Indirect plant-mediated interactions between heterospecific parasitoids that develop in different caterpillar species.

Parasitoids induce physiological changes in their herbivorous hosts that affect how plants respond to herbivory. The signature of parasitoids on induced plant responses to feeding by parasitized herbivores indirectly impacts insect communities interacting with the plant. The effect may extend to parasitoids and cause indirect interaction between parasitoids that develop inside different herbivore hosts sharing the food plant. However, this type of interactions among parasitoid larvae has received very little attention. In this study, we investigated sequential and simultaneous plant-mediated interactions among two host-parasitoid systems feeding on Brassica oleracea plants: Mamestra brassicae parasitized by Microplitis mediator and Pieris rapae parasitized by Cotesia rubecula. We measured the mortality, development time, and weight of unparasitized herbivores and performance of parasitoids that had developed inside the two herbivore species when sharing the food plant either simultaneously or sequentially. Plant induction by parasitized or unparasitized hosts had no significant effect on the performance of the two herbivore host species. In contrast, the two parasitoid species had asymmetrical indirect plant-mediated effects on each other's performance. Cotesia rubecula weight was 15% higher on plants induced by M. mediator-parasitized hosts, compared to control plants. In addition, M. mediator development time was reduced by 30% on plants induced by conspecific but not heterospecific parasitoids, compared to plants induced by its unparasitized host. Contrary to sequential feeding, parasitoids had no effect on each other's performance when feeding simultaneously. These results reveal that indirect plant-mediated interactions among parasitoid larvae could involve any parasitoid species whose hosts share a food plant.

Diverse cropping systems lead to higher larval mortality of the cabbage root fly (Delia radicum).

Root herbivores pose a major threat to agricultural crops. They are difficult to control and their damage often goes unnoticed until the larvae reach their most devastating late instar stages. Crop diversification can reduce pest pressure, generally without compromising yield. We studied how different diversified cropping systems affected the oviposition and abundance of the specialist cabbage root fly Delia radicum, the most important root herbivore in Brassica crops. The cropping systems included a monoculture, pixel cropping, and four variations of strip cropping with varying intra- and interspecific crop diversity, fertilization and spatial configuration. Furthermore, we assessed whether there was a link between D. radicum and other macroinvertebrates associated with the same plants. Cabbage root fly oviposition was higher in strip cropping designs compared to the monoculture and was highest in the most diversified strip cropping design. Despite the large number of eggs, there were no consistent differences in the number of larvae and pupae between the cropping systems, indicative of high mortality of D. radicum eggs and early instars especially in the strip cropping designs. D. radicum larval and pupal abundance positively correlated with soil-dwelling predators and detritivores and negatively correlated with other belowground herbivores. We found no correlations between the presence of aboveground insect herbivores and the number of D. radicum on the roots. Our findings indicate that root herbivore presence is determined by a complex interplay of many factors, spatial configuration of host plants, and other organisms residing near the roots. Supplementary Information: The online version contains supplementary material available at 10.1007/s10340-023-01629-1.

Bringing Fundamental Insights of Induced Resistance to Agricultural Management of Herbivore Pests.

In response to herbivory, most plant species adjust their chemical and morphological phenotype to acquire induced resistance to the attacking herbivore. Induced resistance may be an optimal defence strategy that allows plants to reduce metabolic costs of resistance in the absence of herbivores, allocate resistance to the most valuable plant tissues and tailor its response to the pattern of attack by multiple herbivore species. Moreover, plasticity in resistance decreases the potential that herbivores adapt to specific plant resistance traits and need to deal with a moving target of variable plant quality. Induced resistance additionally allows plants to provide information to other community members to attract natural enemies of its herbivore attacker or inform related neighbouring plants of pending herbivore attack. Despite the clear evolutionary benefits of induced resistance in plants, crop protection strategies to herbivore pests have not exploited the full potential of induced resistance for agriculture. Here, we present evidence that induced resistance offers strong potential to enhance resistance and resilience of crops to (multi-) herbivore attack. Specifically, induced resistance promotes plant plasticity to cope with multiple herbivore species by plasticity in growth and resistance, maximizes biological control by attracting natural enemies and, enhances associational resistance of the plant stand in favour of yield. Induced resistance may be further harnessed by soil quality, microbial communities and associational resistance offered by crop mixtures. In the transition to more sustainable ecology-based cropping systems that have strongly reduced pesticide and fertilizer input, induced resistance may prove to be an invaluable trait in breeding for crop resilience.

