**4.3 Negative effects of insect herbivory on photosynthesis**

In the absence of compensatory mechanisms, insect herbivory causing removal and/or injury of plant tissues most frequently leads to a direct suppression of photosynthetic activity. A seminal report describing the outcome of an extensive examination of the pertinent literature available at the time, indicated that over 50% of all plant-insect interactions, predominantly involving leaf-mining, stem-boring, galling or sucking leaf injury, resulted in a loss of photosynthetic capacity, frequently manifested as decreased photosynthetic rate (Pn) (Welter, 1989). The reduction in chlorophyll content in response to insect damage, frequently reported in plants attacked by phloem feeding insects, has been also reported to result in a decrease in photosynthesis (Kaakeh et al., 1992; Cabrera et al., 1994), with even small reductions leading to a drastic reduction in the photosynthetic rate (Nagaraj et al., 2002). Interestingly, changes in leaf pigment composition caused by insect herbivory were found to have potential application for remote sensing pest detection in Australia. Thus, the reduction in leaf chlorophyll content occurring concomitantly with an increase in photoprotective pigments, known to be a sensitive indicator of plant stress caused by root feeding phylloxera grapevine pests, was exploited for the development of a phylloxera-specific remote detection system (Blanchfield et al., 2006). Additional factors contributing to decreased photosynthesis include changes in the nutrient status of leaves caused by competition between plant sinks and additional sinks created by insect herbivores, mostly sap-feeders or gall-formers (see above), decreased stomatal conductance, which is coupled to reduced WUE and altered water transport, stomatal aperture and/or sucrose transport and loading. Most of these conditions are also known to influence indirect suppression of photosynthesis, as described below.

However, as it has been mentioned already, a plant's response to herbivory is often variable and usually depends on the combined contribution of several factors including the type of tissue injured and the extent tissue damage. An illustrative example for this effect is given by reported data showing that the removal of leaf tissue from soybean by herbivores such as Japanese beetles (*Popillia japonica*), corn earworm caterpillars (*Helicoverpa zea*) (Aldea et al., 2005), cabbage loopers (*Trichoplusia ni*), and green clover-worms (*Plathypena scabra*) (Hammond & Pedigo, 1981; Ostlie & Pedigo, 1984) caused an increase in water loss from damaged tissue, but had a minimal effect on net photosynthesis. Conversely, chewing

activity.

In the absence of compensatory mechanisms, insect herbivory causing removal and/or injury of plant tissues most frequently leads to a direct suppression of photosynthetic activity. A seminal report describing the outcome of an extensive examination of the pertinent literature available at the time, indicated that over 50% of all plant-insect interactions, predominantly involving leaf-mining, stem-boring, galling or sucking leaf injury, resulted in a loss of photosynthetic capacity, frequently manifested as decreased photosynthetic rate (Pn) (Welter, 1989). The reduction in chlorophyll content in response to insect damage, frequently reported in plants attacked by phloem feeding insects, has been also reported to result in a decrease in photosynthesis (Kaakeh et al., 1992; Cabrera et al., 1994), with even small reductions leading to a drastic reduction in the photosynthetic rate (Nagaraj et al., 2002). Interestingly, changes in leaf pigment composition caused by insect herbivory were found to have potential application for remote sensing pest detection in Australia. Thus, the reduction in leaf chlorophyll content occurring concomitantly with an increase in photoprotective pigments, known to be a sensitive indicator of plant stress caused by root feeding phylloxera grapevine pests, was exploited for the development of a phylloxera-specific remote detection system (Blanchfield et al., 2006). Additional factors contributing to decreased photosynthesis include changes in the nutrient status of leaves caused by competition between plant sinks and additional sinks created by insect herbivores, mostly sap-feeders or gall-formers (see above), decreased stomatal conductance, which is coupled to reduced WUE and altered water transport, stomatal aperture and/or sucrose transport and loading. Most of these conditions are also known to influence indirect

However, as it has been mentioned already, a plant's response to herbivory is often variable and usually depends on the combined contribution of several factors including the type of tissue injured and the extent tissue damage. An illustrative example for this effect is given by reported data showing that the removal of leaf tissue from soybean by herbivores such as Japanese beetles (*Popillia japonica*), corn earworm caterpillars (*Helicoverpa zea*) (Aldea et al., 2005), cabbage loopers (*Trichoplusia ni*), and green clover-worms (*Plathypena scabra*) (Hammond & Pedigo, 1981; Ostlie & Pedigo, 1984) caused an increase in water loss from damaged tissue, but had a minimal effect on net photosynthesis. Conversely, chewing

Table 1. Some examples of positive or neutral effects on photosynthesis after herbivory

Elevated CO2 assimilation rate was sufficient to compensate for loss of photosynthetic active tissue. Stomatal conductance and intercellular CO2 were not affected. Mirid salivary secretions treatment also increased photosynthetic

Results Method Reference

GE and fluorescence imaging (FI) Halitschke et al., 2011

Damage type/ feeding guild

Foliagechewing feeder

**4.3 Negative effects of insect herbivory on photosynthesis** 

suppression of photosynthesis, as described below.

