Discussion
The data presented here demonstrates how exposure of tomato plants to an
abiotic stress (salinity), can influence a biotic stress (insect
herbivory). Our findings reveal that the bottom-up effects of salinity
stress exert discernible influences on H. zea biology, including
caterpillar growth rates, tissue selection, and moth oviposition. These
effects are potentially mediated by reduced leaf relative water content,
leaf total protein content, plant nutrient status, and ionic imbalances,
and not entirely via anti-herbivore defense proteins, or the emission of
specific plant volatiles. Usually, co-occurrence of multiple stressors
can result in either individual dominant, additive, or synergistic
effects (Holopainen and Gershenzon, 2010).
Many studies report a positive correlation between water stress and
insect herbivory (Thaler and Bostock, 2004). Drought stress is known to
drive insect outbreaks in certain cases (Mattson and Haack, 1987; Baoet al. , 2019; Lantschner, Aukema and Corley, 2019). The “plant
stress hypothesis” states that insects would find it easier to grow on
stressed plants due to the lower ability of plants to defend themselves.
A study on leaf miners revealed that leaf miners exhibited accelerated
development and shorter developmental times on tomato plants under
elevated salinity (Han et al. , 2016). The carmine spider mite
(Tetranychus cinnabarinus ) showed faster development on
strawberry cultivars under salinity stress (Cakmak and Demiral, 2007).
In a no-choice assay, we observed a decrease in H. zeacaterpillar growth rates with an increasing level of salinity in their
plant diet (Figure 2 ). We did not observe any changes in leaf
area consumption by caterpillars feeding on no salt and salt-treated
substrates (unpublished results). This would indicate that lower
caterpillar growth rates are not due to lower tissue consumption but are
due to other factors. This observation substantiates the “plant vigor
hypothesis,” suggesting H. zea prefers plants grown in
non-saline conditions, as they would be healthier and exhibit a “higher
vigor.” Salt priming of Arabidopsis thaliana seeds led to
elevated ROS and glucosinolates, which was correlated to lower
performance of the generalist common cutworm caterpillar
(Spodoptera litura ) (Xiao et al. , 2019). Another study
demonstrated that Spodoptera exigua larvae were less likely to
damage tomato plants treated with > 50 mM NaCl, consumed
less leaf tissue, displayed decreased pupation rates, and had shorter
lifespans (Marsack and Connolly, 2022). It is evident from many such
studies that the indirect influence of salinity stress on insects is
contingent upon various factors, including the insect feeding guild and
plant species.
Several factors, such as the levels of anti-insect herbivore defense
proteins, leaf water content, leaf nutrient changes, and ionic toxicity,
may collectively contribute to the observed reduction in H. zeacaterpillar growth rates. Dombrowski 2003 reports salt stress-induced
accumulation of proteinase inhibitor II (PIN2 ) transcripts and
amplified tomato responses to wounding via the wound-signalling geneLOXD transcripts. To investigate the interactive effect of salt
stress and insect herbivory on tomato defense responses, we measured the
activity of two jasmonic acid (JA)-inducible plant defense proteins:
polyphenol oxidase (PPO) and trypsin proteinase inhibitor (TPI) (Tanet al. , 2018). However, in spectrophotometric assays, we did not
observe any impact of salt addition on tomato PPO and TPI levels
(Figure 6 ). Similarly, a prior study (Winter et al. ,
2012) failed to detect salt-stress-induced proteinase inhibitor
transcripts or herbivore-induced plant volatiles (HIPVs). It is
possible, however, that 200 mM NaCl depicts a highly stressful
concentration where plants choose to maintain nutrient homeostasis and
growth rather than investing resources in defense metabolites (Abrol,
Vyas and Koul, 2012). Investigation of low concentration salt priming
could provide more insights into this hypothesis (Xiao et al. ,
2019). Gene expression studies showed that GAME4 (glycoalkaloid
metabolism gene 4) and AspPI (aspartic proteinase inhibitor) gene
expression increases significantly under herbivory and high salt
(Supplementary Figure S4) , indicating that salt could prime
plants for a higher induced response to insect herbivory through certain
defenses such as glycoalkaloids or aspartic proteinase inhibitors. A
study in maize showed that although salinity alone did not induce
defense gene expression (Zm-Bx1 and Zm-SerPIN ), joint
salinity and insect herbivory did (Forieri, Hildebrandt and Rostás,
2016). There are some indications of crosstalk between salinity and
insect herbivory pathways, although the precise defenses and genes
involved need to be investigated further.
