycline was comparable to J-06 at all stages (Fig. 4). Survival of the DR10 strains on doxycycline was similar to J-06 at most larval stages but the DR9#4-1xEF1#6 strain showed reduced survival at all stages. (Fig. 4B).
Fig. 4 [Images not available. See PDF.]
Comparison of two doxycycline feeding schedules on the lethality of two component strains. Percentage survival at 8, 24, 48 and 72 h after hatching is shown. The comparative analysis of average survival was carried out between each stage of development. (a) Standard doxycycline feeding protocol for embryo lethal strains. + D = 25 μg/mL doxycycline added to adult and larval diets. − D = Doxycycline is omitted from the parental adult diet and larval diet of offspring. Only the DR10#5AxEF1#16 strain shows significantly reduced viability on the − D regimen. Although DR9#3xEF#1 at − D regimen shows significant differences at 8, 24 and 72 h after hatching, the percentage of mortality does not correspond to the expected 50% if females had died. (b). Survival of embryos and larvae in the modified feeding protocol. Δ + D 25 μg/mL doxycycline added to adult and larval diets. Δ-D Doxycycline is omitted from the parental third larval and adult diets and the larval diet of offspring. The DR9#3xEF1#16 strain shows significantly reduced viability on the Δ-D regimen. J-06 is the parental control colony (black bar). The solid bars correspond to the DR9 and DR10 double component strains with doxycycline and the striped bars without doxycycline. Statistical differences vs J-06 are indicated with letters.
Having identified doxycycline feeding conditions that reduced larval viability, we next determined if lethality was sex-specific by counting the number of males and females that emerged from pupae (Fig. 5, Table S4). On diet with doxycycline, all DH strains produced an equal number of males and females. When doxycycline was omitted from the parental adult diet (i.e. standard regimen, − D), the DR9#3-2BxEF1#6 and DR10#4-3xEF1#6 strains produced mostly males and the DR10#5AxEF1#6 strain produced only males. This suggests that the decrease in survival at 72 h observed with the DR9#3-2BxEF1#6 and DR10#5AxEF1#6 strains (Fig. 4A), was due to female lethality. However, the high proportion of non-eclosed pupae for DR9#3-2BxEF1#6 suggests that some females were not dying until the pupal stage. For the DR10#4-3xEF1#6 strain, almost half the pupae did not eclose suggesting females were not dying until the pupal stage. The few females that survived on a standard diet showed developmental abnormalities and were sterile (Fig. S4).
Fig. 5 [Images not available. See PDF.]
Female-specific lethality of DR9 and DR10 double component strains. The percentage of pupae that emerged as males, females or did not emerge (NE) are shown. + D and Δ + D, diet with 25 μg/mL doxycycline at all stages. − D, doxycycline omitted from parental adult diet and larval offspring diet. Δ-D, doxycycline omitted from parental L3 and adult diets and larval offspring diet. Strains reared in the absence of doxycycline show significant differences between sexes (p-value < 0.001, Wilcoxon test).
On the modified feeding regimen where doxycycline was omitted from the parental L3 and adult diets (Δ-D), both DR9 DH strains produced only males and the DR10#4-3xEF1#6 strain produced mostly males. As with the standard regimen, the high proportion of non-eclosed pupae indicates that females of the DR10#4-3xEF1#6 were not dying until the pupal stage.
