Ongoing ecological and evolutionary consequences by the presence of transgenes in a wild cotton population

In this study, we showed that the expression of cry and cp4-epsps genes in wild cotton altered the secretion of EFN, the associations with different ant species, and the levels of herbivore damage on target plants. Wcry constantly maintained a high production of EFN, regardless of the MeJA treatment, but nectar production was minimal in Wcp4-epsps. These changes in nectar inducibility seem to modify the composition of ant communities, foster the dominance of the generalist and defensive species C. planatus in Bt plants and the presence of ants without defensive role, M. ebeninum, in the herbicide tolerant genotype, while W plants had both defending species (C. planatus, C. rectangularis aulicus and P. gracilis) and invasive ant species (P. longicornis) in the same proportion. Furthermore, herbivore damage and its associated ant community were different according to the introgressed transgene.

Wild and introgressed cotton do not display phenotypic equivalence in natural conditions

In general, it has been assumed that introgressed and wild genotypes should display similar phenotypes in the absence of the selection agents targeted by transgenes. However, when we compared the control group and the three genotypes, we registered different nectar secretion patterns among them (Fig. 1). Similar results have been registered in populations of bt rice and glyphosate-tolerant sunflowers living in natural conditions where introgressed plants are different from their wild relatives⁵.

Transgene expression modified indirect induced defences in wild cotton

Most plants are able to induce responses after herbivore damage and/or phytohormone exogenous application (i.e. jasmonic acid, JA; methyl jasmonate, MeJA; and salicylic acid, SA)^11,28,29. However, unlike wild plants without transgenes, individuals with transgenes were not sensitive to the induction treatment with MeJA for increasing their EFN production (Fig. 1). These results contrast with previous reports on cultivated varieties, such as Bt and glyphosate-resistant (cp4-epsps), in which direct defences such as gossypol terpenoids (160%), hemigossypolone (160%), helicoids 1|4 (213%) and indirect defenses, such as volatile compounds (VOCs) (171.2%) and extrafloral nectar (EFN) (133%), were reported to increase in plants sprinkled with JA and MeJA^21,28,29,30.

The inability of plants with transgenes to have the production of extrafloral nectar induced in them was related to different processes dependent on the identity of the transgenes in question. Whereas Wcry control plants had a high EFN production equivalent to the induced state of W plants, EFN production in Wcp4-epsps plants was inhibited. Contrasting these findings with results obtained under controlled conditions (i.e. greenhouse and crop conditions)^3,21, we suggest that EFN production is linked to genotypes with transgenes and abiotic stress in the coastal dunes, because transgenes are connected to main metabolic pathways that respond to stressful conditions²¹.

Wild cotton with cp4-epsps

In the absence of herbicides acting as a selection agent, wild plants with cp4-epsps exhibited large differences compared to wild plants without them. Their low nectar production (> 8 µg/mL) (Fig. 1) could be linked to the crosstalk between the jasmonate and the salicylate (SA) pathways (Fig. 4, orange and purple section). In G. hirsutum and other species, SA signalling has been proven to negatively affect JA signalling (e.g. Zea mays, Solanum lycopersicum, Nicotiana tabacum and Arabidopsis thaliana)^31,32,33: therefore, we suggest an interference between the SA and JA pathways given previous reports that an over-expression of the cp4-epsps gene modifies the second part of the shikimate pathway (post-chorismate), which leads to the synthesis of essential amino acids as phenylalanine, tryptophan, or tyrosine, the latter being a precursor of benzoic acid BE, and SA^34,35 (Fig. 4, purple section). This evidence highlights that hidden crosstalk effects among different metabolic pathways can scale up and modify plant phenotypes (e.g. extrafloral nectar production).

