Genetic biocontrol approaches to insect pest management offer high species specificity and reduce insecticidal chemical pollution, but these approaches require the release of genetically modified organisms that must perform across a variety of environmental conditions. Because organisms often display phenotypic and gene expression variability when exposed to different environments, it is reasonable to expect that transgenic systems may be prone to environmentally mediated variation in expression and function. In this study, we examined the influence of two abiotic variables, temperature and nutrition, on the penetrance of an early embryonic Tet-off conditional lethality system in the vinegar fly, Drosophila melanogaster. We manipulated parental and offspring environments independently to determine the impact of life stage of exposure on transgene performance by estimating the probability of larval hatching and measuring transcript abundance of the transgenic system components. Our findings revealed that: 1) transgene performance has distinct norms of reaction to temperature and nutrition, 2) the effects of abiotic challenge are greatest when exposure occurs in the embryos expressing the transgene compared to parental exposure, and 3) stress exposures at the extreme limits of permissible conditions can dramatically decrease the penetrance of transgenic lethality. While variation in transcript abundance of the transgenic system was observed in some environments, these changes were not fully congruent with patterns of phenotypic penetrance, suggesting that the observed variation in lethality is likely driven by processes downstream of transcription.
Research Article
Abiotic environments can modify the penetrance of a transgene-based lethality system for genetic biocontrol of insect populations
https://doi.org/10.21203/rs.3.rs-5183317/v1
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conditional lethality
phenotypic plasticity
sterile insect technique
genetically modified insects
- Transgenic insects carrying conditional lethality systems are being integrated to autocidal pest control programs of insect populations
- Efficiency of transgenic conditional lethality is estimated in optimal laboratory conditions but expected to perform in natural environments
- We identified environmentally induced variation in the penetrance of transgenic lethality in response to extreme but permissible thermal and nutritional conditions
Many biological traits are a function of both genotype and environment, and often complex interactions between the two (e.g. Gupta and Lewontin 1982). Thus, it is reasonable to expect the same to be true for engineered traits produced via genetic modifications, because both the host organism and the introduced transgenic system may exhibit environmental dependency. As genetic engineering application progresses from sessile microorganisms and plants (e.g. Doron, Segal, and Shapira 2016; Pellegrino et al. 2018) into mobile animals (Ledford 2015; e.g. Carvalho et al. 2015), the environments across which transgenes must perform has become increasingly variable. Interactions between transgenes and the environment are now well supported by reports of environmentally induced variation in the expression of transgenic traits in bread wheat (Zeller et al. 2010), salmon (Sundström et al. 2007), maize (Trtikova et al. 2015), and the fruit fly (Horn and Wimmer 2003), thus suggesting that proper development of safe genetic engineering applications requires further study of transgene efficacy across diverse environmental conditions.
In the past two decades, several recombinant systems of gene expression have been developed to enhance insect pest management through an autocidal strategy, i.e., the control of a pest population by reducing their reproductive capacity. The Sterile Insect Technique (SIT) is the most successful implementation of the autocidal strategy, and it achieves pest population reduction through repeated infertile mating events between wild females and released sterile males (Knipling 1955). Alternative implementations of the autocidal strategy that integrate genetic engineering have received increased attention, and lethal transgenes are used in these new tactics to generate infertility in lieu of generating random lethal mutations with radiation. Specifically, lethality in the progeny of released males will no longer be due to chromosomal rearrangement induced by radiation exposure (Lindquist 1955; Muller 1927), but instead termination of development will be genetically engineered through the timely expression of a transgene with lethal function (e.g. Heinrich and Scott 2000; Nguyen, Choo, and Baxter 2021), or by overexpression of an otherwise innocuous protein (Phuc et al. 2007). Transgenic technologies will likely expand the scope of SIT and related strategies by reducing operational costs (Alphey, Alphey, and Bonsall 2011). Further, transgenic lethality can be translated to most species susceptible to genetic modification (Teem et al. 2020), and the potential for novel molecular modes of action is expected to reduce the risk of resistance evolution in field pest populations (Alphey, Bonsall, and Alphey 2011).
The expression of most current transgenic lethality systems is regulated by the tetracycline-off (Tet-off) gene-switch repressor (Gossen and Bujard 1992). Two main configurations of conditionally lethal Tet-off systems have been developed for genetic biocontrol operations: 1) a binary driver-effector system (Heinrich and Scott 2000), and 2) a reduced single-element system currently in commercial use named RIDL (Release of Insects Carrying a Dominant Lethal) (Phuc et al. 2007). In both systems, the expression of the gene of interest is induced by the tetracycline transactivator (tTA) and its associated tetracycline response element (TRE), but they differ in the induction of gene expression. In the original driver-effector binary configuration, tTA is encoded in a driver cassette while the effector cassette contains the TRE upstream of the desired sequence to be expressed. In this way, the timing and localization of transgene expression can be specified by an appropriate promoter sequence linked to the tTA in the driver cassette, and the mechanism of lethality (for instance, programmed cell death) can be specified by the protein encoded in the effector cassette. This is different from the RIDL configuration, where tTA is under regulation of TRE and a constitutive promoter sequence, all in a single element, and tTA amplifies its own expression through a positive feedback loop. The control of expression is less specific in RIDL, as the time of induction depends on the background expression levels of the hsp70 minimal promoter sequence (Phuc et al. 2007), and lethality is likely conferred by accumulation of tTA to toxic levels (Bryk et al. 2017). Both system configurations are regular subjects of research. However, given their differences in design, the performance of the two different configurations is likely not equivalent. Thus far, RIDL lethality has only been achieved in post-larval late development, whereas the binary system is effective between embryonic and early larval periods depending on the promoter linked to tTA (Horn and Wimmer 2003; Phuc et al. 2007).