Plant species Herbivore

*Nicotiana attenuata* 

damage.

species

Mirid bugs (*Tupiocoris notatus*)

damage by skeletonizing Mexican bean beetles (*Epilachna varivestis*) caused substantial losses of photosynthesis in the remaining leaf tissue (Peterson et al., 1998). It was hypothesized that the scraping and crushing of interveinal leaf tissue caused by feeding adults and larvae of Mexican bean beetles may have exacerbated localized water stress, ultimately causing tissue desiccation and photosynthesis repression. The timing of damage is also considered to be an important factor. In this regard, early season damage has been usually found to cause more pronounced changes in plants than late season damage, which is in accordance with the assumption that vigorously growing foliage has a greater capacity to respond to various stimuli, including damage. Also, seedlings are generally more susceptible to photosynthetic damage because of a shortage of reserves due to their smaller size or to limitations in nutrient acquisition (Nykänen & Koricheva, 2004; Hódar et al., 2008). A report recording the response of potted fruitless grapevines (*Vitis labrusca* var. Niagara) to early and late season mechanical and insect defoliation was in accordance with this concept by showing that growth, single leaf photosynthesis, and whole-vine photosynthesis were more tolerant to foliar injury late in the season than early in the season (Mercader & Isaacs, 2003). Similar results were obtained from a series of experiments performed to examine a possible trade-off between photosynthesis with defense or reproduction in the common milkweed *Asclepias syriaca*, which is a plant that accumulates toxic cardenolides in a constitutive or inducible manner and is also susceptible to insect damage during its relatively long reproduction period (Delaney et al., 2009). The results of this study showed that leaf Pn impairment after partial leaf defoliation had a seasonal pattern which correlated with *A. syriaca* reproductive phenology but not with cardenolide accumulation. In this regard, the small or absent Pn impairment occurring in leaves of pre-flowering or maturing seed pod plants, contrasted with the moderate to severe leaf Pn impairment detected in leaves of flowering and early seed pod formation plants. Such a behavior led the authors to suggest that a physiological 'cost of reproduction' might be an additional susceptibility factor leading to Pn impairment after herbivory injury on a leaf. Another important aspect to consider is that photosynthesis will be usually more affected when plants are attacked by generalist herbivores, against which they show a higher susceptibility. This is believed to be derived from the lack of a previous and selective co-evolutionary process leading to adaptation (Parker et al., 2006; however, see above). The plant's capacity to tolerate injury, its phenotypic plasticity and the type of environment with which the plant is interacting may be important factors too (Alward & Joern, 1993; Trumble et al., 1993; Delaney & Macedo, 2001; García & Ehrlén, 2002; Zvereva et al., 2010).

The nitrogen status of the plant is also considered to influence the way photosynthesis is affected by herbivory in plants. This is because of the strong positive correlation that is usually observed between photosynthesis rate and nitrogen concentration in plants, predominantly sequestered in the Rubisco enzyme (Field & Mooney, 1986; Evans, 1989; see above). It is not surprising then, that one of the mechanisms offered to explain why the localized decrease in N content negatively affects photosynthesis, a circumstance that has been frequently reported in damaged leaves of woody plants subjected to insect herbivory, is precisely that N deficiency directly affects CO2 assimilation rates by lowering Rubisco levels (Reich et al., 1999; Mediavilla et al., 2001).

Another little studied aspect of plant-insect interations is the effect that insect oviposition might have on photosynthesis. Most of the available data suggest, however, that the effect is predominantly negative. A recent study reported that net photosynthetic rate, J max, and Vc max of pine needles laden with eggs of an herbivorous sawfly were lower than in egg-free

Friend or Foe? Exploring the Factors that Determine the Difference

2006, 2009).

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 181

damage in the leaf, including systemic damage produced in tissue sections not affected directly by the stressor (Chaerle et al., 2007). Moreover, this tool can be combined with thermal imaging, a powerful technique for mapping changes in temperature caused by variations in latent heat flux across leaf surfaces, which can be converted into maps of variable stomatal conductance (Omasa & Takayama, 2003; Jones, 2004; Bajons et al., 2005; Grant et al., 2006). Water limitations in leaves can result, for example, from the disruption in water transport caused by herbivore-damage of water-conducting xylem elements or by midrib vein cutting insects (Tang et al., 2006; Delaney & Higley, 2006; see below). Additional spatial patterning of other components of the photosynthetic machinery, including chlorophyll content and activation of the xanthophyll cycle can be mapped with a technique not frequently employed called hyper-spectral imaging (Nabity et al., 2009). The combined use of these techniques to measure changes in the same leaf in a given experiment, although technically challenging, has provided a deeper insight into the mechanisms by which herbivory indirectly reduces photosynthesis in the remaining undamaged leaf tissue, particularly if combined with physiological data, as reported in several related studies (Zangerl et al., 2002; West et al., 2005; Leinonen & Jones, 2004; Aldea et al., 2006; Tang et al.,

The systemic suppression of photosynthesis in leaf tissues not directly damaged during insect herbivory has been found to extend to an area that greatly exceeds the actual leaf area removed or damaged by the herbivore. For example, the removal of only 5% of the area of an individual wild parsnip leaf by cabbage looper (*T. ni*) caterpillars reduced photosynthesis by 20 %, determined as ΦPSII and the rate of CO2 uptake, in the remaining foliage (Zangerl et al., 2002). The indirect effect was observed to extend to a relatively considerable distance from the cut edges and was still detected for at least 3 days after the caterpillars were removed. Moreover, the size of the indirect effects was positively correlated with defenserelated synthesis of auto-toxic furanocoumarins, suggesting that costs of chemical defense may be one factor that accounts for the deleterious indirect effects of herbivory on plants. Similarly, the decline in photosynthesis in the remaining leaf tissue of damaged oak saplings was equal to the decrease in photosynthesis associated with the actual removal of leaf tissue (Aldea et al., 2006). Chewing damage by cabbage looper larvae also caused substantial reductions in ΦPSII in Arabidospis' leaves at some distance from the tissue removed (Tang et al., 2006). Interestingly, this study demonstrated that the degree of photosynthetic impairment caused by herbivory depended on the nature of the damage inflicted. Therefore, damage caused by first instar larvae, which typically make small holes and avoid veins, led to photosynthetic depression in the remaining leaf tissue near the holes, whereas fourth instars, that make larger perforations while feeding, had little effect on photosynthesis (Tang et al., 2006). The workers concluded that both water stress, induced by the increased rate of water loss near the damaged tissues, and the reduced stomatal conductance produced in the tissues localized at some distance from the injuries, contributed to the inhibition of photosynthesis in the remaining leaf tissues, although subsequent data suggested that induction of defense responses in areas near the holes may have also contributed to the observed decrease in photosynthesis (Tang et al., 2009). The above studies provided evidence suggesting that defense induced auto-toxicity or defense-induced down-regulation of photosynthesis contributed to the indirect repression of photosynthesis. Another contributing factor to indirect suppression of photosynthesis is vasculature tissue severance, which generally leads to a disruption in fluid or nutrient transport, and altered sink