Mating performance and longevity
We next evaluated male fitness parameters that could be important indicators for strain performance in the field. We first measured male mating success. In this assay, transgenic males are placed in a cage with an excess of virgin females for a limited time. If few females mate with transgenic males, then this would indicate that the males have poor mating performance. Males were obtained from the DH strains reared under the standard (-D) or modified (Δ-D) doxycycline feeding regimen. Mating success for males from the DR9#3-2BxEF1#6 and DR10#4-3xEF1#6 strains was similar to J-06 males (Fig. 6A, Table S5). The mating success for males from the other two DH strains, DR9#4-1xEF1#6 and DR10#5AxEF1#6, was significantly less than J-06 (p-value < 0,05 T-test). Males were next evaluated in competition experiments where an equal number of transgenic males and J-06 males are placed in a cage with the same number of J-06 virgin females (i.e. a 1:1:1 ratio). After mating for a limited time, females were removed and mating choice assessed by examining the fluorescence of larval offspring. The mating competitiveness index (MCI) was calculated as the number mated to transgenic divided by the total number of females who mated (Table S6). Males from the DR10#4-3xEF1#6 strain obtained by either doxycycline feeding regimen were fully competitive with J-06 males (Fig. 6B). With males from the DR9#4-1xEF1#6 strain, the preference for J-06 males was borderline statistically significant (MCI = 0.37, 95% CI 0.249–0.496, p = 0.051). Males from the other DH strains, DR9#3-2BxEF1#6 and DR10#5AxEF1#6, were outcompeted by J-06 males. We next measured the longevity of males from the transgenic strains reared under the standard or modified doxycycline feeding regimen. With the exception of males from the DR9#3-2BxEF1#6 strain, male longevity was comparable to J-06 (Fig. 6C). DR9#3-2BxEF1#6 males reared on either doxycycline feeding regimen showed a significant decrease in longevity (p-value < 0.001).
Fig. 6 [Images not available. See PDF.]
Mating success, competitiveness, and longevity of double component males. Two component strains were reared without doxycycline in the parental adult diet and larval offspring diets (D) or in addition doxycycline was omitted from the parental L3 diet (Δ-D). (a) Male mating success. Percentage females that mated when 5 transgenic males were placed with 15 J-06 females for 4 h. DR9#4-1xEF1#6 and DR10#5AxEF1#6 were less competent when compared to J-06 (p-value < 0.05, T-test). (b) Male competitiveness. Mating success of 10 transgenic lines males and 10 J-06 males for 10 J-06 females over 20 h. The male competitiveness index (MCI) values are shown. An MCI of 0.5 indicates that the transgenic strain males are equally competitive with the control strain. Asterisks indicate the MCI was significantly less than the null-hypothesized value of 0.5. With males from the DR9#4-1xEF1#6 strain, the preference for J-06 males was borderline statistically significant (MCI = 0.37, 95% confidence interval of 0.249–0.496). The DR10#4-3xEF1#6 males were equally competitive with the J-06 control line on the either antibiotic feeding regimen [(-D) or (Δ-D)]. (c) Male survival measured as days post adult emergence. The mean values of three replicate experiments are shown. There were no significant differences in the longevity of any of the strains compared to J-06 regardless on the diet regimen, except for DR9#3-2BxEF1#6 on the modified feeding protocol Δ-D (p < 0.001, Multiple Comparisons of Means, Tukey Contrasts)).
Discussion
The primary goal of this study was to assess whether utilizing the Chtra sex-specific intron to provide female-specific expression of both genetic components (tTA and Lshid) would improve male fitness and performance compared to previously developed two-component conditional female-embryo lethal strains20. A second goal was to determine if promoters from the Chhalo and g6451 genes, identified previously as embryo-specific22, could provide the desired embryo-specific expression profile for tTA. The tTA lines generated were examined for stage of expression and splicing accuracy. The two component DR9 (Chhalo-tTA) and DR10 (g6451-tTA) strains were evaluated for production on doxycycline, conditional female lethality at different stages and male fitness.
Molecular analyses confirmed that tTA transcripts were alternatively spliced, consistent with expectations if the Chtra intron is being spliced in a sex-specific manner. These analyses also found that the g6451 gene contained an additional intron in the 5’UTR that was not previously annotated22. The Chhalo promoter was primarily active in embryos, with limited expression at later stages and none in the ovaries, making it well-suited for early-acting female lethality. In contrast, the g6451 promoter was active throughout development, including in the ovaries, which is in contrast with earlier RNAseq data that found significant expression only in embryos22. RT_PCR analysis with primer pairs specific for the g6451 gene showed expression in embryos and later stages, consistent with the observed activity of the g6451 promoter. It is unclear why the earlier RNAseq analysis failed to detect significant expression of the g6451 gene at later stages.