Consistent with prior studies, we observed a substantial reduction in
leaf relative water contents following salt treatment (Figure
5 ) (Cuartero and Fernández-Muñoz, 1998; Wang, Haseeb and Zhang, 2021).
A study (Lin, Paudel, et al. , 2021) reported M. sextalarvae consumed lower amounts of plant tissue under severe drought
stress. M. sexta larvae showed compensatory feeding in later
instars by insects feeding more on low-water-containing diets (probably
to make up for water deficiencies) (Ojeda-Avila, Woods and Raguso,
2003). While lower water contents in diet could account for lower
caterpillar growth rates, additional factors such as altered nutrient
availability and ionic toxicity can have additive or synergistic
effects.
According to a study in soybean, salt-stressed plants are
photosynthetically less productive and would lead to lower insect growth
due to their lower nutritional quality (Najjar et al. , 2018).
Many plants, in their bid to tolerate salt stress, typically possess the
capacity to selectively exclude Na+ and
Cl- ions while facilitating water uptake (Munns,
2005). For example, the halophyte Hordeum marinum starts
accumulating Na+ and Cl- ions only
after exposure to 450 mM NaCl (Garthwaite, von Bothmer and Colmer,
2005). However, a study on transgenic salt-tolerant tomato plants
reported salt accumulation in tomato leaves at much lower concentrations
(Zhang and Blumwald, 2001). Additionally, another study demonstrated the
accumulation of Na+ in older tomato leaves, possibly
as a protective mechanism to shield younger leaves from salt stress
(Khelil, Menu and Ricard, 2007). A study under low salt conditions (5
mM) showed an increase in Na+, Cl-,
C, S, Zn2+, and Cu2+ in A.
thaliana shoots (Hongqiao et al. , 2021). In line with these
studies, we detected an accumulation of Na+,
Zn2+, Ca2+, Mg2+,
and Mn2+ ions in salt-stressed leaves (Figure
9, Supplementary Figure S3 ). Notably, the micronutrients
Zn2+ and Mn2+ are components of
superoxide dismutases (Cu-Zn SOD and Mn-SOD) that catalyze
H2O2 formation from superoxide radicals.
The observed accumulation of Zn2+ and
Mn2+ ions with increasing salt treatment may be
indicative of their role in enhancing NaCl tolerance, as demonstrated by
prior research in A. thaliana (Wang et al. , 2004; Liuet al. , 2015). Like some other studies, our study also shows that
K+ uptake is hampered possibly through high-affinity
K+ transporters, whereas Ca2+ and
Mg2+ levels increase with salt application, possibly
as salt tolerance mechanisms by the plant (Carter et al. , 2005;
Fuchs et al. , 2005; Valdez-Aguilar, Grieve and Poss, 2009;
Nieves-Cordones et al. , 2010; Koksal et al. , 2016; Akter
and Oue, 2018). It is interesting to note that many insects seek salts
actively, and the accumulation of sodium can offset the negative effects
of plant secondary metabolites on insect herbivores (Joern, Provin and
Behmer, 2012; Kaspari, 2020; Marroquin, Holmes and Salazar, 2023).