In principle, conditional lethality systems are self-limiting because the environmental persistence of transgenic insects is not possible without high doses of tetracycline to suppress expression, which is highly unlikely outside rearing facilities. Therefore, Tet-off conditional lethality systems are considered to be the safest systems among the multiple alternatives developed for genetic biocontrol and are attractive options for commercial or public implementation (Mumford 2012). However, despite careful design, performance instability is a common feature in genetic engineering, and certain factors are recognized to influence the penetrance of a transgenic phenotype. For instance, genomic position effect suppression related to the insertion site locus of the transgene (e.g. Allen, Norris, and Surani 1990; Grewal and Klar 1996). Thus, even though conditional lethality systems are designed to be self-limited, 100% efficacy is rarely achieved. For example, between 2-4% of male RIDL mosquitos’ progeny survived without tetracycline under laboratory conditions (Phuc et al. 2007), indicating incomplete penetrance of the transgene construct. There is yet to be a regulatory consensus on an appropriate performance threshold for transgenic lethality, and RIDL was approved for field tests despite its incomplete efficacy (Carvalho et al. 2015; Harris et al. 2012; Lacroix et al. 2012). Because the expression and activity of lethal transgenic systems depends on the host’s cellular machinery, additional sources of perturbation in biological systems, like environmentally mediated variation, may also increase the incidence of failure events of the transgenic lethality systems and lead to survival of transgenic insects in the field.
The probability of failure in transgene expression can be understood from a framework of phenotypic variation, which in natural systems is usually considered to be the product of genetic, environmental, and developmental factors. In fact, significant variation in the penetrance of transgenic lethality due to varying genomic backgrounds has been reported (Knudsen et al. 2020). In addition to genetic factors that influence the penetrance of a transgenic trait, lethal transgenes are expected to encounter dynamic changes in cellular context due to environmental stress experienced by the host organisms. Under such a dynamic cytological environment, fluctuations in the penetrance of transgenic phenotypes are likely due to changes in transgene expression or other downstream processes, such as post-translational modifications to effector proteins. In some cases, environmentally induced phenotypic variation is due to adaptive plasticity that has evolved to maintain homeostasis under challenging conditions, and this plasticity may also influence subsequent generations (Sgrò, Terblanche, and Hoffmann 2016). In simple terms, environmental perturbations to fundamental cellular processes may lead to changes in transgene expression or activity and thus the penetrance of transgenic traits. However, our understanding of how environmental conditions may affect transgene trait penetrance is currently limited to a few examples, and general rules for understanding environmental effects on transgene penetrance have yet to be described.
A clearer understanding of transgene/environment interactions can provide relevant information for improving transgenic insect strains, as well as the development of a methodological framework to assess the potential risks associated with incomplete penetrance of transgenic traits in the field. Here, we used the common vinegar fly Drosophila (Sophophora) melanogaster to study the influence of temperature and nutritional quality on the penetrance of conditional lethality induced by a Tet-off system in the classic binary configuration. Specifically, we used a construct designed for early embryonic expression of the driver element specified by the serendipity alpha (sry α) promoter sequence 1 (Ibnsouda et al. 1993) and with a resultant lethal effect specified in the effector element by head involution defective Alanine-5 (hidAla5) (Bergmann et al. 1998), the phosphorylation-desensitized mutant of the endogenous pro-apoptotic hid gene (Grether et al. 1995).
Specifically, we exposed embryos carrying these constructs or their parents to a range of temperatures and diets to identify the extent to which environmental conditions affect the penetrance of transgenic lethality. We expected offspring survival under these conditions to be related to the transcript abundance of transgene components, because the penetrance of developmental mutations is often the result of variable transcription (e.g. Green et al. 2020; Raj et al. 2010). Our results show that while transgenic lethality remained high under most treatments evaluated, (1) environmental conditions at the extremes of permissible abiotic conditions can decrease the penetrance of transgenic lethality, (2) the norm of reaction for transgenic lethality depends on the type and timing of environmental variation (temperature vs. nutrition), and (3) variation in transcript abundance of the transgene is insufficient to explain reduced penetrance of the transgenic lethality trait, suggesting that environmental variation in penetrance is acting through mechanisms downstream of transcript abundance such as translation or post-translational modification.