The productivity of the four assembled transgenic sexing strains (TSS) on diet supplemented with doxycycline was comparable to J-06 except for the DR9#4xEF1#6 strain that showed reduced survival at all larval stages. The four TSS showed promising male-only production when doxycycline was omitted from the parental adult and larval offspring diets. The DR9 strains predominantly produced males under a standard feeding regimen, though female lethality occurred at late larval or pupal stages. However, with a modified feeding regimen where doxycycline was omitted from the parental third instar and adult and larval offspring diets, female lethality shifted to early developmental stages, aligning with the project’s original objective. With the standard feeding regimen, it appears that sufficient doxycycline from the third larval feeding is transferred by the mother to the developing egg and is inhibiting the female lethal system in embryos. Similar results were obtained with DR6 and DR7 two component strains20.
The DR10 two component strains also produced mostly or only males but female lethality was at later developmental stages. One DR10 strain, DR10#5xEF1#6, proved unrecoverable under the modified doxycycline regimen, possibly due to excessive tTA expression. On the standard feeding regimen with doxycycline omitted from the parental adult diet and larval diet, DR10#5xEF1#6 produced only males. Female lethality occurred at the late larval stages. For the other DR10 strain, DR10#4-3xEF1#6, few females were produced on either the standard or modified feeding regimen. The females died at the pupal stage and the few that eclosed were deformed and were sterile.
Male fitness assays revealed mixed results. With the male mating success assays, which measures the ability of males to successfully mate with virgin females, males from two strains were comparable to J-06. Males from the other two strains showed a small but significant decrease in mating success. With the male mating competition assay, two strains were competitive with J-06. For one of the strains, DR10#4-3xEF1#6, males also showed high levels of mating success. Males from the other competitive strain, DR9#4-1xEF1#6, showed reduced levels of mating success. For three of the strains male longevity was comparable to J-06. The exception was DR9#3-2BxEF1#6, which showed significantly reduced lifespan. Thus, males from the DR10#4-3xEF1#6 strain showed the most promise from the fitness assays as they were equally competitive with J-06 males and showed comparable male mating success and longevity.
In conclusion, no single strain perfectly met all the desired criteria. The DR9 TSS were closer to the original goal of early female lethality, especially under a modified feeding regimen, but suffered from either poor male competitiveness and longevity (DR9#3-2xEF1#6) or reduced mating success and productivity on doxycycline (DR9#4-1xEF1#6). Of the two DR10 TSS, the DR10#4-3xEF1#6 could potentially be incorporated into the SIT program as it produces mostly males on either standard or modified feeding regimen and the males produced have good fitness characteristics. However, female lethality was at the pupal stage, which will not produce savings on larval diet. In this regard the DR10#4-3xEF1#6 strain is comparable to the tTA overexpression sexing strains that showed good performance metrics but late-stage lethality13. While any savings are desirable, we previously estimated savings gained by using an early female lethal system would be minor compared to economic gains from a male-only release22. Further, late-acting lethality could be beneficial if fertile males are released rather than sterile males28. Modeling indicates this could provide more efficient genetic suppression than SIT. The modeling has also shown that, if there is significant density dependence at the larval stage, late-acting lethality is more effective than an early lethal system29,30. Although our efforts to date have been focused on using early embryo promoters to drive tTA expression18,21, these promoters are often active in ovaries21, which can impact adult female fertility. Our success here in building a TSS using a promoter that is active at early and later stages, suggests that it may be worthwhile to explore the potential of using promoters from genes that are active only in larval stages, particularly early larvae. This could produce female lethality at larval stages, which would nevertheless save some diet costs as most diet is used in the third and final larval feeding. If the promoter is not active at the pupal or adult stage, this could minimize any impact on adult female fitness.
One advantage of TSS is reduced risk should fertile flies escape containment compared to the wild type strain used currently31. Without tetracycline in the diet, female offspring of any escaped individuals would not survive and thus a population would be unable to establish. This could be particularly important with the development of a screwworm rearing factory in Mexico and a release center in Texas32. An additional advantage of TSS is protection against failure of irradiation, which, although very rare, has occurred in the past during the screwworm eradication campaign33. Consequently, because TSS provide several advantages, we plan to seek regulatory approval to field test radiation sterilized males for one or more screwworm transgenic sexing strains.