control plants that were not attacked. The negative effect was deemed to have occurred as the result of an egg deposition process that involved wounding of the plant tissue by the sawflies' ovipositor prior to the laying of eggs into its ovipositional wound (Schröder et al., 2005). In a more recent report, the oviposition and wounding effects were separated by employing two pentatomid insects (*Murgantia histrionica* and *Nezara viridula*) having different feeding habits but known not to cut or otherwise physically damage the host substrate during the oviposition procedure. In this process, the eggs are laid in clusters on the leaf surface and adhere to it by a sticky oviduct secretion (Bin et al., 1993; Colazza et al., 2004). Nevertheless, a surprisingly large inhibition of photosynthesis was detected in leaves of *Brassica oleracea*, one of the plant models employed together with common bean, in response to oviposition by *M. histrionica*, even when oviposition was not associated with feeding activity. High resolution chlorophyll fluorescence imaging revealed that the damage to photochemistry caused by feeding and oviposition was restricted to the affected areas, whereas an increase in photochemical yield detected temporarily in the neighboring intact areas of the attacked leaves, indicated the onset of a compensatory response. To date, the way(s) in which insect oviposition affects photosynthesis, occurring either with or without ovipositional plant wounding, remain(s) unknown. However, reduced diffusion rates of CO2 in the mesophyll cells was suggested as a possible mechanism leading to photosynthesis inhibition during oviposition in the absence of plant wounding (Velikova et al., 2010).

#### **4.4 Indirect effects of insect herbivory on photosynthesis**

Leaf area removal not only affects photosynthesis in the damaged tissue but may have a *hidden* or indirect effect in tissues not directly damaged by the herbivore which undergo an additional reduction in photosynthetic capacity and alterations in transpiration (Welter, 1989; Zangerl et al., 2002; Aldea et al., 2006; Berger et al., 2007; Bilgin et al., 2008; Nabity et al., 2009). The discovery that herbivory-induced alterations to photosynthesis and transpiration propagate into remaining undamaged leaf tissue was greatly favored by the development of imaging techniques tools. These proved capable of performing spatiallyresolved measurements of the component processes of photosynthesis across leaf surfaces in order to provide direct estimates of the magnitude of local and systemic damage in a quantitative, multi-layered or complementary and non-invasive way. The ability to measure chlorophyll fluorescence by imaging techniques was a pivotal development, considering that this is by far the most important indicator of photosynthetic stress and damage to the photosynthetic apparatus. Thus, chlorophyll fluorescence provides a precise measure of the quantum yield of photosystem II in light-adapted leaves (ΦPSII), which is, in turn, related to the rate of carbon fixation (Genty et al., 1989), and may be used to calculate the photosynthetic electron transport rate driving photosynthesis and photorespiration (Di Marco et al., 1990). Chlorophyll fluorescence data can also be used to assess damage to the photochemical aspect of photosynthesis by measurements of the quantum yield in darkadapted leaves (by determining the ratio between variable and maximal fluorescence, Fv/Fm) and by the amount of photochemical energy lost as heat (by measuring the nonphotochemical quenching of fluorescence, NPQ) (Genty & Harbinson, 1996). Fluorescence imaging, which more often than not correlates with photosynthetic capacity measured by gas exchange, further improved the already superior suitability of this technique to assess damage to the photosynthetic apparatus, by providing a topographical panorama of

control plants that were not attacked. The negative effect was deemed to have occurred as the result of an egg deposition process that involved wounding of the plant tissue by the sawflies' ovipositor prior to the laying of eggs into its ovipositional wound (Schröder et al., 2005). In a more recent report, the oviposition and wounding effects were separated by employing two pentatomid insects (*Murgantia histrionica* and *Nezara viridula*) having different feeding habits but known not to cut or otherwise physically damage the host substrate during the oviposition procedure. In this process, the eggs are laid in clusters on the leaf surface and adhere to it by a sticky oviduct secretion (Bin et al., 1993; Colazza et al., 2004). Nevertheless, a surprisingly large inhibition of photosynthesis was detected in leaves of *Brassica oleracea*, one of the plant models employed together with common bean, in response to oviposition by *M. histrionica*, even when oviposition was not associated with feeding activity. High resolution chlorophyll fluorescence imaging revealed that the damage to photochemistry caused by feeding and oviposition was restricted to the affected areas, whereas an increase in photochemical yield detected temporarily in the neighboring intact areas of the attacked leaves, indicated the onset of a compensatory response. To date, the way(s) in which insect oviposition affects photosynthesis, occurring either with or without ovipositional plant wounding, remain(s) unknown. However, reduced diffusion rates of CO2 in the mesophyll cells was suggested as a possible mechanism leading to photosynthesis inhibition during oviposition in the absence of plant wounding (Velikova et