Transgenic system and animal husbandry
We studied the transgenic D. melanogaster strain Double Homozygous-1 (DH-1) that carries a binary driver/effector Tet-off transgenic system that induces lethality during early embryogenesis by ectopic expression of a mutant pro-apoptotic factor (Zhao, Schetelig, and Handler 2020). DH-1 is in the mutant white1118 genetic background and was created via inbreeding two strains carrying independently inserted piggyBac vectors carrying either the driver or the effector cassettes. The driver vector, pB{PUb-DsRed.T3, s1-tTA}, contains tTA regulated by the sry α s1 promoter sequence and the DsRed.T3 fluorescent protein regulated by the constitutive polyubiquitin (PUb) promoter. The effector vector, pB{3xP3-ECFP-5’HS4>5’HS4-TREp-hidAla5-5’HS4>5’HS4}, encodes the pro-apoptotic protein HIDAla5 regulated by the TRE sequence, a heptameric tandem repeat of the tetracycline operator sequence (Gossen and Bujard 1992). Both hidAla5 and TRE are flanked by 5’HS4 insulator sequences and carry an additional Enhanced Cyan Fluorescent Protein (ECFP) regulated by the 3xP3 promoter. The Tet-off system is induced in the syncytial blastoderm during cellularization between 120 and 170 min at 22°C after fertilization (Schweisguth, Vincent, and Lepesant 1991). In the absence of tetracycline, tTA dimers bind to TRE in the effector element, induce the ectopic expression of hidAla5 and, consequently, activate hid-dependent programmed cell death resulting in embryonic lethality. When present, tetracycline molecules allosterically inhibit dimerization of tTA monomers preventing their induction of hidAla5 expression. We applied 64 ug/ml tetracycline to the rearing media of DH-1 to maintain the strain and ensure flies were exposed to sufficient antibiotic to suppress the system.
In addition, we used the wild type D. melanogaster Oregon-R strain as the simulated target pest population. Specifically, we crossed adult unmated Oregon-R females with transgenic DH-1 males in a tetracycline-free environment to simulate the mating events intended in the autocidal strategy in the field. To determine the effect of our environmental treatments on the probability of hatching in wild type flies, we also conducted parallel experiments with Oregon-R flies exposed to the same environmental conditions.
Embryo collection
We simulated the most common SIT strategy where only males are released, and therefore we evaluated embryos carrying a single paternally inherited copy of the transgenic system. Because no alleles of the piggyBac insertion loci are present in the wild-type genome, the transgenic system exists in these embryos in hemizygous state. For the remainder of the text, we will refer to embryos hemizygous for the lethal transgene as ‘transgenic’ and those homozygous for the unmodified genotype as ‘wild-type’. Variation in transgenic lethality was quantified as the probability of hatching in transgenic embryos, all of which were expected to die because the parental generation was not supplied with tetracycline after adult emergence as would occur in a field operation. In addition, we evaluated the probability of wild-type embryos hatching under the same experimental treatments as a control to determine the general effect of our treatments. Our metric of probability of hatching is derived from binary data (hatch/no hatch) and can be interpreted as the hatching rate often used in agricultural science and SIT literature (Klassen and Curtis 2005).
Transgenic and wild-type embryos were reared under the following standard protocol. Standard conditions were 25°C, 12:12 L:D cycle and rearing on a cornmeal-yeast-molasses diet. These conditions were modified accordingly for each experimental treatment as described below. Parental flies were sorted by sex using brief CO2 anesthesia 1-2 h after adult eclosion and kept on tetracycline-free food vials for five days. On the fifth day after adult emergence, 200 adult males and 200 adult females were allowed to mate for 24 h at 25°C in embryo collection cages (Genesee Scientific Corp., El Cajon, CA, USA) containing grape agar (Genesee Scientific Corp., El Cajon, CA, USA) and a small amount of baker’s yeast paste to promote oogenesis and oviposition. On the sixth day after adult emergence, freshly oviposited embryos were collected from the oviposition cages and incubated for scoring.
To estimate the probability of hatching, embryos were harvested in 1 h intervals using a collection basket fitted with nylon mesh (Genesee Scientific Corp., El Cajon, CA, USA), then washed with 75% ethanol for 5 seconds, and rinsed with distilled water. These embryos were transferred to petri dishes and incubated on black filter paper (Ahlstrom Munksjo, Mt. Holly Springs, PA, USA) saturated with distilled water until hatching or death. To estimate transcript abundance, embryo samples for were collected in 30 min periods and incubated for 150 min to allow embryos to reach the late blastoderm stage, except in experiments where embryonic temperature was varied, in which the timing was adjusted (see below). After completing the incubation period, embryos used for measuring gene expression were recovered from the oviposition substrate with a fine paint brush, suspended in 30 µL of distilled water inside 1.5 mL screwcap tubes, and frozen to -80°C. In standard conditions, sampling time was 165 minutes on average, which was chosen as the sampling point between the induction of the Tet-off lethality system and the end of blastoderm cellularization and provided a transcriptional snapshot of both driver and effector elements in the same biological sample.
