Inbreeding compromises host plant defense gene expression and improves herbivore survival

Abstract

Inbreeding commonly occurs in flowering plants and often results in a decline in the plant's defense response. Insects prefer to feed and oviposit on inbred plants more than outbred plants – suggesting that selecting inbred host plants offers them fitness benefits. Until recently, no studies have examined the effects of host plant inbreeding on insect fitness traits such as growth and dispersal ability. In a recent article, we documented that tobacco hornworm (Manduca sexta L.) larvae that fed on inbred horsenettle (Solanum carolinense L.) plants exhibited accelerated larval growth and increased adult flight capacity compared to larvae that fed on outbred plants. Here we report that M. sexta mortality decreased by 38.2% when larvae were reared on inbred horsenettle plants compared to larvae reared on outbreds. Additionally, inbred plants showed a notable reduction in the average relative expression levels of LIPOXYGENEASE-D (LoxD) and 12-OXOPHYTODIENOATE REDUCTASE-3 (OPR3), two genes in the jasmonic acid signaling pathway that are upregulated in response to herbivore damage. Our study presents evidence that furthers our understanding of the biochemical mechanism responsible for differences in insect performance on inbred vs. outbred host plants.

Herbivorous insects acquire the nutrients necessary for growth and development from the plant tissues and fluids that they feed on. Consequently, plants defend themselves from herbivore attack by producing a variety of structural (spines and trichomes)¹ and chemical defenses (toxic secondary metabolites).² Plant defenses reduce the ability of herbivores to obtain nutrients from the plant's tissues; thus, plants with weakened defenses provide improved food quality for herbivores–this can offer benefits to the herbivore in terms of better growth and faster development.^3,4 Plants with diminished defense capability may suffer greater herbivore damage and exhibit lower overall fitness under conditions of herbivore stress than well defended plants.

Inbreeding, a common genetic process in plants,⁵ can produce individuals with reduced fitness (inbreeding depression).^6,7 Compared to outbred plants, inbred plants are more susceptible to herbivores –under both field and lab conditions,^8-10 and herbivores perform better when they feed on inbred plants.^11-13 Previous studies using horsenettle (Solanum carolinense L.), a perennial weed native to the United States, showed that tobacco hornworm caterpillars (Manduca sexta L.) preferred to feed on inbred plants more than outbreds.^10,12 In another choice experiment, adult M. sexta females oviposited more frequently on inbred horsenettle plants compared to outbreds.¹⁴ The predilection for inbred plants exhibited by the insects suggests that they are gaining fitness benefits by choosing inbred host plants rather than outbred plants.

In a very recent study, we compared larval growth and adult flight physiology of M. sexta when the larvae were reared on inbred horsenettle plants vs. larvae that were reared on outbred plants. We showed that larvae reared on inbred plants grew faster and developed into larger pupae compared to larvae reared on outbred plants.¹⁵ Furthermore, adult mass-specific flight metabolic rates were higher in adults that were reared on inbred plants, and differences in flight metabolic rates were also associated with changes to the alternative splicing profiles of Troponin t, a flight muscle protein that regulates muscle contraction.^16,15 Our results show that feeding on inbred host plants produces changes in the insect that cascade through larval and pupal development to affect adult flight muscle protein composition and flight muscle power output–indicating that M. sexta larvae reared on inbred horsenettle plants acquired nutrients at a faster rate, and/or ingested lower levels of plant produced toxins than larvae reared on outbred plants.

Our recent results strongly suggest a link between differences in the biochemistry of inbred and outbred horsenettle plants,¹⁷ and changes to insect herbivore growth, oviposition, and flight capacity. A possible explanation as to why inbred plants serve as better hosts for developing insects could be that inbred plants suffer from a limited ability to upregulate genes in important defense biochemical pathways (e.g., jasmonic acid signaling/biosynthesis pathway)–thus weakening their defense response to herbivory. This hypothesis is supported by a preliminary microarray study comparing the response of inbred and outbred herbivore damaged horsenettle genets–which revealed that the jasmonic acid (JA) pathway (a key biochemical pathway employed to defend against chewing herbivores such as M. sexta) was compromised due to inbreeding.¹⁸ Results from Kariyat et al.¹⁸ suggest that variation in the gene expression levels of plant produced defense compounds, due to inbreeding, could be responsible for changes to growth,¹⁵ oviposition,¹⁴ and flight capacity¹⁵ of M. sexta. However, no studies have directly compared relative expression levels (RA) of genes in the JA pathway between inbred and outbred horsenettle plants, or linked differences in defense gene RAs, due to inbreeding, to changes in herbivore performance.

To test this hypothesis, we compared the survival ratios of M. sexta reared on inbred and outbred horsenettle plants, and we compared the RAs of 2 genes in the JA pathway in samples of inbred and outbred plants damaged by third instar M. sexta larvae. Inbred and outbred plants used for our experiments were previously (2002) derived from 16 maternal plants collected from a natural population located near State College, PA, USA (40° 47′ 29″ N, 77° 51′ 31″ W). Two ramets were produced from each of the original 16 field-collected plants; flowers from one ramet were cross-pollinated (outbred), while flowers from the second ramet were self-pollinated (inbred).^19,20 Plants used in this experiment were produced by propagating horizontal root cuttings from one inbred and one outbred plant from 3 maternal families (designated B1, B3, and B4; for details see ref. 20). Twelve horsenettle plants from each genet (3 maternal families × 2 breeding types) were placed into separate rearing cages (71 cm × 57 cm × 66 cm; L × W × H) in a greenhouse (16:8 L:D; 25:22°C; 65% RH); plants were 6-8 weeks old and were not flowering. Seventy two M. sexta neonate larvae were placed randomly on the living plants; 36 larvae were placed on inbred plants, and 36 larvae were placed on outbred plants. Larvae could move and feed freely on the plants within the confines of the cages, and plants were replaced as need. Larval body mass measurements were recorded at 2 time periods during each instar, early (shortly after molting) and late (during molt sleep).²¹ Adults were used for our flight metabolism and flight muscle gene expression experiments.¹⁵