Leaf area removal not only affects photosynthesis in the damaged tissue but may have a *hidden* or indirect effect in tissues not directly damaged by the herbivore which undergo an additional reduction in photosynthetic capacity and alterations in transpiration (Welter, 1989; Zangerl et al., 2002; Aldea et al., 2006; Berger et al., 2007; Bilgin et al., 2008; Nabity et al., 2009). The discovery that herbivory-induced alterations to photosynthesis and transpiration propagate into remaining undamaged leaf tissue was greatly favored by the development of imaging techniques tools. These proved capable of performing spatiallyresolved measurements of the component processes of photosynthesis across leaf surfaces in order to provide direct estimates of the magnitude of local and systemic damage in a quantitative, multi-layered or complementary and non-invasive way. The ability to measure chlorophyll fluorescence by imaging techniques was a pivotal development, considering that this is by far the most important indicator of photosynthetic stress and damage to the photosynthetic apparatus. Thus, chlorophyll fluorescence provides a precise measure of the quantum yield of photosystem II in light-adapted leaves (ΦPSII), which is, in turn, related to the rate of carbon fixation (Genty et al., 1989), and may be used to calculate the photosynthetic electron transport rate driving photosynthesis and photorespiration (Di Marco et al., 1990). Chlorophyll fluorescence data can also be used to assess damage to the photochemical aspect of photosynthesis by measurements of the quantum yield in darkadapted leaves (by determining the ratio between variable and maximal fluorescence, Fv/Fm) and by the amount of photochemical energy lost as heat (by measuring the nonphotochemical quenching of fluorescence, NPQ) (Genty & Harbinson, 1996). Fluorescence imaging, which more often than not correlates with photosynthetic capacity measured by gas exchange, further improved the already superior suitability of this technique to assess damage to the photosynthetic apparatus, by providing a topographical panorama of

al., 2010).

**4.4 Indirect effects of insect herbivory on photosynthesis** 

damage in the leaf, including systemic damage produced in tissue sections not affected directly by the stressor (Chaerle et al., 2007). Moreover, this tool can be combined with thermal imaging, a powerful technique for mapping changes in temperature caused by variations in latent heat flux across leaf surfaces, which can be converted into maps of variable stomatal conductance (Omasa & Takayama, 2003; Jones, 2004; Bajons et al., 2005; Grant et al., 2006). Water limitations in leaves can result, for example, from the disruption in water transport caused by herbivore-damage of water-conducting xylem elements or by midrib vein cutting insects (Tang et al., 2006; Delaney & Higley, 2006; see below). Additional spatial patterning of other components of the photosynthetic machinery, including chlorophyll content and activation of the xanthophyll cycle can be mapped with a technique not frequently employed called hyper-spectral imaging (Nabity et al., 2009). The combined use of these techniques to measure changes in the same leaf in a given experiment, although technically challenging, has provided a deeper insight into the mechanisms by which herbivory indirectly reduces photosynthesis in the remaining undamaged leaf tissue, particularly if combined with physiological data, as reported in several related studies (Zangerl et al., 2002; West et al., 2005; Leinonen & Jones, 2004; Aldea et al., 2006; Tang et al., 2006, 2009).

The systemic suppression of photosynthesis in leaf tissues not directly damaged during insect herbivory has been found to extend to an area that greatly exceeds the actual leaf area removed or damaged by the herbivore. For example, the removal of only 5% of the area of an individual wild parsnip leaf by cabbage looper (*T. ni*) caterpillars reduced photosynthesis by 20 %, determined as ΦPSII and the rate of CO2 uptake, in the remaining foliage (Zangerl et al., 2002). The indirect effect was observed to extend to a relatively considerable distance from the cut edges and was still detected for at least 3 days after the caterpillars were removed. Moreover, the size of the indirect effects was positively correlated with defenserelated synthesis of auto-toxic furanocoumarins, suggesting that costs of chemical defense may be one factor that accounts for the deleterious indirect effects of herbivory on plants. Similarly, the decline in photosynthesis in the remaining leaf tissue of damaged oak saplings was equal to the decrease in photosynthesis associated with the actual removal of leaf tissue (Aldea et al., 2006). Chewing damage by cabbage looper larvae also caused substantial reductions in ΦPSII in Arabidospis' leaves at some distance from the tissue removed (Tang et al., 2006). Interestingly, this study demonstrated that the degree of photosynthetic impairment caused by herbivory depended on the nature of the damage inflicted. Therefore, damage caused by first instar larvae, which typically make small holes and avoid veins, led to photosynthetic depression in the remaining leaf tissue near the holes, whereas fourth instars, that make larger perforations while feeding, had little effect on photosynthesis (Tang et al., 2006). The workers concluded that both water stress, induced by the increased rate of water loss near the damaged tissues, and the reduced stomatal conductance produced in the tissues localized at some distance from the injuries, contributed to the inhibition of photosynthesis in the remaining leaf tissues, although subsequent data suggested that induction of defense responses in areas near the holes may have also contributed to the observed decrease in photosynthesis (Tang et al., 2009). The above studies provided evidence suggesting that defense induced auto-toxicity or defense-induced down-regulation of photosynthesis contributed to the indirect repression of photosynthesis. Another contributing factor to indirect suppression of photosynthesis is vasculature tissue severance, which generally leads to a disruption in fluid or nutrient transport, and altered sink