Experimental treatments
Our experimental design quantified variation in the penetrance of transgenic lethality in distinct thermal and nutritional environments at three different life-stages of exposure. For both thermal and nutritional experiments, the influence of abiotic treatments was determined relative to a preselected standard condition, either 25°C for temperature or Intermediate P:C diet for nutritional value experiments. In the case of our thermal manipulations, multiple temperatures were selected at regular intervals across the permissive temperature range for growth of D. melanogaster (12-32°C) (Petavy et al. 2001). Using the geometric framework of nutrition (Simpson and Raubenheimer 2012), we designed a series of nutritional-quality treatments across the protein:carbohydrate (P:C) space in two axes: first isocaloric diets with varying P:C ratios (High, Intermediate, Low P:C) and second dietary restriction treatments with reduced caloric content (Intermediate P:C, Quarter, Desiccation) (Figure 2d). The specific treatments applied to each life stage are described in detail below.
Both transgenic and wild-type flies experienced the above-mentioned environmental conditions in one of three predetermined life stages (Figure 1). We defined the filial embryonic stage as the period experienced by the embryos from oviposition until death or larval hatching. Nutritional quality treatments were omitted for our embryo-only treatments given that embryos do not feed and exclusively depend on their parental nutritional supply. Next, the parental adulthood stage was defined as the period when both wild and released transgenic adult parents are in the field together. We also included the maternal pre-adult stage, from the first larval instar to adult eclosion, to simulate the environment of each parent during their immature stages, meaning exposure to experimental treatments for wild-type females to simulate varying developmental conditions in the natural environment and standard laboratory conditions for transgenic males to simulate the rearing facilities.
Each experimental treatment consisted of one abiotic condition applied to a specific exposure stage. For the embryo-only treatments, embryos were exposed to one of six temperatures (16, 19, 22, 25, 28, and 31°C) from the time of oviposition to hatching or death. Because the rate of embryogenesis changes with temperature, we adjusted the time of phenotyping and transcript abundance sampling using the D. melanogaster temperature-dependent developmental rate equation of (Kuntz and Eisen 2014):
Where t is development time in hours and T is temperature in degrees Celsius. The incubation period for gene expression samples was calculated as the time equivalent to 180 minutes (30 min collection + 150 min treatment) at 25°C to determine the temperature-specific stage of late blastoderm cellularization. Similarly, for the estimation of the probability of hatching we calculated a time interval equivalent to 24 hours at 25°C to determine the expected time required for the completion of embryogenesis.
Experimental conditions for adult-only treatments were applied for five days between adult emergence and mating of the parental flies, but mating was always performed at 25°C to avoid potential confounding effects with changes in mating behavior (Schnebel and Grossfield 1984). For parental adulthood temperature treatments, five temperatures were evaluated (19, 22, 25, 28, and 31°C). Five levels were also evaluated for nutritional quality, namely three levels of isocaloric diets that differ in the P:C ratio (High P:C 7.4:7.2, Intermediate P:C 4.7:11.4, and Low P:C 2.0:15.7) with formulation as used by (Matzkin et al. 2011), one caloric restriction level named ‘Quarter’ (P:C 1.6:5.4) equivalent to 25% of caloric content of the Intermediate P:C diet, and one ‘Desiccation’ level that consisted of limiting the access to water and food for 24 hours prior to mating.
We also investigated the effect of temperature shock in the parental adults on conditional lethality. Thermal shock treatments consisted of 1 hour exposure to either 0°C (cold-shock) or 34°C (heat-shock), and these were compared to a no-shock treatment kept at 25°C as a baseline control. Thermal shock treatments were applied to 6-day old parental adults of both genotypes just after the 24 h mating period. Embryo samples for the estimation of the probability of hatching were collected at 1, 2, 4, 24, and 48 h after thermal shock. Transcript abundance samples were collected at 2 and 24 h after thermal shock.
For the maternal juvenile development conditions, four temperatures (19, 22, 25, 28°C) and four nutritional quality levels (High P:C, Intermediate P:C, Low P:C, caloric restriction) were applied from the first instar of the maternal larval stage to maternal adult emergence, after which females were kept at 25°C. Transgenic parental males developed in standard conditions (25°C and cornmeal-yeast-molasses diet).
Phenotyping transgenic lethality
A hatching event was scored when an embryo presented a visibly disrupted chorion. This hatching definition identifies individuals that deviate from early embryonic lethality and is a stringent measure of performance of the lethal transgenic system. Developed mouth hooks and coordinated movements are required for first instar D. melanogaster larva to break the vitelline membrane and chorion (Siekhaus and Fuller 1999), hence disruption of these layers indicates that the expected transgene mode of action (i.e., early embryonic lethality) was disrupted. Further, our measurement of lethality is amenable to high-throughput screening, because dishes of eggs can be photographed and scored afterwards, as opposed to direct observations of live larvae.