Parasitism causes changes in caterpillar odours and associated bacterial communities with consequences for host-location by a hyperparasitoid.

Microorganisms living in and on macroorganisms may produce microbial volatile compounds (mVOCs) that characterise organismal odours. The mVOCs might thereby provide a reliable cue to carnivorous enemies in locating their host or prey. Parasitism by parasitoid wasps might alter the microbiome of their caterpillar host, affecting organismal odours and interactions with insects of higher trophic levels such as hyperparasitoids. Hyperparasitoids parasitise larvae or pupae of parasitoids, which are often concealed or inconspicuous. Odours of parasitised caterpillars aid them to locate their host, but the origin of these odours and its relationship to the caterpillar microbiome are unknown. Here, we analysed the odours and microbiome of the large cabbage white caterpillar Pieris brassicae in relation to parasitism by its endoparasitoid Cotesia glomerata. We identified how bacterial presence in and on the caterpillars is correlated with caterpillar odours and tested the attractiveness of parasitised and unparasitised caterpillars to the hyperparasitoid Baryscapus galactopus. We manipulated the presence of the external microbiome and the transient internal microbiome of caterpillars to identify the microbial origin of odours. We found that parasitism by C. glomerata led to the production of five characteristic volatile products and significantly affected the internal and external microbiome of the caterpillar, which were both found to have a significant correlation with caterpillar odours. The preference of the hyperparasitoid was correlated with the presence of the external microbiome. Likely, the changes in external microbiome and body odour after parasitism were driven by the resident internal microbiome of caterpillars, where the bacterium Wolbachia sp. was only present after parasitism. Micro-injection of Wolbachia in unparasitised caterpillars increased hyperparasitoid attraction to the caterpillars compared to untreated caterpillars, while no differences were found compared to parasitised caterpillars. In conclusion, our results indicate that host-parasite interactions can affect multi-trophic interactions and hyperparasitoid olfaction through alterations of the microbiome.

Family dinner: Transcriptional plasticity of five Noctuidae (Lepidoptera) feeding on three host plant species.

Polyphagous insects often show specialization in feeding on different host plants in terms of survival and growth and, therefore, can be considered minor or major pests of particular hosts. Whether polyphagous insects employ a common transcriptional response to cope with defenses from diverse host plants is under-studied. We focused on patterns of transcriptional plasticity in polyphagous moths (Noctuidae), of which many species are notorious pests, in relation to herbivore performance on different host plants. We compared the transcriptional plasticity of five polyphagous moth species feeding and developing on three different host plant species. Using a comparative phylogenetic framework, we evaluated if successful herbivory, as measured by larval performance, is determined by a shared or lineage-specific transcriptional response. The upregulated transcriptional activity, or gene expression pattern, of larvae feeding on the different host plants and artificial control diet was highly plastic and moth species-specific. Specialization, defined as high herbivore success for specific host plants, was not generally linked to a lower number of induced genes. Moths that were more distantly related and showing high herbivore success for certain host plants showed shared expression of multiple homologous genes, indicating convergence. We further observed specific transcriptional responses within phylogenetic lineages. These expression patterns for specific host plant species are likely caused by shared evolutionary histories, for example, symplesiomorphic patterns, and could therefore not be associated with herbivore success alone. Multiple gene families, with roles in plant digestion and detoxification, were widely expressed in response to host plant feeding but again showed highly moth species-specific. Consequently, high herbivore success for specific host plants is also driven by species-specific transcriptional plasticity. Thus, potential pest moths display a complex and species-specific transcriptional plasticity.

Evolution of koinobiont parasitoid host regulation and consequences for indirect plant defence.