Methods
Fly rearing and embryo injections
Individuals of the strain Jamaica-06 of C. hominivorax were obtained from the mass-rearing plant in Pacora, Panama (COPEG). The J-06 individuals were reared in the biosecure USDA-ARS facility as described previously26. Larvae of C. hominivorax were fed on an artificial diet at 37 °C and 70% humidity until they reached the third instar larvae stage. After this period, the larvae were passed to another room at 33 °C and 60% humidity for 6 days to complete the pupal period and subsequently, the collected pupae were placed in cages within a colony room at 25.5 °C, 55% humidity and 12:12 h light/dark cycle.
To make transgenic lines, pre-blastoderm embryos were injected with a mixture of DR9 or DR10 plasmid (800 ng/μL), Lchsp83-pBac helper plasmid (400 ng/μL), and pBac RNA helper (400 ng/μL) using methods developed previously for piggyBac mediated germline transformation of L. cuprina14,24. The G0 adults that developed from injected embryos were crossed to J-06 females and males. Transgenic offspring were identified during the larvae stage by whole body green fluorescence using a M165FC fluorescent stereomicroscope (Leica Microsystems) and bred to homozygosity by selecting brightly fluorescent larvae. Similarly, TSS were established by selecting for larvae that were brightly fluorescent for the ZsGreen and DsRed fluorescent proteins. After establishment, the TSS are screened periodically for red and green fluorescence. At the time of submission, the TSS had been maintained for 52 generations with no evidence of non-fluorescent larvae, confirming double homozygosity.
Plasmid construction
The DR9 and DR10 plasmids (Fig. 1A) employ promoters from the Chhalo and g6451 genes to drive tTA expression. To make the constructs, the tetO21-hsp70 enhancer-promoter from the tTA overexpression plasmid pBS-FL314 was replaced with a synthesized fragment from the Chhalo or g6451 genes, which each contained the entire 5’UTR and upstream sequences. The early promoter-tTA-SV40pA gene cassettes were excised by digestion with NotI and XhoI and ligated with the piggyBac vector pB[Lchsp83-ZsGreen]24 that had been cut with the same enzymes. The constructs were confirmed by whole plasmid DNA sequencing. The plasmid sequences of DR9 and DR10 have been deposited at GenBank with the accession numbers PV841818 and PV841817, respectively.
Gene expression analysis
Total RNA was isolated from 0–1, 2–3 and 4–6 h embryos, wandering third instar larvae, pupae at the P2 stage (approximately 48 h after starting pupation) and ovaries using TRIzol (Invitrogen California, USA) and RNeasy® Mini kit (Qiagen Hilden, Germany) according to the manufacturer’s instructions21.
Transcripts of the genes of interest were detected by reverse-transcriptase PCR (RT-PCR), using qMAX™ cDNA Synthesis Kits (Accuris Instruments New Jersey, USA) to produce cDNA and the DreamTaq PCR Master Mix (Thermo Scientific Massachusetts, USA). Amplification conditions were as follows: initial denaturation at 95 °C for 3 min followed 30 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min. Final extension was 72 °C for 5 min. Oligonucleotides for detection of tTAv, Chhalo and g6451 RNAs and for sex-specific expression of tTAv in the driver lines are listed in Table S7.
Oligonucleotides for the ChGapdh gene were used as a control as previously described34.
Amplification products were visualized in 1% agarose gel with TAE buffer. The bands were excised and purified with the NucleoSpin Gel & PCR Clean-up (Macherey- Nagel), following the manufacturer’s instructions. The PCR products were cloned into pGEM-T easy vector (Promega Wisconsin, USA) followed by Sanger DNA sequencing. Sequence alignment was carried out using Clustal Omega 1.2.2 from Geneious Prime 2024.0.
Early lethality assessment and female lethality tests
Virgin females homozygous for the EF1#6 effector line10 were crossed with DR9 or DR10 homozygous males to generate double heterozygous flies, which were inbred and their progeny screened to select only individuals homozygous for both the driver and effector construct (double homozygous) by epifluorescence microscopy based on fluorescence intensity. Strains were maintained in an artificial larvae diet with 25 μg/mL of doxycycline and the adults fed water with 25 μg/mL of doxycycline.