Estimation of transgene expression
To estimate expression levels of the transgenic system across our experimental conditions, we measured the transcript abundance of tTA, hidAla5, and the housekeeping gene RP49 as an endogenous control with real-time qPCR. Frozen embryos were homogenized in lysis buffer with a Fast-Prep24 TM 5G Bead Homogenizer (MP Biomedical, Santa Ana, CA, USA) with 40-60 0.5 mm zirconia/silica beads (Biospec Products, Bartlesville, OK, USA). RNA was extracted with the RNeasy mini kit (Qiagen, Germantown, MD, US) following the manufacturer’s protocol, and cDNA was synthesized from 100 ng RNA using the QScript cDNA Synthesis Kit (QuantaBio, Beverly, MA, USA). Real-time qPCR reactions were prepared with ten-fold dilutions of the cDNA using PerfeCTa SYBR Green Fastmix (QuantaBio, Beverly, MA) and were measured in triplicate on a QuantaStudioTM 6 Flex real-time PCR System (Applied Biosystems, Foster City, CA, USA) following instructions provided by the manufacturer. Melting curves were inspected for single peaks to ensure no primer dimers were generated. The primers designed for each gene demonstrated good linearity (R2 > 0.99) and efficiency (1.98 < E < 2.05) using an 8-point standard curve. Fold changes of tTA and hidAla5 were calculated with the method (Livak and Schmittgen 2001) using RP49 as the reference gene. Primer sequences are provided in Table 1.
Replication design and sample sizes
A total of six experiments were analyzed, three involving chronic exposure to temperature treatments at three distinct life stages, two involving the chronic exposure to nutritional treatments at two different life stages, and one involving thermal shock in adults. Each of the first five experiments that included chronic exposure were performed twice, while the thermal shock experiment was performed three times. Each experiment was performed as a weekly sequence of random blocks, each block consisting of three different treatments, and each treatment was evaluated as a single cohort consisting of two oviposition cages, one per embryo genotype. From each oviposition cage, the samples used to estimate the probability of hatching were taken at three sequential one-hour embryo collections that varied in size (Table 2). Therefore, the replication design in the five experiments with chronic exposure included a total of six biological replicates per treatment per genotype given that each treatment was performed twice with three embryo samples collected on each occasion. In the thermal shock experiment, a single sample was collected per cohort, thus a total of three biological samples per treatment per sampling time was collected except for 48-h treatments which only had two observations. In the first five experiments with chronic exposures, two gene expression samples of 20 embryos were taken in intervals of 30-minutes for each of the two experimental replicates, giving a total of four biological replicates per treatment, but in a few cases one outlier was removed leaving three observations. In the case of the thermal shock experiment, one sample of 20 embryos was recovered per treatment (no-shock, heat-shock, and cold-shock) at 2- and 24-h evaluation times in 30 min intervals for each of the three experimental replicates, resulting in three biological replicates per group.
Statistical analyses
All statistical analyses were performed in R v4.1.0 (R Core Team 2021) with lme4 (v1.1-29) and emmeans (v1.8.0) and graphed with ggplot2 (3.5.1). The probability of hatching was estimated with logistic regression using a generalized mixed effects model in each of the six experiments by fitting the joint dataset of transgenic and control embryos. The fixed effects in the five chronic exposure experiments were Genotype and Temperature (or Nutrition depending on the experiment) and their interaction. The random effects were modeled with a repeated measures approach using random intercepts for the three sequentially collected samples (termed Repeated Measure) and random slopes for Cohort. The fixed effects in the thermal shock experiment model were Genotype, Shock (hot, cold, no shock), and their interaction. The random effects were also modeled with a repeated measures approach using random intercepts for Time after shock and random slopes for Replicate. In each model, the explanatory variable of interest, either Temperature, Nutrition quality, or Shock, was modeled as a nominal categorical variable.
Statistical inference was assessed for each hatching model using a “treatment v. control” approach using custom contrasts to define post hoc pairwise comparisons between experimental and reference conditions. Specifically, we applied Dunnet’s test approach to assess differences between treatments and controls in a generalized mixed model. The reference treatment was 25°C in the case of experiments of thermal exposure and the ‘Intermediate P:C’ diet in the case of nutritional quality. In the case of thermal shock experiments, the reference treatment used for pairwise comparisons was No-shock at each time point evaluated. In each case, p-values were adjusted for multiple comparisons using the multivariate t method.
Statistical inference for transcript abundance differences in the abiotic condition by life stage datasets was estimated with a categorical linear model fit using ΔCT values as a function of treatment levels of Temperature or Nutrition variables. The differences between treatments were calculated relative to a reference treatment, which were 25°C for temperature treatments and Intermediate P:C diet in the case of nutritional quality treatments. Being a simple linear model, significant differences between the treatment and the control groups were determined with Dunnet’s post hoc test (Dunnett 1955). In the case of parental shock treatments, a linear model was specified with the variables Time after shock, Shock, and their interaction. Significance was assessed in Heat shock v. No-shock and Cold shock v. No shock pairwise comparisons for each Time after shock level with a post-hoc test using custom contrasts, again, in the spirit of Dunnet’s test approach.