Tritrophic interactions among plants, herbivorous insects and their parasitoids have been well studied in the past four decades. Recently, a new angle has been uncovered: koinobiont parasitoids, that allow their host to keep feeding on the plant for a certain amount of time after parasitism, indirectly alter plant responses against herbivory via the many physiological changes induced in their herbivorous hosts. By affecting plant responses, parasitoids may indirectly affect the whole community of insects interacting with plants induced by parasitized herbivores and have extended effects on plant fitness. These important findings have renewed research interests on parasitoid manipulation of their host development. Parasitoids typically arrest their host development before the last instar, resulting in a lower final weight compared to unparasitized hosts. Yet, some parasitoids prolong their host development, leading to larger herbivores that consume more plant material than unparasitized ones. Furthermore, parasitoid host regulation is plastic and one parasitoid species may arrest or promote its host growth depending on the number of eggs laid, host developmental stage and species as well as environmental conditions. The consequences of plasticity in parasitoid host regulation for plant-insect interactions have received very little attention over the last two decades, particularly concerning parasitoids that promote their host growth. In this review, we first synthesize the mechanisms used by parasitoids to regulate host growth and food consumption. Then, we identify the evolutionary and environmental factors that influence the direction of parasitoid host regulation in terms of arrestment or promotion of host growth. In addition, we discuss the implication of different host regulation types for the parasitoid's role as agent of plant indirect defence. Finally, we argue that the recent research interests about parasitoid plant-mediated interactions would strongly benefit from revival of research on the mechanisms, ecology and evolution of host regulation in parasitoids.

Plant defense strategies against attack by multiple herbivores.

Plants may effectively tailor defenses by recognizing their attackers and reprogramming their physiology. Although most plants are under attack by a large diversity of herbivores, surprisingly little is known about the physiological capabilities of plants to deal with attack by multiple herbivores. Studies on dual herbivore attack identified that defense against one attacker may cause energetic and physiological constraints to deal with a second attacker. How these constraints shape plant plasticity in defense to their full community of attackers is a major knowledge gap in plant science. Here, we provide a framework for plant defense to multiherbivore attack by defining the repertoire of plastic defense strategies that may allow plants to optimize their defenses against a multitude of stressors.

The Ecology of Hyperparasitoids.

Hyperparasitoids are some of the most diverse members of insect food webs. True hyperparasitoids parasitize the larvae of other parasitoids, reaching these larvae with their ovipositor through the herbivore that hosts the parasitoid larva. During pupation, primary parasitoids also may be attacked by pseudohyperparasitoids that lay their eggs on the parasitoid (pre)pupae. By attacking primary parasitoids, hyperparasitoids may affect herbivore population dynamics, and they have been identified as a major challenge in biological control. Over the past decades, research, especially on aphid- and caterpillar-associated hyperparasitoids, has revealed that hyperparasitoids challenge rules on nutrient use efficiency in trophic chains, account for herbivore outbreaks, or stabilize competitive interactions in lower trophic levels, and they may use cues derived from complex interaction networks to locate their hosts. This review focuses on the fascinating ecology of hyperparasitoids related to how they exploit and locate their often inconspicuous hosts and the insect community processes in which hyperparasitoids are prominent players.

Plasticity in induced resistance to sequential attack by multiple herbivores in Brassica nigra.

In nature, plants interact with multiple insect herbivores that may arrive simultaneously or sequentially. There is extensive knowledge on how plants defend themselves against single or dual attack. However, we lack information on how plants defend against the attack of multiple herbivores that arrive sequentially. In this study, we investigated whether Brassica nigra L. plants are able to defend themselves against caterpillars of the late-arriving herbivore Plutella xylostella L., when plants had been previously exposed to sequential attack by four other herbivores (P. xylostella, Athalia rosae, Myzus persicae and Brevicoryne brassicae). We manipulated the order of arrival and the history of attack by four herbivores to investigate which patterns in sequential herbivory determine resistance against the fifth attacker. We recorded that history of sequential herbivore attack differentially affected the capability of B. nigra plants to defend themselves against caterpillars of P. xylostella. Caterpillars gained less weight on plants attacked by a sequence of four episodes of attack by P. xylostella compared to performance on plants that were not previously damaged by herbivores. The number of times the plant was attacked by herbivores of the same feeding guild, the identity of the first attacker, the identity and the guild of the last attacker as well as the order of attackers within the sequence of multiple herbivores influenced the growth of the subsequent herbivory. In conclusion, this study shows that history of sequential attack is an important factor determining plant resistance to herbivores.