Assessment of female lethality of double homozygous strains was as previously described10,18, which consisted of removing the antibiotic from the parental generation in the adult water or also the late larval diet.
To assess the stage of early lethality, embryo masses was collected on ground beef and 100 mg of embryos were weighed and separated with 4% w/v sodium hydroxide solution for 2 min and washed with distilled water and placed in 10 ml of distilled water.
Egg hatch rate was obtained from the count of empty eggs approximately 8 h after hatching, by placing 200 individual embryos onto damp black filter paper that was on top of a damp paper towel in two Petri dishes. The Petri dishes were incubated at 37 °C overnight and, the following morning, the number of empty eggs were counted and the percentage of eggs that hatched was calculated. To obtain the survival rate of larvae at 24 h, 48 h and 72 h, one ml of embryos in suspension were placed in each Petri dish with black filter paper and fed daily. The initial number of eggs in each Petri dish was counted and recorded. After 24 h three of the petri dishes were randomly chosen and the number of hatched larvae was counted. After the larval count was performed, the petri dish was discarded. The same procedure was performed at 48 and 72 h after oviposition with the remaining Petri dishes. Three replicates per stage larvae per over at least three generations were recorded.
To assess pupal emergence, 100 pupae were placed in a closed container and adults were allowed to emerge for 3 days after the emergence of the first insect. Males and females were counted and the percentage of emergence and sex ratio were calculated. Three or more independent tests were performed for each strain.
Fitness and fertility essays
The sex ratio of adults, and fitness under mass-rearing conditions of each transgenic strain (with and without doxycycline) and J-06 control colonies were performed with minor modifications as previously described10,13.
To assess fitness under mass rearing conditions, approximately 75 ml of eggs were seeded in 30 ml of fresh larval diet in a large polystyrene tray and incubated at 37 °C and 70% humidity for 3 days, adding more food daily, until they reached the third instar larvae stage. On the fourth day of development, the larvae were placed in a room at 33 °C and 60% humidity for pupation into containers of sawdust. On the eighth day, the pupae were sieved out of the sawdust and 100 pupae were counted and placed in a plastic container with a lid. Three days after emergence, the number of females and males were counted to obtain sex ratio as previously described13.
Male mating success was evaluated by placing 4-day-old virgin flies (5 males and 15 J-06 females) in a rearing cage and allowing them to mate undisturbed for 4 h. After mating, the flies were anesthetized with carbon dioxide, and the males were discarded. Within 48 h females were killed by placing the cage in a − 20 °C freezer for 20–30 min, and then their spermatheca were removed. Mating success was confirmed if sperm were observed in the spermatheca. For each strain, flies were taken from three independent cages and replicated in three subsequent generations.
For male competition assays, 10 J-06 males and 10 transgenic males were placed in a cage with 10 J-06 females for 24 h. All flies were virgin before testing and 4 days old. After the mating time, the flies were anesthetized with carbon dioxide, and the males were discarded. At 6 days old, individual females were placed in 15 mL glass vials with a cotton stopper and 2 g of room temperature lean ground beef scented with oviposition stimulant and left for 2 h in a 37 °C incubator to oviposit. Individual tubes with eggs were left on the meat and placed in a 37 °C incubator until the next day for screening after hatching. Offspring were observed for fluorescence under a fluorescent stereomicroscope (Leica M165FC stereoscope). The male sexual competitiveness was determined by means of the Male Competitiveness Index (MCI), which consists of dividing the total number of transgenic male matings by the sum of matings to transgenic and to J06 males13. For each strain, flies were taken from three independent cages and replicated in three subsequent generations.
Adult male longevity was assessed as previously described26. The assay consisted of placing 25 pupae each in three cages per treatment with ample food and water. Dead adults were removed and counted daily and continued until all flies had died. Survival was observed in three cages for each treatment over three subsequent generations. In addition, two cages with 25 pupae of the J-06 control line were placed and the same procedure was repeated as the transgenic strains, where only males were counted and females were discarded.