To determine if host plant inbreeding affected the vitality of the insects, we compared the survival ratios (two proportion z test) of M. sexta larvae that were reared on inbred vs. outbred host plants. We found that the survival ratios of inbred reared larvae were significantly higher than those reared on outbred plants (Fig. 1). When larvae fed on inbred horsenettle plants, 22.2% more survived to the pupa stage (z = 1.95, P = 0.026), and 26.1% more survived to the adult stage (z = 1.61, P = 0.054) compared to larvae that fed on outbred plants. In other words, the mortality rate of inbred fed larvae was 38.5% lower than larvae that fed on outbreds. This result provides evidence that host plant inbreeding impacts insect herbivore survival, and corroborates our earlier findings that plant inbreeding affects M. sexta growth and physiological performance.¹⁵

Figure 1.

Proportion of Manduca sexta larvae that developed into pupae and adult moths when reared on inbred and outbred horsenettle host plants. Bars compare ratios of inbred fed larvae (N = 36) and outbred fed larvae (N = 36) surviving to subsequent development stages.

To examine if insect survival differences were related to variation in expression of host plant defense genes, we quantified the expression levels of two genes in the JA pathway: LIPOXYGENEASE-D (LoxD) and 12-OXOPHYTODIENOATE REDUCTASE-3 (OPR3)–both genes are upregulated in response to herbivore damage.²² Third instar M. sexta larvae were allowed to feed on their host plants for ∼24 hours, after which leaf material was collected from damaged plants and control (undamaged) plants from maternal family B3. Leaves were immediately flash frozen in liquid nitrogen and stored at −80°C for subsequent RNA extraction. Gene expression levels were quantified using SYBR Green based real-time quantitative PCR (qRT-PCR)²³ with an Applied Biosystems® 7500 fast real-time PCR system (Life Technologies, Grand Island, NY).

Our results (Fig. 2) indicate that the average RAs of these major defense genes are lower in inbred plants, compared to outbred plants. Inbred plants showed a 5.2 fold reduction in their average RA levels of LoxD, and a 4.2 fold reduction in their average RA levels of OPR3; although, small samples sizes (N = 2) prevented accurate statistical comparisons of RAs from inbred and outbred plants. Nevertheless, our data displays a trend which points to lower expression levels of these two genes in the inbred plants. This trend is supported by a microarray study that showed herbivore damaged inbred horsenettle plants exhibited a ≥2 fold decrease in expression levels of gene clusters related to herbivore defense.¹⁸ In addition to the pivotal role that JA plays in plant direct chemical defense, JA is also involved in the synthesis of plant volatiles, a major element of indirect defense. Previous field trials, using M. sexta damaged horsenettle plants, showed that inbreeding curtails the plant's production of key terpenoid volatiles²⁴–this negatively impacts the plant's ability to attract predators and/or parasitoids. Furthermore, JA is the main signaling molecule for plant structural defense induction (e.g. leaf trichomes), another defense trait negatively affected by inbreeding.²⁵ Compromised anti-herbivore defense gene expression may explain why other studies have shown that inbred host plants are more likely to be attacked by herbivores than outbred plants and herbivores often perform better when they feed on inbred plants.^26,15 It also might explain why inbred plants are preferred in choice experiments by both M. sexta larvae^12,10 and ovipositing females.¹⁴

Figure 2.

Average (mean ± SE) relative expression levels (RA) of 2 genes in the jasmonic acid signaling/biosynthesis pathway. RA levels recorded from leaves that were collected from inbred and outbred horsenettle plants damaged by 3^rd instar Manduca sexta larvae. Bars represent RAs of Lipoxygenase-D (LoxD) and 12-Oxophytodienoate reductase-3 (OPR3). Numbers above bars show average RAs calculated from ΔΔ-Ct values (N = 2).

Inbreeding in horsenettle causes significant reductions in the plant's induced defense responses^18,23 and resistance to herbivory.^8-10 We used only minimal manipulation, a single generation of host plant inbreeding, to produce differences (inbred vs. outbred) in the host plants of an indigenous plant-insect system (horsenettle-tobacco hornworm). Our findings provide further evidence that plant inbreeding can produce biochemical changes in host plants that can impact the health and vigor of animals at a higher trophic level. Moreover, these data are consistent with previous microarray results that indicate a possible mechanism that explains why insect herbivores exhibit increased survival, growth, and flight metabolic output when reared on inbred plants.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We sincerely thank Scott DiLoreto and Roxanne Lease for providing greenhouse space to grow plants and rear caterpillars. We also thank Sarah Scanlon for help planting horsenettle rhizomes and Michelle Peiffer for providing qPCR primers and technical advice.

Funding

This work was supported by National Science Foundation grants IOS-0950416 and DEB-1120476 to JHM and DEB-1050998 to AGS.