Friend or Foe? Exploring the Factors that Determine the Difference

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 183

were in agreement with the high water losses associated with skeletonizing damage, by showing that the cut edges of soybean leaves damaged by Japanese beetles known to follow this mode of herbivory, suffered a very substantial dehydration (Aldea et al., 2005). However, their data showed that although damage to the inter-veinal tissue increased transpiration by 150 % for up to 4 days post-injury, it had no detectable effect on CO2 exchange, and even induced a short-lived increase in photosynthetic efficiency in undamaged tissue of damaged leaves. Such a contradictory outcome was deemed to have happened as a result of a transient decoupling of photosynthetic electron transport from carbon assimilation caused by insect damage (Aldea et al., 2005). Regarding the above, it is not surprising to know that plants can increase WUE as a strategy to ameliorate the negative effects of herbivory, as was recently found in apple tress infested by leaf-mining moths (Pincebourde et al., 2006). Thus, WUE was found to be about 200% higher in the mined apple leaf tissues in comparison to intact leaf portions, prompting the proposal that minimizing water losses reduces the negative impact on photosynthesis derived from herbivore attacks, by avoiding severe reductions in the CO2 assimilated to water loss ratio. Autotoxicity by resident plant metabolites having potential biocidal properties that can directly affect the host plant may represent an important fitness cost. This adds to the investment in energy and C and N sources already employed for their synthesis, which could have otherwise been used for growth and reproduction (Zangerl & Bazzaz, 1993). Autotoxicity has been recorded in cases where secondary compounds having biocidal properties that severely affect the photosynthetic machinery of the plant are either released from specialized storage tissues that confine them (e.g. glands, trichomes or oil tubes) or accumulate as a consequence of leaf damage. An early study investigating autotoxicity in defense-related metabolites, linked nicotine toxicity to the reduction in photosynthesis in a number Solanaceous plants (Baldwin & Callahan, 1993). Some time later, the suppression of ΦPSII in regions of the leaf near the tissue removed by caterpillars was related to an increased production of toxic furanocoumarins (Zangerl et al., 2002; see above). A subsequent study tested the autotoxicity of several essential oil components, including several monoterpenes and sesquiterpenes and myristicin, an essential oil component derived from the phenylpropanoid pathway, in three plant species known to produce them (i.e. *Pastinaca sativa*, *Petroselinum crispum*, and *Citrus jambhiri*) (Gog et al., 2005). The toxic effects, which were assessed by quantifying reductions in photosynthetic capacity as measured by chlorophyll fluorescence imaging, were examined both by exogenous applications of pure compounds and by the release, by slicing, of endogenous essential oils known to contain these compounds, among others. Monoterpenes, but not the caryophyllene and farnesene sesquiterpenes or myristicin, produced a rapid and spatially extensive decline in photosynthetic capacity that was detected within a time frame of 200 s. On the other hand, the release of endogenous essential oils significantly reduced photosynthetic activity in all three plant species examined, an effect that was more pronounced in *P. sativa* and *P. crispum*. The auto-toxic effect of monoterpenes was assumed to be related to the loss of cell and organelle integrity associated with their known capacity to disrupt membranes (Harrewijn et al., 2001; Maffei et al., 2001). Conversely, coumarins and furanocoumarins have been long known to negatively affect photosynthesis in several plant species. Photo-phosphorylation uncoupling, energy transfer inhibition and/or Hill reaction inhibition have been identified as the probable mechanisms responsible for their suppression of photosynthesis in higher plants (Macías et al., 1999; Veiga et al., 2007).

demand. The collected data indicates that the probable mechanisms responsible for reducing photosynthesis in remaining leaf tissues are multifaceted, ranging from disruptions in fluid or nutrient transport to self-inflicted reductions in metabolic processes. However, the magnitude of their contribution to indirect impairment of photosynthesis will vary depending in large part on the type of feeding damage and the mode of defense deployed by the plant under attack (Nabity et al., 2009). In this respect, chewing damage and fungal and gall infections were found to differentially affect the component processes of photosynthesis of nearby leaf tissue in several hard-wood tree species, with fungal infections and galls causing large depressions (>25%) of photosynthetic effciency (as ΦPSII) over extended areas of the leaf around the visible damage, whereas chewing damage resulted in minor (≈7%) depressions of ΦPSII that were restricted to a 1 mm perimeter around the perforations. Although similar in their effect on electron transport through PSII, the indirect effects of fungal and gall infections on photosynthesis were found to operate through different mechanisms. A reduction in stomatal conductance with an associated decline in intercellular CO2 concentration may have contributed to the depression of ΦPSII around fungal spots but not in gall surrounding areas. On the other hand, the mild and localized suppressions of the photosynthetic efficiency in tissue surrounding chewing damage was attributed to the desiccation of tissue along the edges of damage, similarly to other reports (Aldea et al., 2005; Tang et al., 2006). One sobering conclusion reached by this study was that the indirect, negative, effects of photosynthesis caused by biotic stress on tress were exacerbated by elevated CO2, exposing yet another damaging element of the ongoing global trend towards higher CO2 levels in the earth's atmosphere.

Defoliation injury which severs venation indiscriminately (e.g by fouth instar, but not first instar, *T. ni* larvae; see above) can damage xylem and/or phloem, leading to altered water transport, stomatal aperture, and sucrose transport and loading. All these changes can strongly contribute to reduce photosynthesis in the remaining leaf tissue. Severing veins and inter-veinal tissue also alters the hydraulic construction of leaves as the result of an exponential reduction in resistance occurring with increasing damage (Nardini & Salleo, 2005). Long- or short-term leaf desiccation can also occur in the absence of alternative pathways for water transport. If insect feeding is subtle enough to avoid outright cell rupture (e.g. by phloem-feeders), modulation of nutrients sequestered by feeding will alter plant osmotica or sink/source relationships (Girousse et al., 2005; Dorchin et al., 2006). Feeding may physically obstruct fluid flow with insect mouthparts (stylets) or cell fragments and alter photosynthesis and water balance in remaining leaf tissue (Reddall et al., 2004; Delaney & Higley, 2006). A particular mechanism of plant vasculature disruption is midrib vein cutting, a little-known type of specialized herbivory suggested to have evolved as a strategy to avoid trapping leaf latex or toxic cardenolide defenses in plant species mostly restricted to the Asclepiadaceae (milkweed), Apocynaceae (dogbane), Polygonaceae and Fabaceae families. This type of damage was found to impair several leaf gas exchange parameters, but only downstream from the injury location. Photosynthesis impairment caused by midrib herbivory was more severe than manually imposed and actual insect defoliation, was relatively long-lasting and became most severe as the injury location came closer to the petiole (Delaney & Higley, 2006). As mentioned above, a form of defoliation in soybean plants known as skeletonization, is characterized by the removal of patches of tissue, reduced photosynthesis in remaining tissue on damaged leaves and on adjacent undamaged leaflets (Peterson et al., 1998). A related study reported results that