Wild cotton with cry

Wild cotton plants with cry genes continuously produced EFN as a constitutive defence (Fig. 1), in equivalent quantities as the induced state of W plants. EFN production is regulated by the octadecanoid signalling pathway, which can be activated by herbivore damage, mechanical damage, and phytohormones, such as JA and MeJA^21,28 (Fig. 4, green section). However, for cotton, a specific elicitor is not necessary³⁶. Four key genes for the synthesis of JA and MeJA have been described: AOS, AOC, HPL, and COI1³⁷. In Bt maize, studies comparing GM corn and its isogenic lines report an increase of 24% in phenols and 63% of DIMBOA (2,4-dihidroxi-7-metoxi-1,4-benzoxazin-3-ona; natural defences against lepidopteran herbivores)¹¹. This is consistent with observations of a synergy between maize direct defences and Bt genes, after exogenous applications of JA (Fig. 4, orange section). Considering the latter, we suggest that Wcry cotton may present a similar response, as the genes activating the JA pathway are GhAOS and GhCOI1 (homologs to maize JA biosynthesis genes: ZmAOS and ZmCOI1), in addition to Ghppo1, which confers natural resistance to lepidopteran pest, such as H. armigera³⁸. The interaction of cry with other genes could modify the production of EFN in Wcry plants.

Effect of the transgenes’ expression on ants associated to wild cotton

We identified eight species of ants harvesting EFN (Table 2), but with distinctive communities as a function of the plant genotype. This result suggests that the change in quantity, and possibly the composition and quality of EFN, can influence the ant community associated with G. hirsutum^39,40,41.

Changes in plant reward production could potentially compromise the attraction of natural enemies of herbivores⁴². In our study, the availability of EFN was modified. Although species richness was the same as in W plants (Table 2), the most abundant ant species associated with Wcp4-epsps plants, M. ebeninum, is considered a generalist species. Moreover, due to the lack of aggressive behaviour, this species does not represent an effective biotic defence⁴³. The high abundance of this non-defensive species could be associated with the greater herbivore damage observed in Wcp4-epsps plants (Fig. 2). In contrast, W or Wcry plants showed a greater abundance of more aggressive ant species such as C. planatus, C. rectangulatus, and P. brunneus and significantly less herbivore damage.

In Wcry cotton, the community of patrolling ants was mainly dominated by C. planatus, in both treatments (control and induction). Interestingly, although the amount of nectar did not vary between treatments, the abundance of ants was significantly different. The dominance of a single ant species could have benefited the plants with increased indirect defence, reducing herbivore damage and promoting a greater seed production per plant, as described in Turnera ulmifolia⁴⁴, Schomburgkia tibicinis⁴⁵, and Opuntia stricta⁴². However, considering the aggressive and dominant behaviour of C. planatus, there may be ecological costs through antagonistic relationships with pollinators. Ants can interrupt pollination and affect plant fitness^25,46,47. The outcome of these mutualistic and antagonistic interactions requires further study.

Effects of transgenes on herbivore damage

Considering that the type of mutualism that cotton sustains with ants is defensive, we suggest that the change we observed in the composition of ants is likely to have influenced herbivore damage in the different genotypes, which in turn has the potential to reduce fitness as shown by other studies of cotton^48,49,50. However, a study carried out on wild upland cotton reported that plants tolerate intermediate levels of leaf damage inflicted by leaf-chewing insects (< 50%)⁵¹. Thus, the increased herbivore damage observed in Wcp4-epsps plants (< 10%; 4.402 ± 0.863) does not necessarily jeopardize plant growth or reproductive success. For example, in the absence of glyphosate, transgenic hybrids of rice and soybean show changes in their phenology (i.e. early flowering and shorter germination times) and an increase in their fitness (i.e. larger fruit and seed sets) compared to their wild relatives^4,5. It has been suggested that the over-expression of the epsps gene is responsible for these compensation effects on plant fitness, in view of the biotic and abiotic stressors that activate the shikimate pathway^5,15. Further investigation to find out if this is the case for Wcp4-epsps cotton is currently being developed under natural conditions.