However, a high level of Na+ ions could disrupt the
insect’s osmotic balance, interfering with the excretory, nervous,
digestive, and respiratory systems (Silver and Donini, 2021).Helicoverpa armigera larvae grew better on an artificial diet
with a lower amount of sodium, indicating that higher sodium
concentrations could be toxic to insects (Xiao et al. , 2010). We
also observed lower caterpillar growth on a salt-containing artificial
diet (Supplementary Figure S5 ), which indicates that higher
salt levels (Na+ and Cl- ions) in
the diet are detrimental to insect growth.
We do not disregard the influence of other chemical defenses or
essential nutrients that are not measured here on H. zeacaterpillar performances. For instance, glycoalkaloids such as tomatine
increase in salt-stressed plants and could potentially lead to decreased
caterpillar growth and deter caterpillar feeding (Friedman, 2002; Hanet al. , 2016; Dong et al. , 2020). Studies also
report that salinity affects the production of phenolics, terpenoids,
and cyanogenic glycosides in different plants, all of which can affect
insect growth (Wahid and Ghazanfar, 2006; Mahmoudi et al. , 2010;
Ballhorn and Elias, 2014; Wang et al. , 2015; Marroquin, Holmes
and Salazar, 2023). Tomato plants under salt stress would need to
significantly increase their alkaloid production within three days for
this hypothesis to hold. Interestingly, we observed that salt treatment
alone did not lead to upregulation of GAME4 , an upstream gene in
the steroidal alkaloid biosynthesis pathway (Dzakovich, Francis and
Cooperstone, 2022). We did, however, see a higher induced response ofGAME4 in herbivore-damaged salt-stressed plants, which indicates
some salinity-based priming against insect herbivory. Analyzing alkaloid
contents would help validate this hypothesis. However, Thaler & Bostock
2004 report that salt stress by itself does not alter JA-inducible
responses, which could be why we did not see any differences in gene
expression and enzyme levels between salt treatments in no herbivory
tissue.
Lastly, it is important to note that the effects of salt stress on
insect herbivores can vary depending on the insect species and the
severity of the salt stress. Some insect species can be more
salt-tolerant than others. Our findings collectively emphasize that in
salt-stressed plants, factors such as nutrient imbalances, ionic
toxicity, and lower relative water contents (alone or in conjunction)
(Martinez et al. , 2012) cause lower H. zea herbivore
performances than alterations to anti-insect herbivore defense protein
levels.
In petri dish-based choice assays, H. zea caterpillars avoided
plants treated with salt (Figure 3 ). The initial behavior of
the insect caterpillars revealed a random selection of leaves for
feeding, as evident by the “first establish” parameter. This indicates
that cut leaf-disc-based volatile or visual cues do not influence insect
behavior. However, as time progressed, a discernible shift occurred,
with caterpillars increasingly favoring leaves treated with lower salt
concentrations for consumption. This caterpillar feeding preference was
seen in caterpillar leaf area consumption as well as the “first
finish” parameter. The observed change in caterpillar feeding
preference complemented caterpillar growth rates, suggesting that
caterpillars preferred to feed on no-salt-containing diets, which would
result in a higher growth rate. Insect preference for plants under lower
salinity is also observed in other studies (Quais et al. , 2020;
Ali et al. , 2021; Marsack and Connolly, 2022).
Like the caterpillars, we observed a clear preference of H. zeamoths for no-salt-treated plants (Figure 4 ). Conversely, a
study on monarch butterflies showed no difference in female oviposition
between no-salt and salt-treated plants (Mitchell et al. , 2019).