Statistical analysis
Statistical analyses were mostly completed using the R statistics program (R Core Team 2016). Comparison of early lethality between the strains against J-06 in the two different feeding protocols was done using one-way ANOVA test to identify significant differences between the groups. Subsequently, Tukey multiple pairwise-comparisons was performed, to determine the mean difference between J-06 and specific strains. Pairwise comparisons of sex ratio (male and female) from the female lethality test were done using the Wilcoxon signed-rank test.
For the male mating success assays, comparison between transgenic males and J-06 males was performed using one-way ANOVA followed by multiple pairwise Student´s T-tests against a J-06 as reference group. For the male mating competitiveness assays, the mating competitiveness index (MCI) was calculated as the number mated to transgenic divided by the total number of females who mated. All data were analyzed in SAS (Version 9.4, Cary, NC). Counts were pooled across replicates and the overall MCI was calculated separately for each strain. The MCI for each strain was tested against the null-hypothesized value of 0.5 using a two-tailed z-test for a proportion. Results were considered to be statistically significant when p < 0.05. Longevity analysis was completed using the survival package (ver. 3.7; Therneau, 2023) by Cox proportional hazards regression model with multiple comparisons of means by Tukey contrasts and plotted with ggplot2 (ver. 3.5.1; Wickham 2016).
Acknowledgements
The authors would like to thank Rosaura Sánchez, Nicolas Mendoza, Hermogenez González, Odilis Rodríguez and Kenneth Castillo for their contributions to rearing strains. We thank Emily Griffith for statistical analysis of the mating competition data. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. This research was supported in part by the U.S. Department of Agriculture, Agricultural Research Service. USDA is an equal opportunity provider and employer.
Author contributions
MJS and AVL designed research. AVL, AS, ADA, GQ, MV, AM performed the experiments, AVL, AA, GQ, MV, AM collected and analyzed the data. AVL and MJS wrote the first draft of the manuscript. All authors edited the manuscript. MJS obtained funding for this project. All authors read, edited and approved the final manuscript.
Funding
This research was supported by cooperative agreements between USDA-APHIS and NCSU awarded to MJS (award numbers AP21IS000000C003 and AP22IS000000C005), and by the Research Capacity Fund (HATCH) project award no. 1020092 from the U.S. Department of Agriculture’s National Institute of Food and Agriculture. Aidamalia Vargas Lowman is a member of the National Research System (SNI) of SENACYT, Republic of Panama.
Data availability
The datasets generated for this study can be found in the manuscript and in the supplementary material. The nucleotide sequences for the plasmids developed in this study have been deposited at Genbank (accession numbers PV841818 and PV841817).
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
1. Hall, MJ; Wall, RL; Stevens, JR. Traumatic myiasis: A neglected disease in a changing world. Annu. Rev. Entomol.; 2016; 61, pp. 159-176. [DOI: https://dx.doi.org/10.1146/annurev-ento-010715-023655] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26667275]
2. Mastrangelo, T; Welch, JB. An overview of the components of AW-IPM campaigns against the new world screwworm. Insects; 2012; 3, pp. 930-955. [DOI: https://dx.doi.org/10.3390/insects3040930] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26466720][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553557]
3. Vargas-Teran, M., Spradbery, J. P., Hofmann, H. C. & Tweddle, N. E. in Sterile Insect Technique. Principles and Practice in Area-Wide Integrated Pest Management (eds V.A. Dyck, J. Hendrichs, & A. S. Robinson) 949–978 (CRC Press, 2021).