In this study we quantified the efficacy of an early-embryonic conditional lethality system in D. melanogaster across a range of temperature and nutritional treatments. The probability of transgenic embryo hatching across the reference levels for all experiments (25°C, Intermediate P:C, No-shock) was 1.76% on average (SD=1.79, n=44) with a median of 1.29%, which can also be interpreted as the probability of failure of the conditional lethality system under standard laboratory conditions.
Environmentally induced variation in the penetrance of transgenic lethality
Among the three life stages of exposure evaluated (Figure 2a-c), only some of the temperature treatments that were applied directly to the embryos decreased the penetrance of transgenic lethality relative to the 25°C reference treatment (Table 3). In this case where temperature treatments were applied only to embryos, the probability of failure estimates displayed an inverted performance curve with a minimum survival at 25°C (0.64 % [SE=0.1]) but increasing probability of hatching to as high as 2.78 % [SE=0.36] at 19°C and 3.00 % [SE = 0.4] at 31°C (Figure 2a).
Modifying the nutritional environment of the parental flies also induced variation in the penetrance of transgenic lethality. Individuals fed the Quarter-strength diet (1.8P:4.5C compared to the control 7.1P:17.9C) either as adults (Figure 2e) or as maternal larvae (Figure 2f) had decreased penetrance of transgenic lethality. Specifically, adult parental exposure to the Quarter-strength diet increased the probability of failure from 2.31% [SE = 0.42] in the reference diet to 16.45% [SE = 0.8], while pre-adult maternal exposure increased failure of embryonic lethality to 8.16% [SE = 0.48].
Beyond the effects of the Quarter-strength, caloric-restriction diet, isocaloric imbalanced P:C diets also caused variation in the penetrance of transgenic lethality (Figure 2e,f). Relative to the reference Intermediate P:C diet (2.31%, SE=0.42), both High P:C (3.92%, SE = 0.53) and Low P:C (11.16%, SE = 0.82) diets significantly decreased the penetrance of transgenic lethality following parental adulthood exposure (Table 4). On the other hand, both unbalanced diets increased the penetrance of transgenic lethality when experienced exclusively by maternal pre-adult exposure, but in this case only Low P:C (0.70%, SE = 0.15) induced a significant difference relative to the reference Intermediate P:C diet (2.88%, SE = 0.48). The probability of hatching of wild-type embryos increased on P:C ratios both higher and lower than the intermediate P:C reference diet when exposed during parental adulthood, but this was not the case with pre-adult maternal exposure.
We further studied the influence of parental exposure to heat (34°C) and cold (4°C) shock treatments to identify potential impacts on their offspring’s penetrance of transgenic lethality. Our results indicate that parental thermal shock influenced the survival of both transgenic and wild-type embryos (Figure 4a). Both parental heat and cold shocks significantly reduced the penetrance of transgenic lethality with a modest effect size (Table 5), and in each case, at just a single time point after exposure. Specifically, the effect of cold shock was first detectable in transgenic embryos collected 4 hours after shock with a 8.5% (SE=1.4) probability of hatching relative to the 3.1% (SE = 1.0) of the no-shock control at the same sampling time; in turn, heat shock induced a 6.2% (SE=1.5) probability of transgenic embryo hatching in embryos collected at 24 hours after shock relative to the 0.4% (SE = 0.2) of the no-shock treatment at the same sampling time. The transient influence of thermal shock in the probability of hatching of transgenic embryos was distinct from that in wild-type embryos, where the first embryo collection after shock presented the largest decrease in the probability of hatching and slowly recovered over time (Figure 4a).
Transcriptional variation of the transgenic lethality system is induced by environmental stress
We hypothesized that variation in the penetrance of transgenic lethality would be at least partly due to interference in the molecular events required for the complete expression and function of the lethal transgene (Figure 3c). We tested this hypothesis at the transcriptional level by measuring the transcript abundance of the driver and effector elements of the Tet-off system in embryos across our different treatments. In general, there was little relationship between transcript abundance and observed lethality, with the following exception. Embryos subjected to pre-adult maternal exposure to Low P:C ratio diet (Figure 3k) showed a decrease in penetrance of transgenic lethality together with a decrease in transcriptional abundance of the effector element hidAla5 when compared to Intermediate P:C diet (t(9.25) = 2.9, p = 0.04).
While most of the transcriptional abundance results did not show significant variation, distinct patterns in transgene expression were observed in thermal treatments experienced directly by the embryos or by their parents during adulthood (Figure 3). As mentioned above, this variation in transcript abundance did not track the observed variation in lethality. For example, with embryonic exposure to 31°C, the penetrance of transgenic lethality decreased (Table 3), but the transcript abundance of the driver element tTA increased (Figure 3a), each relative to the 25°C control treatment. Furthermore, there was also significant variation in transgene expression in embryos following parental adulthood exposure, even though no significant differences were observed in the penetrance of transgenic lethality (Figure 3d,e). For example, a decrease in transcript abundance for the effector element hidAla5 was observed at 19 and 31°C, while the driver element tTA either increased (19°C) or became undetectable (31°C).