demand. The collected data indicates that the probable mechanisms responsible for reducing photosynthesis in remaining leaf tissues are multifaceted, ranging from disruptions in fluid or nutrient transport to self-inflicted reductions in metabolic processes. However, the magnitude of their contribution to indirect impairment of photosynthesis will vary depending in large part on the type of feeding damage and the mode of defense deployed by the plant under attack (Nabity et al., 2009). In this respect, chewing damage and fungal and gall infections were found to differentially affect the component processes of photosynthesis of nearby leaf tissue in several hard-wood tree species, with fungal infections and galls causing large depressions (>25%) of photosynthetic effciency (as ΦPSII) over extended areas of the leaf around the visible damage, whereas chewing damage resulted in minor (≈7%) depressions of ΦPSII that were restricted to a 1 mm perimeter around the perforations. Although similar in their effect on electron transport through PSII, the indirect effects of fungal and gall infections on photosynthesis were found to operate through different mechanisms. A reduction in stomatal conductance with an associated decline in intercellular CO2 concentration may have contributed to the depression of ΦPSII around fungal spots but not in gall surrounding areas. On the other hand, the mild and localized suppressions of the photosynthetic efficiency in tissue surrounding chewing damage was attributed to the desiccation of tissue along the edges of damage, similarly to other reports (Aldea et al., 2005; Tang et al., 2006). One sobering conclusion reached by this study was that the indirect, negative, effects of photosynthesis caused by biotic stress on tress were exacerbated by elevated CO2, exposing yet another damaging element of the

ongoing global trend towards higher CO2 levels in the earth's atmosphere.

Defoliation injury which severs venation indiscriminately (e.g by fouth instar, but not first instar, *T. ni* larvae; see above) can damage xylem and/or phloem, leading to altered water transport, stomatal aperture, and sucrose transport and loading. All these changes can strongly contribute to reduce photosynthesis in the remaining leaf tissue. Severing veins and inter-veinal tissue also alters the hydraulic construction of leaves as the result of an exponential reduction in resistance occurring with increasing damage (Nardini & Salleo, 2005). Long- or short-term leaf desiccation can also occur in the absence of alternative pathways for water transport. If insect feeding is subtle enough to avoid outright cell rupture (e.g. by phloem-feeders), modulation of nutrients sequestered by feeding will alter plant osmotica or sink/source relationships (Girousse et al., 2005; Dorchin et al., 2006). Feeding may physically obstruct fluid flow with insect mouthparts (stylets) or cell fragments and alter photosynthesis and water balance in remaining leaf tissue (Reddall et al., 2004; Delaney & Higley, 2006). A particular mechanism of plant vasculature disruption is midrib vein cutting, a little-known type of specialized herbivory suggested to have evolved as a strategy to avoid trapping leaf latex or toxic cardenolide defenses in plant species mostly restricted to the Asclepiadaceae (milkweed), Apocynaceae (dogbane), Polygonaceae and Fabaceae families. This type of damage was found to impair several leaf gas exchange parameters, but only downstream from the injury location. Photosynthesis impairment caused by midrib herbivory was more severe than manually imposed and actual insect defoliation, was relatively long-lasting and became most severe as the injury location came closer to the petiole (Delaney & Higley, 2006). As mentioned above, a form of defoliation in soybean plants known as skeletonization, is characterized by the removal of patches of tissue, reduced photosynthesis in remaining tissue on damaged leaves and on adjacent undamaged leaflets (Peterson et al., 1998). A related study reported results that were in agreement with the high water losses associated with skeletonizing damage, by showing that the cut edges of soybean leaves damaged by Japanese beetles known to follow this mode of herbivory, suffered a very substantial dehydration (Aldea et al., 2005). However, their data showed that although damage to the inter-veinal tissue increased transpiration by 150 % for up to 4 days post-injury, it had no detectable effect on CO2 exchange, and even induced a short-lived increase in photosynthetic efficiency in undamaged tissue of damaged leaves. Such a contradictory outcome was deemed to have happened as a result of a transient decoupling of photosynthetic electron transport from carbon assimilation caused by insect damage (Aldea et al., 2005). Regarding the above, it is not surprising to know that plants can increase WUE as a strategy to ameliorate the negative effects of herbivory, as was recently found in apple tress infested by leaf-mining moths (Pincebourde et al., 2006). Thus, WUE was found to be about 200% higher in the mined apple leaf tissues in comparison to intact leaf portions, prompting the proposal that minimizing water losses reduces the negative impact on photosynthesis derived from herbivore attacks, by avoiding severe reductions in the CO2 assimilated to water loss ratio. Autotoxicity by resident plant metabolites having potential biocidal properties that can