4. Wyss, JH. Screwworm eradication in the Americas. Ann. N. Y. Acad. Sci.; 2000; 916, pp. 186-193.2000NYASA.916.186W [DOI: https://dx.doi.org/10.1111/j.1749-6632.2000.tb05289.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11193620]
5. Alexander, JL. Screwworms. J. Am. Vet. Med. Assoc.; 2006; 228, pp. 357-367. [DOI: https://dx.doi.org/10.2460/javma.228.3.357] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16448356]
6. Scott, MJ; Concha, C; Welch, JB; Phillips, PL; Skoda, SR. Review of research advances in the screwworm eradication program over the past 25 years. Entomol. Exp. Appl.; 2017; 164, pp. 226-236. [DOI: https://dx.doi.org/10.1111/eea.12607]
7. Valdez-Espinoza, UM et al. The reemergence of the New World screwworm and its potential distribution in North America. Sci. Rep.; 2025; 15, 23819.2025NatSR.1523819V [DOI: https://dx.doi.org/10.1038/s41598-025-04804-9] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/40610581][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12229696]
8. Zaldivar-Gomez, A; Gomez-Vazquez, JP; Iniesta-Valencia, AJ; Figueroa-Martínez, LG; Rico-Chávez, O. Estimation the reinvasion of New World Screwworm (Cochliomyia hominivorax) in Central America: The role of animal movement in disease dispersal and control measures. Veter. Parasitol. Region. Stud. Rep.; 2025; 59, [DOI: https://dx.doi.org/10.1016/j.vprsr.2025.101220] 101220.
9. Rendon, P; McInnis, D; Lance, D; Stewart, J. Medfly (Diptera: Tephritidae) genetic sexing: Large-scale field comparison of males-only and bisexual sterile fly releases in Guatemala. J. Econ. Entomol.; 2004; 97, pp. 1547-1553. [DOI: https://dx.doi.org/10.1603/0022-0493-97.5.1547] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15568342]
10. Concha, C et al. An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT. BMC Genet.; 2020; 21, 143. [DOI: https://dx.doi.org/10.1186/s12863-020-00948-x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33339501][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747452]
11. Gossen, M; Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA; 1992; 89, pp. 5547-5551.1992PNAS..89.5547G [DOI: https://dx.doi.org/10.1073/pnas.89.12.5547] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/1319065][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC49329]
12. Li, F; Vensko, SP, 2nd; Belikoff, EJ; Scott, MJ. Conservation and sex-specific splicing of the transformer gene in the Calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata. PLoS ONE; 2013; 8, 2013PLoSO..856303L [DOI: https://dx.doi.org/10.1371/journal.pone.0056303] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23409170][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567074]e56303.
13. Concha, C et al. A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique. BMC Biol.; 2016; 14, 72. [DOI: https://dx.doi.org/10.1186/s12915-016-0296-8] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27576512][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004303]
14. Li, F; Wantuch, HA; Linger, RJ; Belikoff, EJ; Scott, MJ. Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina. Insect Biochem. Mol. Biol.; 2014; 51, pp. 80-88. [DOI: https://dx.doi.org/10.1016/j.ibmb.2014.06.001] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24928635]
15. Gong, P et al. A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly. Nat. Biotechnol.; 2005; 23, pp. 453-456. [DOI: https://dx.doi.org/10.1038/nbt1071] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15750586]
16. Schetelig, MF; Handler, AM. A transgenic embryonic sexing system for Anastrepha suspensa (Diptera: Tephritidae). Insect Biochem. Mol. Biol.; 2012; 42, pp. 790-795. [DOI: https://dx.doi.org/10.1016/j.ibmb.2012.07.007] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22858603]
17. Ogaugwu, CE; Wimmer, EA. Molecular cloning and expression of nanos in the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Gene Expr. Patterns; 2013; 13, pp. 183-188. [DOI: https://dx.doi.org/10.1016/j.gep.2013.03.002] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23567755]
18. Yan, Y; Scott, MJ. A transgenic embryonic sexing system for the Australian sheep blow fly Lucilia cuprina. Sci. Rep.; 2015; 5, 16090.2015NatSR..516090Y [DOI: https://dx.doi.org/10.1038/srep16090] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26537204][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633611]
19. Edman, RM et al. Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos. Insect Mol. Biol.; 2015; 24, pp. 58-70. [DOI: https://dx.doi.org/10.1111/imb.12135] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25225046]
20. Arp, A. P. et al. Development and evaluation of screwworm, Cochliomyia hominivorax, transgenic sexing strains with embryonic gene promoters for a genetic control program. bioRxiv, 2025.2005.2001.651745. https://doi.org/10.1101/2025.05.01.651745 (2025).