Impact of parasitoid-associated polydnaviruses on plant-mediated herbivore interactions.

Insect herbivores interact via plant-mediated interactions in which one herbivore species induces changes in plant quality that affects the performance of a second phytophagous insect that shares the food plant. These interactions are often asymmetric due to specificity in induced plant responses to herbivore attack, amount of plant damage, elicitors in herbivore saliva and plant organ damaged by herbivores. Parasitoids and their symbiotic polydnaviruses alter herbivore physiology and behaviour and may influence how plants respond to parasitized herbivores. We argue that these phenomena affect plant-mediated interactions between herbivores. We identify that the extended phenotype of parasitoid polydnaviruses is an important knowledge gap in interaction networks of insect communities.

Flowers prepare thyselves: leaf and root herbivores induce specific changes in floral phytochemistry with consequences for plant interactions with florivores.

The phenotypic plasticity of flowering plants in response to herbivore damage to vegetative tissues can affect plant interactions with flower-feeding organisms. Such induced systemic responses are probably regulated by defence-related phytohormones that signal flowers to alter secondary chemistry that affects resistance to florivores. Current knowledge on the effects of damage to vegetative tissues on plant interactions with florivores and the underlying mechanisms is limited. We compared the preference and performance of two florivores on flowering Brassica nigra plants damaged by one of three herbivores feeding from roots or leaves. To investigate the underlying mechanisms, we quantified expression patterns of marker genes for defence-related phytohormonal pathways, and concentrations of phytohormones and glucosinolates in buds and flowers. Florivores displayed contrasting preferences for plants damaged by herbivores feeding on roots and leaves. Chewing florivores performed better on plants damaged by folivores, but worse on plants damaged by the root herbivore. Chewing root and foliar herbivory led to specific induced changes in the phytohormone profile of buds and flowers. This resulted in increased glucosinolate concentrations for leaf-damaged plants, and decreased glucosinolate concentrations for root-damaged plants. The outcome of herbivore-herbivore interactions spanning from vegetative tissues to floral tissues is unique for the inducing root/leaf herbivore and receiving florivore combination.

Plant age at the time of ozone exposure affects flowering patterns, biotic interactions and reproduction of wild mustard.

Exposure of plants to environmental stressors can modify their metabolism, interactions with other organisms and reproductive success. Tropospheric ozone is a source of plant stress. We investigated how an acute exposure to ozone at different times of plant development affects reproductive performance, as well as the flowering patterns and the interactions with pollinators and herbivores, of wild mustard plants. The number of open flowers was higher on plants exposed to ozone at earlier ages than on the respective controls, while plants exposed at later ages showed a tendency for decreased number of open flowers. The changes in the number of flowers provided a good explanation for the ozone-induced effects on reproductive performance and on pollinator visitation. Ozone exposure at earlier ages also led to either earlier or extended flowering periods. Moreover, ozone tended to increase herbivore abundance, with responses depending on herbivore taxa and the plant age at the time of ozone exposure. These results suggest that the effects of ozone exposure depend on the developmental stage of the plant, affecting the flowering patterns in different directions, with consequences for pollination and reproduction of annual crops and wild species.

Plant defence to sequential attack is adapted to prevalent herbivores.

Plants have evolved plastic defence strategies to deal with the uncertainty of when, by which species and in which order attack by herbivores will take place1-3. However, the responses to current herbivore attack may come with a cost of compromising resistance to other, later arriving herbivores. Due to antagonistic cross-talk between physiological regulation of plant resistance to phloem-feeding and leaf-chewing herbivores4-8, the feeding guild of the initial herbivore is considered to be the primary factor determining whether resistance to subsequent attack is compromised. We show that, by investigating 90 pairwise insect-herbivore interactions among ten different herbivore species, resistance of the annual plant Brassica nigra to a later arriving herbivore species is not explained by feeding guild of the initial attacker. Instead, the prevalence of herbivore species that arrive on induced plants as approximated by three years of season-long insect community assessments in the field explained cross-resistance. Plants maintained resistance to prevalent herbivores in common patterns of herbivore arrival and compromises in resistance especially occurred for rare patterns of herbivore attack. We conclude that plants tailor induced defence strategies to deal with common patterns of sequential herbivore attack and anticipate arrival of the most prevalent herbivores.