directly affect the host plant may represent an important fitness cost. This adds to the investment in energy and C and N sources already employed for their synthesis, which could have otherwise been used for growth and reproduction (Zangerl & Bazzaz, 1993). Autotoxicity has been recorded in cases where secondary compounds having biocidal properties that severely affect the photosynthetic machinery of the plant are either released from specialized storage tissues that confine them (e.g. glands, trichomes or oil tubes) or accumulate as a consequence of leaf damage. An early study investigating autotoxicity in defense-related metabolites, linked nicotine toxicity to the reduction in photosynthesis in a number Solanaceous plants (Baldwin & Callahan, 1993). Some time later, the suppression of ΦPSII in regions of the leaf near the tissue removed by caterpillars was related to an increased production of toxic furanocoumarins (Zangerl et al., 2002; see above). A subsequent study tested the autotoxicity of several essential oil components, including several monoterpenes and sesquiterpenes and myristicin, an essential oil component derived from the phenylpropanoid pathway, in three plant species known to produce them (i.e. *Pastinaca sativa*, *Petroselinum crispum*, and *Citrus jambhiri*) (Gog et al., 2005). The toxic effects, which were assessed by quantifying reductions in photosynthetic capacity as measured by chlorophyll fluorescence imaging, were examined both by exogenous applications of pure compounds and by the release, by slicing, of endogenous essential oils known to contain these compounds, among others. Monoterpenes, but not the caryophyllene and farnesene sesquiterpenes or myristicin, produced a rapid and spatially extensive decline in photosynthetic capacity that was detected within a time frame of 200 s. On the other hand, the release of endogenous essential oils significantly reduced photosynthetic activity in all three plant species examined, an effect that was more pronounced in *P. sativa* and *P. crispum*. The auto-toxic effect of monoterpenes was assumed to be related to the loss of cell and organelle integrity associated with their known capacity to disrupt membranes (Harrewijn et al., 2001; Maffei et al., 2001). Conversely, coumarins and furanocoumarins have been long known to negatively affect photosynthesis in several plant species. Photo-phosphorylation uncoupling, energy transfer inhibition and/or Hill reaction inhibition have been identified as the probable mechanisms responsible for their suppression of photosynthesis in higher plants (Macías et al., 1999; Veiga et al., 2007).

Friend or Foe? Exploring the Factors that Determine the Difference

Damage type /feeding guild

Foliagechewing, single celland phloem feeders

Foliagefeeder

Foliagechewing feeder

Phloem feeder

Phloem feeder

Plant species Herbivore

*Nicotiana attenuata, N. longiflora*

Wild parsnip (*Pastinaca sativa*)

Tobacco (*Nicotiana tabacum*); soybean (*Glycine max*)

Soybean (*Glycine max*)

Sorghum (*Sorghum bicolor*)

species

Horn worm (*Manduca sexta*); *Tupiocoris notatus*; aphid (*Myzus nicotianae*)

Cabbage looper

(*T. ni*)

Tobacco budworm (*Heliothis virescens*); oblique-banded leaf roller (*Choristoneura rosaceana*)

Soybean aphid (*Aphis glycines*); two-spotted spider mite (*Tetranychus urticae*)

Greenbugs (*Schizaphis graminae*)

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 185

Up-regulation of defenserelated genes and downregulation of primary metabolism and

photosynthesis-related genes. CO2 assimilation and photosystem II efficiency reduced by 16% and 8% respectively in the remaining tissue of damaged leaves.

Decreased efficiency of photosystem II that extended beyond the area directly

Insect locomotion and herbivory across leaf surfaces reduced photosynthesis and increased production of ROS and signaling

damaged.

molecule 4-aminobutyrate.

rates was not the consequence

Reduction up to 50% in photosynthetic

photoelectron transport

was not impaired. Spider mites decreased photosynthesis, stomatal conductance, transpiration and clorophyll content.

Photosynthesis-related

suppressed strongly

genes were

by MeJA, and to a lesser extent by SA and aphids.

of stomatal limitation and

Results Method Reference(s)

GE, fluorescen ce imaging (FI) and Microarray analyses (MA)

GE and chlorophyll fluorescence (ChlF)

ChlF, GE and CIR

MA Zhu-

Salzman et al., 2004

ChlF Bown

Hermsmeier et al., 2001; Izaguirre et al., 2003; Voelckel & Baldwin 2004a,b; Voelckel et al., 2004; Halitschke et al., 2003, 2011

Zangerl et al., 2002

et al., 2002

Macedo et al., 2003a; Haile & Higley, 2003

A number of selected examples in which insect herbivory has been shown to directly or indirectly influence photosynthesis in a negative way, including many already described above, are shown in Table2.


Friend or Foe? Exploring the Factors that Determine the Difference Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 185

184 Artificial Photosynthesis

A number of selected examples in which insect herbivory has been shown to directly or indirectly influence photosynthesis in a negative way, including many already described

leaf.

Sap feeder Chlorophyll content and

infestation.

affected.

yield.

reduced.

Photosynthetic rates per unit area of damaged leaves were reduced by spittlebug feeding, but not by beetle or aphid feeding. Spittlebug feeding did not cause stomatal closure, but impaired C fixation within the

photosynthesis decreased 75 and 45% respectively after

Photosynthetic depression and transpiration increase were quantitatively related to initial aphid infestation densities and to the length of feeding

Adults and larvae reduced photosynthetic rates of the remaining tissue of the injured leaflet. A significant linear relationship between photosynthetic rate and percentage injury was observed. Light reactions of photosynthesis were not

Photosynthetic rate was decreased 50%; associated with reductions in chlorophyll fluorescence and fluorescence

No changes were found in stomatal conductance, intercellular CO2 concentration, and leaf chlorophyll content.