In contrast, Wcry plants exhibited the lowest herbivore damage, although not significantly different from wild plants without transgenes (Fig. 3). The expression of cry genes, conferring a new defensive trait against lepidopterans, may represent an advantage for plants under selection when target herbivores are present. For example, within the natural distribution of wild sunflowers, genotypes with cry genes showed less herbivore damage than their wild relatives (WR), increasing their seed production by 55%⁴. Nonetheless, in G. hirsutum we did not find differences in herbivore damage when compared to the W and Wcry genotypes, probably because lepidopteran species targeted by Bt cotton were not present in our study site, hence, cry expression might not have a role in increasing fitness. However, indirect effects could be present through its interaction with native herbivores. For example, experiments under laboratory and experimental conditions conducted on maize with cry genes, have shown non-lethal effects in the physical condition of non-target caterpillars (e.g. smaller size, lower weight, lower survival, and more larval instars)⁵². Likewise, if the nutritious quality of herbivore tissues changes after consuming Bt toxins, these effects could cascade to higher trophic levels^53,54, increasing mortality and decreasing longevity or development of predators. This has been shown for chewing predators (e.g. Chrysoperla carnea and lady beetles, Coccinellidae) because they ingest the gut of the prey, where most of the toxins are concentrated⁵⁴. Parasitoids have also been found to be affected after the consumption of Bt toxins contained in their preys⁵⁵. When the wasp Microplitis mediator (parasitoid of H. armigera) was fed with larvae containing cry toxins, they extended its egg and larval development time by 1–2 days, significantly decreasing its pupal weight by 35%, and its overall longevity when the toxin concentrations were high (4–8 μg g⁻¹). Hence, a question that warrants further investigation is how Wcry cotton genotypes in the wild affect herbivore and predator communities and their interactions.

By means of an integrative methodology, we evaluated the effect of the cry and cp4-epsps gene expression in wild cotton plants. As a result, we obtained the following noteworthy results: (1) differential response in the induced defence mechanism (extrafloral nectar production) concordat to plant genotype; and (2) modification of biotic interactions between introgressed cotton and relevant organisms, under natural conditions.

Although several hypotheses have been raised regarding the consequences of GMO release into the environment (e.g. gene flow, hybridization, and introgression), these have only been tested under controlled conditions. We found that some theory-based concerns can be confirmed when performing functional experimental designs in the wild. First, it is possible to investigate ecological and evolutionary impacts of new genes under natural conditions by studying community processes, such as changes in tri-trophic interactions. Second, we detected physiological and possible metabolic alterations generated by the expression of transgenes in wild cotton plants without the pressure of selection agents (pests and herbicides) targeted by those genes. Third, until 2008, 4 wild metapopulations of upland cotton showed evidence of introgression with GM cotton², and recently, introgression in the Baja California Sur, BCSM, Central Pacific, CPM, and Yucatan Peninsula YPM metapopulations has been reported³. In this study, we reaffirm the presence of transgenes in the YPM, but it’s important to consider that the establishment of these has been fast. Whereas during the first monitoring in 2008, YPM metapopulation didn’t register the presence of transgenes, in 2018, 60.64% of 61 plants had them (Table 1).

In natural ecosystems, changes at different scales (i.e. genetic, individual, and community), following the introduction of novel genes lead to endless research possibilities. Through them we can integrate broad information regarding biological control, agro-biotechnologies, and conservation biology, with promising further applications. Although we do not know the routes of transgene dispersion, we provide evidence of some of the mechanisms that could favour the establishment and persistence of these new genes, mainly due to their interaction with key defence metabolic pathways. However, transgene frequency in wild populations and the associated ecological consequences must continue to be monitored and evaluated so as to contribute information that allows us to make decisions for the conservation of the primary genetic pool and the ecological and evolutionary processes that have shaped its diversity.

Alterations in the defence mechanisms of wild relatives in one of the most important crops for humanity represents critical evidence on the threats of introgressed genes to biological and cultural heritage for the following generations. Such negative consequences would be enough to envision GMO liberation into the environment, short and long distance from its origin centre, from a different perspective. At this point, we are in a watershed moment to: (1) develop the necessary research to mitigate the ecological, evolutionary caused by the introgression of GMOs into the wild-to-domesticated complex of the species used, as well as reformulating risk assessments for protection goals; (2) fill gaps in our knowledge of the complex dispersion routes of transgenes from crop to wild relatives and native varieties; and finally, (3) prioritize the in-situ conservation of the primary gene pool without transgenes. All these efforts would contribute to the integration of the available disperse information so we may understand the consequences of transgenic plants coexisting with their relatives. As we demonstrated in the current study, the presence of these genes can cause intrinsic changes in wild populations of cotton (allelic frequency), and changes in their ecological interactions. If we want to conserve in-situ the primary gene pool of wild relatives, we must work to identify the ecological and evolutionary processes affected by the existence and permanence of these transgenes within their populations. Upon the detection of these genes, mitigation strategies to reduce the magnitude of the damage can be promptly designed.

Source: Ecology - nature.com