Parasitism by endoparasitoid wasps alters the internal but not the external microbiome in host caterpillars.

BACKGROUND: The microbiome of many insects consists of a diverse community of microorganisms that can play critical roles in the functioning and overall health of their hosts. Although the microbial communities of insects have been studied thoroughly over the past decade, little is still known about how biotic interactions affect the microbial community structure in and on the bodies of insects. In insects that are attacked by parasites or parasitoids, it can be expected that the microbiome of the host insect is affected by the presence of these parasitic organisms that develop in close association with their host. In this study, we used high-throughput amplicon sequencing targeting both bacteria and fungi to test the hypothesis that parasitism by the endoparasitoid Cotesia glomerata affected the microbiome of its host Pieris brassicae. Healthy and parasitized caterpillars were collected from both natural populations and a laboratory culture. RESULTS: Significant differences in bacterial community structure were found between field-collected caterpillars and laboratory-reared caterpillars, and between the external and the internal microbiome of the caterpillars. Parasitism significantly altered the internal microbiome of caterpillars, but not the external microbiome. The internal microbiome of all parasitized caterpillars and of the parasitoid larvae in the caterpillar hosts was dominated by a Wolbachia strain, which was completely absent in healthy caterpillars, suggesting that the strain was transferred to the caterpillars during oviposition by the parasitoids. CONCLUSION: We conclude that biotic interactions such as parasitism have pronounced effects on the microbiome of an insect host and possibly affect interactions with higher-order insects.

Plant-phenotypic changes induced by parasitoid ichnoviruses enhance the performance of both unparasitized and parasitized caterpillars.

There is increasing awareness that interactions between plants and insects can be mediated by microbial symbionts. Nonetheless, evidence showing that symbionts associated with organisms beyond the second trophic level affect plant-insect interactions are restricted to a few cases belonging to parasitoid-associated bracoviruses. Insect parasitoids harbour a wide array of symbionts which, like bracoviruses, can be injected into their herbivorous hosts to manipulate their physiology and behaviour. Yet, the function of these symbionts in plant-based trophic webs remains largely overlooked. Here, we provide the first evidence of a parasitoid-associated symbiont belonging to the group of ichnoviruses which affects the strength of plant-insect interactions. A comparative proteomic analysis shows that, upon parasitoid injection of calyx fluid containing ichnovirus particles, the composition of salivary glands of caterpillars changes both qualitatively (presence of two viral-encoded proteins) and quantitatively (abundance of several caterpillar-resident enzymes, including elicitors such as glucose oxidase). In turn, plant phenotypic changes triggered by the altered composition of caterpillar oral secretions affect the performance of herbivores. Ichnovirus manipulation of plant responses to herbivory leads to benefits for their parasitoid partners in terms of reduced developmental time within the parasitized caterpillar. Interestingly, plant-mediated ichnovirus-induced effects also enhance the performances of unparasitized herbivores which in natural conditions may feed alongside parasitized ones. We discuss these findings in the context of ecological costs imposed to the plant by the viral symbiont of the parasitoid. Our results provide intriguing novel findings about the role played by carnivore-associated symbionts on plant-insect-parasitoid systems and underline the importance of placing mutualistic associations in an ecological perspective.

Intraspecific variation in plant-associated herbivore communities is phylogenetically structured in Brassicaceae.

As a result of co-evolution between plants and herbivores, related plants often interact with similar herbivore communities. Variation in plant-herbivore interactions is determined by variation in underlying functional traits and by ecological and stochastic processes. Hence, typically, only a subset of possible interactions is realised on individual plants. We show that insect herbivore communities assembling on individual plants are structured by plant phylogeny among 12 species in two phylogenetic lineages of Brassicaceae. This community sorting to plant phylogeny was retained when splitting the community according to herbivore feeding guilds. Relative abundance of herbivores as well as the size of the community structured community dissimilarity among plant species. Importantly, the amount of intraspecific variation in realised plant-herbivore interactions is also phylogenetically structured. We argue that variability in realised interactions that are not directly structured by plant traits is ecologically relevant and must be considered in the evolution of plant defences.