Suppressed photosynthetic rate after infestation, especially at lower leaf positions. Chlorophyll content and total plant dry weight were also

Results Method Reference(s)

Gas exchange (GE)

Meyer & Whitlow, 1992

et al., 1994

et al., 1998

et al., 1998

Lin et al., 1999

Watanabe & Kitagawa, 2000

GE Cabrera

GE Shannag

GE Peterson

GE and chlorophyll content (ChlC)

Carbon isotope ratios (CIR)

Damage type /feeding guild

Phloem, foliage and xylem-sap feeders

Phloem feeder

Foliagescraping chewing feeder (skeletoniz er)

Phloem feeder

Phloem feeder

above, are shown in Table2.

species

Aphid (*Uroleucon caligatum*); beetle (*Trirhabda* sp.); spittlebug (*Philaenus spumarius*)

Aphid (*Schizophis graminum*)

Aphid (*Aphis gossypii*)

Mexican bean beetles (*Epilachna varivestis*)

Silverleaf whitefly (*Bemisia argentifolii*)

Planthopper (*Nilaparvata lugens*)

Plant species Herbivore

Goldenrod (*Solidago altissima*)

Barley (*Hordeum vulgare*)

Cotton (*Gossypium hirsutum*)

Soybean (*Glycine max*); dry bean (*Phaseolus vulgaris*)

Cotton (*Gossypium hirsutum*)

Rice (*Oryza sativa*)


Friend or Foe? Exploring the Factors that Determine the Difference

Damage type /feeding guild

Foliagechewing feeder

Foliagechewing feeder

Phloem feeder

Phloem feeder

Plant species Herbivore

*Arabidopsis thaliana* 

Common milkweed (*Asclepias syriaca*)

Tomato (*Solanum lycopersicum*)

Wheat (*Triticum aestivum*)

Savoy cabbage (*Brassica oleracea*); Common bean *Phaseolus vulgaris* 

herbivory damage.

species

(*T. ni*)

Milkweed tussock (*Euchaetes egle*); monarch butterfly larvae (*Danaus plexippus*); salt marsh tiger moth (*Estigmene* 

*acrea*)

*tabaci*)

*noxia*);

aphid (*Rhopalosiphum* 

*padi*)

Whitefly (*Bemisia* 

Russian wheat aphid (*Diuraphis* 

bird cherry-oat

harlequin cabbage bug (*Murgantia histrionica*); *Nezara viridula*

Cabbage looper

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 187

Decreased efficiency of photosystem II determined by the mode of feeding by different larvae instars and water stress associated with herbivore damage.

Corresponding induction of defense gene expression (*cynnamate-4-hydroxylase*) with photosynthesis reduction, but photosynthetic damage spread further into surrounding

Partial tissue consumption by insect herbivores caused photosynthetic impairment on remaining tissue. Reduction in photosynthetic rates lasted ˃5 days. Neighboring uninjured leaves had a small degree (10%) of compensatory photosynthesis.

Complete photosynthetic recovery observed at one day

General repression of photosynthetic genes in an apparent infestation-stagedependent mode.

Both aphids negatively affected net photosynthesis; *D. noxia* had a greater impact than *R. padi*. Reduction was not related to the light reaction

via pigment losses.

rapidly and substantially by feeding and oviposition in the attacked areas. Stomatal conductance did not decrease with photosynthesis. Oviposition did not induce photoinhibitory damage.

Sap feeder Photosynthesis decreased

Table 2. Some examples of direct or indirect negative effects on photosynthesis after

post-injury.

wounded areas.

Results Method Reference(s)

GE, ChlF and gene expression in transgenic plants harboring a C4H:GUS fusion

Tang et al., 2006, 2009

GE Delaney

SSH Délano-

GE and ChlC

ChlF and GE

Frier & Estrada-Hernández 2009; Estrada-Hernández et al., 2009

Macedo et al., 2009

Velikova et al., 2010

et al., 2008


Friend or Foe? Exploring the Factors that Determine the Difference Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 187

186 Artificial Photosynthesis

Reduced light-saturated photosynthesis occurred only with high infestation levels. No significant reductions in photosynthetic rates were detected at all initial infestation levels.

Photosynthesis declined with crop age and was faster in mite-infested leaves. A minor

photosynthesis was observed in bottom leaves due to greater light penetration in canopies severely defoliated by mite

Feeding activity reduces leaf net CO2 exchange rates; even when green patches maintain levels close to those in intact leaves. Stomatal conductance and hence transpiration

enhancement of

damage.

rates were highly affected.

feeding.

Photosynthesis gene

Decreased efficiency of photosystem II extended further form visible damage. Gall damage had the greatest depression and chewing rarely

affected efficiency of photosystem II surrounding tissue over small distances.

expression (e.g. photosystem I and II, chlorophyll a-b binding proteins and ferredoxin) was down-regulated by budworm

Results Method Reference(s)

GE and photosynthetic photon flux density

Reddall et al., 2004, 2007

GE Pincebourde

MA Ralph

ChlF and thermal imagining (TI)

et al., 2006

et al., 2006

Aldea et al., 2006

Damage type /feeding guild

Mesophyll feeders or foliagechewing feeder

Mesophyll feeders

Floem feeders, stem boring or foliagechewing feeder

Foliagefeeder or gall inductor

Plant species Herbivore

Cotton (*Gossypium hirsutum*)

Apple trees (*Malus domestica*)

Conifer (*Picea sitchensis*)

Hardwood saplings (*Carya tomentosa, C. glabra, Quercus alba, Q. velutina Ulmus ulata*, *Acer rubrum*)

species

Two-spotted spider mite (*Tetranychus urticae*)

European red mites (*Panonychus ulmi*); Spotted tentiform (*Phyllonorrycter blancardella)*

Spruce budworms (*Choristoneura occidentalis*) white pine weevils (*Pissodes strobi*)

Polyphemus (*Antheraea polyphemus*); redhumped caterpillar (*Schizura concinna*); wasps (*Caryomyia, Eriophyes, Cecidomyia* and

*Cynipid* spp.)


Table 2. Some examples of direct or indirect negative effects on photosynthesis after herbivory damage.

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298.

346.

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