21. Yan, Y et al. Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters. Mol. Genet. Genom.; 2020; 295, pp. 287-298. [DOI: https://dx.doi.org/10.1007/s00438-019-01622-3]
22. Scott, MJ et al. Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs. Commun. Biol.; 2020; 3, 424. [DOI: https://dx.doi.org/10.1038/s42003-020-01152-4] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32753684][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403345]
23. Mazumdar, A; Mazumdar, M. How one becomes many: blastoderm cellularization in Drosophila melanogaster. BioEssays; 2002; 24, pp. 1012-1022. [DOI: https://dx.doi.org/10.1002/bies.10184] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12386932]
24. Concha, C et al. Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae). Insect Biochem. Mol. Biol.; 2011; 41, pp. 70-75. [DOI: https://dx.doi.org/10.1016/j.ibmb.2010.09.006] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20869440]
25. Concha, C; Scott, MJ. Sexual development in Lucilia cuprina (Diptera, Calliphoridae) is controlled by the transformer gene. Genetics; 2009; 182, pp. 785-798. [DOI: https://dx.doi.org/10.1534/genetics.109.100982] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19433631][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710159]
26. Arp, AP et al. Doxycycline is a viable alternative to tetracycline for use in insect Tet-Off transgenic sexing systems, as assessed in the blowflies Cochliomyia hominivorax and Lucilia cuprina (Diptera: Calliphoridae). J. Econ. Entomol.; 2024; 117, pp. 650-659. [DOI: https://dx.doi.org/10.1093/jee/toae023] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38401129]
27. Chaudhury, MF; Chen, H; Sagel, A; Skoda, SR. Effects of new dietary ingredients used in artificial diet for screwworm larvae (Diptera: Calliphoridae). J. Econ. Entomol.; 2015; 108, pp. 1429-1434. [DOI: https://dx.doi.org/10.1093/jee/tov039] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26470272]
28. Schliekelman, P; Gould, F. Pest control by the release of insects carrying a female-killing allele on multiple loci. J. Econ. Entomol.; 2000; 93, pp. 1566-1579. [DOI: https://dx.doi.org/10.1603/0022-0493-93.6.1566] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11142283]
29. Vella, MR; Gould, F; Lloyd, AL. Mathematical modeling of genetic pest management through female-specific lethality: Is one locus better than two?. Evol. Appl; 2021; 14, pp. 1612-1622. [DOI: https://dx.doi.org/10.1111/eva.13228] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34178107][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210802]
30. Gentile, JE; Rund, SS; Madey, GR. Modelling sterile insect technique to control the population of Anopheles gambiae. Malar. J.; 2015; 14, 92. [DOI: https://dx.doi.org/10.1186/s12936-015-0587-5] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25889145][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351850]
31. Thomas, DD; Donnelly, CA; Wood, RJ; Alphey, LS. Insect population control using a dominant, repressible, lethal genetic system. Science; 2000; 287, pp. 2474-2476.2000Sci..287.2474T [DOI: https://dx.doi.org/10.1126/science.287.5462.2474] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10741964]
32. Release, P. USDA Announces Sweeping Plans to Protect the United States from New World Screwworm, https://www.usda.gov/about-usda/news/press-releases/2025/08/15/usda-announces-sweeping-plans-protect-united-states-new-world-screwworm (2025).
33. Dyck, V. A. et al. in Sterile Insect Technique. Principles and Practice in Area-Wide Integrated Pest Management (eds V.A. Dyck, J. Hendrichs, & A. S. Robinson) 525–545 (Springer, 2005).
34. Paulo, DF et al. Disruption of the odorant coreceptor Orco impairs foraging and host finding behaviors in the New World screwworm fly. Sci. Rep.; 2021; 11, 11379.2021NatSR.1111379P [DOI: https://dx.doi.org/10.1038/s41598-021-90649-x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34059738][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167109]
© The Author(s) 2025. This work is published under http://creativecommons.org/licenses/by-nc-nd/4.0/ (the "License"). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.