In accordance with the transient influence of thermal shock in the penetrance of transgenic lethality, the transcript abundance of the effector element hidAla5 significantly increased in response to heat shock, which induced a 2.8 fold increase between 2 and 24 hour embryo collections (ΔΔCT = 1.5, t(6) = 2.8, p = 0.029). As in previous results, the direction of the varying transgene expression was opposite to our predictions because the transcript abundance of hidAla5 increased over time while the penetrance of transgenic lethality decreased during that interval.
In this study we evaluated the effects of the abiotic environment on the penetrance of an early-embryonic Tet-off conditional lethality system intended for autocidal genetic biocontrol. As expected, the performance of the transgenic system was high under optimal conditions, but our results indicate that some suboptimal thermal or nutritional scenarios can reduce the penetrance of the transgenic lethal phenotype. Most of these suboptimal environmental scenarios increased the probability of survival resulting in larval hatching, indicating a failure of the engineered lethality during early embryogenesis. Therefore, we surmise that the host organisms’ environment can reduce the penetrance of the transgenic lethal system it carries and thus increase the potential for transgene system failure in the field.
Abiotic conditions at the extremes of the permissible range had the strongest effects on penetrance of embryonic lethality, and the magnitude of their influence increased with more recent exposure relative to the induction of the lethal transgenic system. In the case of temperature, the penetrance of transgenic lethality decreased at both extremes of the thermal range evaluated, essentially an inverse thermal performance curve. In contrast, as expected, the norms of reaction for hatching in wild-type embryos displayed the canonical shape of a thermal performance curve, with the highest hatching probabilities near the optimal temperature of 25°C and decreasing hatching at the higher and lower ends of the temperature range (Huey and Stevenson 1979).
Ours is not the only work to show that temperature can affect the penetrance of transgenic systems. Horn and Wimmer (2003) evaluated the same conditional lethality system as ours at three temperatures and counted the number of active larvae at each temperature. As in our study, the penetrance of transgenic lethality was temperature-dependent, although there was a negative linear trend between survival and temperature, with highest survival at 18°C and no survival at 29°C. Our early embryonic criteria of lethality penetrance is distinct from the moving larvaeused by Horn and Wimmer (2003), which may partly explain the difference in results. However, our study also used a slightly different genetic construct: 95 additional base pairs in the sry α promoter sequence (s1 instead of s2) (Ibnsouda et al. 1993) and lacked flanking 5′ HS4 insulator elements in the driver cassette that were present in Horn and Wimmer (2003), thus leading to potential differences in the temperature sensitivity of the constructs’ performance.
In addition to confirming a transgene/environment interaction with respect to temperature, our results indicate that cross-generational variation in the penetrance of transgenic lethality can be influenced by parental nutrition, in particular caloric restriction. Understanding the connection between nutrition and transgenic lethality is highly relevant because resource limitation is prevalent in natural environments (Boonstra 2013). Most notably, the largest decreases in the penetrance of transgenic lethality resulted from caloric restriction under adult parental exposure and maternal pre-adult exposure to our quarter-strength. Dietary restriction induces physiological and biochemical changes in exposed organisms, including increases in lifespan (Partridge, Piper, and Mair 2005). But effects of caloric restriction can also be detected in offspring in the form of increased tolerance to heat shock, oxidative stress, and starvation (Lee et al. 2023), suggesting a potential role for these stress responses in the expression of transgenic phenotypes. Many environmental stressors modulate the activity of apoptotic signaling (Franco et al. 2009), and the hidAla5effector transgene encodes a pro-apoptotic protein. Thus, we hypothesize that environmentally mediated reductions in transgene penetrance may be explained by modification of apoptosis signaling.
Diets with below or above optimal P:C ratio also induced a change in the penetrance of transgenic lethality despite containing the same calories as the reference diet, thus suggesting that macromolecule composition can influence the penetrance of transgenic lethality. However, these results only provide clues about the relationships between transgene penetrance and diet composition. In D. melanogaster, fitness components are known to be influenced by dietary composition, most notably lifespan and reproductive success (see Lee et al. 2008), which may provide a clue about the mechanisms driving the decrease in efficiency of the lethal transgenic system. However, the contribution of each macronutrient to transgenic penetrance may be confounded by our experimental protocol, because when a diet is available at libitum, ingestion of unbalanced foods is often compensated by increased or decreased feeding to meet the required intake of a critical nutrient (usually protein), leading to over or under ingestion of other nutrients and decreased fitness (Le Couteur et al. 2016). Thus, our results indicate that future research on specific facets of diet composition may prove fruitful in identifying mechanisms behind transgene variation due to nutrition.
Estimating plasticity in the function of Tet-off systems conferring embryonic lethality can be used to create performance standards for transgenic insect strains, but the limitations of lab-based approaches for estimating risks in the field must be considered. When applied for risk forecasting purposes, biological interpretations from different definitions of failure of transgenic lethality may lead to divergent conclusions. For instance, if the penetrance of embryonic lethality remains high at elevated temperatures, as indicated by the lack of physiological escapers reported in Horn and Wimmer (2003), environments with lower temperatures would have an increased risk of failure. On the contrary, the inverted u-shaped performance curve for temperature observed in our experiments suggests a higher risk of failure in both cooler and warmer environments or seasons. Therefore, our results indicate that the composition of the transgenic system, or even the metric for evaluation of transgene penetrance can affect later interpretations of the probability of failure of any transgenic system and highlight the importance of including precise definitions of failure when comparing transgenic systems, even between those with the same modes-of-action.