The “optimal oviposition theory” or “mother knows best hypothesis”
suggests that female moths exhibit a preference for laying their eggs on
plant hosts that will maximize the fitness and performance of their
offspring (Jaenike, 1978; Valladares and Lawton, 1991; Courtney and
Kibota, 2017). Female moths utilize various parameters to determine
suitable host plants for oviposition. For many noctuid lepidopterans,
olfactory cues have been identified as crucial in locating and selecting
host plants (Jost and Pitre, 2002; Rojas, Virgen and Cruz-López, 2003;
McCallum et al. , 2011). These cues include volatile compounds
emitted by plants, which can either attract or repel moths based on
their preferences and adaptations (De Moraes, Mescher and Tumlinson,
2001; Bruce, Wadhams and Woodcock, 2005). For example, isothiocyanates
act as oviposition stimulants for the diamondback moth Plutella
xylostella (Renwick et al. , 2006), and pyrrolizidine alkaloids
stimulate oviposition in the cinnabar moth (Tyria jacobaeae )
(Macel and Vrieling, 2003). Studies report that salt stress changes the
emission of three terpenes, (Z)-beta-ocimene, 2-carene, and
β-phellandrene, which have been quantitatively correlated with salt
concentration (Tomescu et al. , 2017). Green leaf volatiles such
as (Z)-3-hexenal and terpenoids like β-phellandrene and α-pinene have
been implicated in attracting lepidopteran moths for oviposition (Huanget al. , 2020; Agbessenou et al. , 2022; Sisay et
al. , 2023). Like the abovementioned studies, we also observed decreased
levels of β-phellandrene as well as 2-carene, α-phellandrene,
β-caryophyllene, and α-humulene in salt-treated plants (Figure
10 ). Insect responses to plant volatiles depend on the insect species,
the specific volatile compound in the volatile blend, and the
concentration of relevant volatiles. Decreased constitutive emissions of
β-phellandrene might cause lower H. zea moth oviposition on
salt-treated plants. We did not observe any differences in total
volatiles emitted per gram of fresh weight between stressed and
unstressed plants, as corroborated by some other studies (Loreto and
Delfine, 2000; Teuber et al. , 2008; Forieri, Hildebrandt and
Rostás, 2016). However, the total volatiles emitted by the whole plant
could be a more ecologically relevant unit.
Furthermore, moths may also rely on visual and tactile cues, including
leaf shape, color, and texture, as indicators of plant quality for
egg-laying (Rojas and Wyatt, 1999; Jakobsson et al. , 2017;
Keerthi et al. , 2023). Plant physical characteristics, such as
the presence and density of trichomes and leaf hairs, can likewise
influence moth oviposition choices. For example, oviposition by P.
xylostella on A. thaliana decreases with an increasing density
of leaf trichomes (Handley, Ekbom and Ågren, 2005). The presence of
glandular trichomes on a wild potato species (Solanum
berthaultii ) acts as a deterrent to oviposition by the potato tuber
moth (Phthorimaea operculella ) (Malakar and Tingey, 2000).
However, some studies have reported that moths tend to lay eggs on plant
parts exhibiting higher trichome densities and concentrated defense
chemicals (Thomson, 1987). Additionally, the nutritional composition of
host plants, including nitrogen levels, sugar content, and secondary
metabolites, plays a vital role in shaping moth oviposition decisions
(Heisswolf, Obermaier and Poethke, 2005; Chen, Ruberson and Olson, 2008;
Gripenberg et al. , 2010). We did not observe any changes to leaf
trichome density under salt stress (unpublished results). However, salt
stress, leading to lower water availability, may potentially increase
the concentration of chemicals in trichomes. Trichomes could also lose
turgor and decrease in size, thus influencing moth oviposition. The
influence of trichome-level changes on H. zea oviposition
decisions cannot be ignored and remain to be investigated.
Lastly, moth host finding and consequently oviposition can also be
altered due to local changes in relative humidity (Godfrey and Holtzer,
1991; Wolfin et al. , 2018). Salt stress would lead to stomatal
closure and reduced transpiration, which would in turn reduce local
humidity. We observed statistically non-significant decreases in leaf
relative humidity under salt treatment for 5 days (preliminary data).
These differences, although statistically non-significant, could still
be ecologically significant for an ovipositing H. zea moth.