Insect species richness affects plant responses to multi-herbivore attack.

Plants are often attacked by multiple insect herbivores. How plants deal with an increasing richness of attackers from a single or multiple feeding guilds is poorly understood. We subjected black mustard (Brassica nigra) plants to 51 treatments representing attack by an increasing species richness (one, two or four species) of either phloem feeders, leaf chewers, or a mix of both feeding guilds when keeping total density of attackers constant and studied how this affects plant resistance to subsequent attack by caterpillars of the diamondback moth (Plutella xylostella). Increased richness in phloem-feeding attackers compromised resistance to P. xylostella. By contrast, leaf chewers induced a stronger resistance to subsequent attack by caterpillars of P. xylostella while species richness did not play a significant role for chewing herbivore induced responses. Attack by a mix of herbivores from different feeding guilds resulted in plant resistance similar to resistance levels of plants that were not previously exposed to herbivory. We conclude that B. nigra plants channel their defence responses stronger towards a feeding-guild specific response when under multi-species attack by herbivores of the same feeding guild, but integrate responses when simultaneously confronted with a mix of herbivores from different feeding guilds.

Predictability of Biotic Stress Structures Plant Defence Evolution.

To achieve ecological and reproductive success, plants need to mitigate a multitude of stressors. The stressors encountered by plants are highly dynamic but typically vary predictably due to seasonality or correlations among stressors. As plants face physiological and ecological constraints in responses to stress, it can be beneficial for plants to evolve the ability to incorporate predictable patterns of stress in their life histories. Here, we discuss how plants predict adverse conditions, which plant strategies integrate predictability of biotic stress, and how such strategies can evolve. We propose that plants commonly optimise responses to correlated sequences or combinations of herbivores and pathogens, and that the predictability of these patterns is a key factor governing plant strategies in dynamic environments.

Insect egg-killing: a new front on the evolutionary arms-race between brassicaceous plants and pierid butterflies.

Evolutionary arms-races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front-line of plant defence is the killing of herbivorous insect eggs. We test whether an egg-killing plant trait has an evolutionary basis in such a plant-insect arms-race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)-like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg-killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade-specific induction of strong, egg-killing HR-like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR-like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg-killing. Our findings suggest that the plants' egg-killing trait is a new front on the evolutionary arms-race between Brassicaceae and pierid butterflies beyond the well-studied plant toxins that have evolved against their caterpillars.

Effects of ozone stress on flowering phenology, plant-pollinator interactions and plant reproductive success.

Tropospheric ozone is a highly oxidative pollutant with the potential to alter plant metabolism. The direct effects of ozone on plant phenotype may alter interactions with other organisms, such as pollinators, and, consequently, affect plant reproductive success. In a set of greenhouse experiments, we tested whether exposure of plants to a high level of ozone affected their phenological development, their attractiveness to four different pollinators (mason bees, honeybees, hoverflies and bumblebees) and, ultimately, their reproductive success. Exposure of plants to ozone accelerated flowering, particularly on plants that were growing in autumn, when light and temperature cues, that commonly promote flowering, were weaker. Simultaneously, there was a tendency for ozone-exposed plants to disinvest in vegetative growth. Plant exposure to ozone did not substantially affect pollinator preference, but bumblebees had a tendency to visit more flowers on ozone-exposed plants, an effect that was driven by the fact that these plants tended to have more open flowers, meaning a stronger attraction signal. Honeybees spent more time per flower on ozone-exposed plants than on control plants. Acceleration of flower production and the behavioural responses of pollinators to ozone-exposed plants resulted in retained reproductive fitness of plants pollinated by bumblebees, honeybees and mason bees, despite the negative effects of ozone on plant growth. Plants that were pollinated by hoverflies had a reduction in reproductive fitness in response to ozone. In a natural setting, acceleration of flowering by ozone might foster desynchronization between plant and pollinator activities. This can have a strong impact on plants with short flowering periods and on plants that, unlike wild mustard, lack compensatory mechanisms to cope with the absence of pollinator activity in the beginning of flowering.