Our results provide limited support for the hypothesis that the environment influences transgene penetrance by directly modulating transcriptional activation. For the most part, there was no correlation between phenotypic variation in penetrance of embryonic lethality and transcriptional variation (i.e., increased penetrance with decreased transcription) or between driver and effector components (i.e., one element increased while the other decreased or showed no change). Perhaps, the causal correlations expected between transgene expression and phenotypic penetrance would be detectable once fine-grained temporal development of the transgenic system’s expression is considered in the analysis, similar to the study of Tet-off systems in microorganisms in which the use of dynamic models allowed the Tet-off system’s response to be separated from global transcriptional fluctuations (Pedraza and van Oudenaarden 2005). Nonetheless, there are multiple functional aspects of the lethality system that we did not assay (induction time, cellular localization, potential interaction with endogenous elements, etc.) that may explain the reduced penetrance of transgenic embryonic lethality, but these thorough mechanistic experiments were beyond the scope of the current work.
While thorough characterization of the molecular components of a conditional lethality system may have merit in terms of system validation, it may not be sufficient to explain the environmentally induced plasticity of transgenic lethality. This is because the penetrance of the pro-apoptotic effector gene depends on both the expression of the recombinant protein as well as its interaction with endogenous elements participating in the apoptotic cascade. Given that penetrance of lethality was not well correlated with transcript levels of the transgene components in our study, modulation of the expression and/or activity of other apoptosis machinery is a plausible mechanism for environmentally induced plasticity in embryonic death. Several genetic elements directly interact with hid, including Drosophila inhibitor of apoptosis 1 (DIAP1) gene, which is negatively regulated by HID in the control of caspase-dependent apoptosis (Wang et al. 1999). Another candidate is Caspase-8, which is necessary for HID-induced programmed cell death, and it is activated by direct interaction with HID, such that deficiency of Caspase-8 (dredd) in flies suppresses HID-induced apoptosis (Quinn et al. 2000; Varghese et al. 2002). Taken together, the lack of relationship between transcript abundance and transgene penetrance necessitates additional experiments to identify the molecular mechanisms that underscore transgenic lethality and may contribute to transgene performance in natural environments.
Prediction of the probability of failure of a transgenic lethal system in the field is an important piece of information when conducting environmental risk assessment of genetically modified organisms. Ideally, performance estimates generated in standard laboratory conditions would provide sufficient information to predict the risk of failure for a planned release program, but our results indicate that some environmental conditions insects frequently encounter can modify the efficiency of transgenic lethality systems. This discovery highlights the inherent complexity of the genetically engineered autocidal strategy, which requires complex interactions between host genome, the transgenes, other biochemical and cellular mechanisms, and the environment (Pfennig 2021). The complexity of the system does not necessarily determine its lack of predictability, and it remains to be determined whether the observed environmentally induced plasticity of the transgenic system here evaluated is a pervasive characteristic of every transgenic lethality system. However, our results urge additional descriptions of environmental influence in the penetrance of transgenic phenotypes across the catalogue of transgenic insect strains intended for environmental release. Future research will be needed to both understand the innerworkings of transgenic function under environmental stress and provide insights to improve the robustness of transgene function across varying environments to extend the shelf life of transgenic approaches developed for insect pest management.
FRPG, AMH, DAH, and NMT conceived and designed research. FRPG conducted experiments, analyzed data and wrote the first version of the manuscript. AMH and JPB contributed with reagents and/or analytical tools. All authors critically reviewed previous versions of the manuscript, read and approved the final manuscript.
Acknowledgements
We would like to express our deepest gratitude to the high school and undergraduate research assistants Cisco Hadder, Sophia Zhou, Anabelle Wilson, Faith Boles, Leah Carpenter, and Katie Collins for their efforts in data collection. We also thank Clare Rittschof, Joe Zhou, Catherine Linnen and Doug Harrison for their valuable comments on earlier versions of the manuscript.
Funding
This work was supported by Biotechnology Risk Assessment Grants Program grant 2017-33522-27068 and Hatch Project 1010996 from the USDA National Institute of Food and Agriculture.
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
Author contributions
Conception and design of the study was performed by Fernan R. Perez-Galvez, Nicholas M. Teets, and Daniel A. Hahn. Fernan R. Perez-Galvez conducted experiments, analyzed data and wrote the first version of the manuscript. Alfred M. Handler and Justin P. Bredlau contributed with reagents and/or analytical tools. All authors critically reviewed previous versions of the manuscript, read and approved the final manuscript.
Data availability
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.
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Tables 1 to 5 are available in the Supplementary Files section
No competing interests reported.