Comparative transcriptomic analysis of chemoreceptors in two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus.

Chemoreception through odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs) represents the functions of key proteins in the chemical ecology of insects. Recent studies have identified chemoreceptors in coleopterans, facilitating the evolutionary analysis of not only ORs but also IRs and GRs. Thus, Cerambycidae, Tenebrionidae and Curculionidae have received increased attention. However, knowledge of the chemoreceptors from Scarabaeidae is still limited, particularly for those that are sympatric. Considering the roles of chemoreceptors, this analysis could shed light on evolutionary processes in the context of sympatry. Therefore, the aim of this study was to identify and compare the repertoires of ORs, GRs and IRs between two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus. Here, construction of the antennal transcriptomes of both scarab beetle species and analyses of their phylogeny, molecular evolution and relative expression were performed. Thus, 119 new candidate chemoreceptors were identified for the first time, including 17 transcripts for B. prasinus (1 GR, 3 IRs and 13 ORs) and 102 for H. elegans (22 GRs, 14 IRs and 66 ORs). Orthologs between the two scarab beetle species were found, revealing specific expansions as well as absence in some clades. Purifying selection appears to have occurred on H. elegans and B. prasinus ORs. Further efforts will be focused on target identification to characterize kairomone and/or pheromone receptors.

Herbivory Damage Increased VOCs in Wild Relatives of Murtilla Plants Compared to Their First Offspring.

Murtilla (Ugni molinae) is a shrub native to Chile that has undergone an incipient domestication process aimed at increasing its productivity. The reduction in intrinsic chemical defenses due to the domestication process has resulted in a decrease in the plant's ability to defend itself against mechanical or insect damage. In response to this damage, plants release volatile organic compounds (VOCs) as a means of defense. To understand how domestication has impacted the production of VOCs in the first offspring of murtilla, we hypothesized that their levels would be reduced due to the induction of mechanical and herbivore damage. To test this hypothesis, we collected VOCs from four offspring ecotypes and three wild relatives of murtilla. We induced mechanical and herbivore damage in the plants and then enclosed them in a glass chamber, where we captured the VOCs. We identified 12 compounds using GC-MS. Our results showed that wild relative ecotypes had a higher VOC release rate of 624.6 µg/cm2/day. Herbivore damage was the treatment that produced the highest release of VOCs, with 439.3 µg/cm2/day in wild relatives. These findings suggest that herbivory triggers defenses through the emission of VOCs, and that domestication has influenced the production of these compounds in murtilla. Overall, this study contributes to bridging the gap in the incipient domestication history of murtilla and highlights the importance of considering the impact of domestication on a plant's chemical defenses.

Arbuscular mycorrhizal fungi enhance monoterpene production in red clover (Trifolium pratense L.): a potential tool for pest control.

The possibility of modifying terpene production in plants is a defensive strategy that has been studied in conjunction with their biosynthetic pathways. A biotic factor such as Arbuscular Mycorrhizal Fungi (AMF) could modify terpene production in Trifolium pratense L. In this work, the enzymatic production of monoterpenes in Superqueli INIA cultivar with two AMF was evaluated via HeadSpace-Gas Chromatography (HS-GC). A significant increase of (S)-limonene was found in plants inoculated with Claroideoglomus claroideum as well as with the AMF mix (genera Scutellospora, Acaulospora and Glomus). Moreover, significant increases in other monoterpenes such as (-)-ß-pinene, myrcene, linalool, were observed. Results showed higher monoterpene production capacities in the Superqueli-INIA cultivar, suggesting the participation of monoterpene synthases (MTS). The significant rise of (S)-limonene in red clover plants inoculated with AMF suggests this strategy could be implemented in an agronomical manage for controlling the H. obscurus, the primary pest.

Infection of perennial ryegrass (Lolium perenne) by an endophyte fungus (Neotyphodium lolii) decreases the abundance and diversity of predators and parasitoids

ABSTRACT Perennial ryegrass is one of the most important food sources in animal production. However, several pests affect this crop, and one of the primary control strategies is the symbiotic relationships between ryegrass endophyte fungi. This fungus produces alkaloids that exhibit toxic activity against arthropods. Furthermore, the effect of fungi may extend to higher trophic levels, including predators (spiders and/or insects), decreasing their abundance and diversity. Given the importance of spiders and insects as predators, whether the symbiotic interaction between perennial ryegrass and endophyte fungus reduces the abundance and diversity of predators pose an important question. To address this question, natural enemies in perennial ryegrass were collected and analyzed over a year, and the percentage of endophyte fungus was evaluated by the presence of hyphae from two ryegrass cultivars, Jumbo (E-) and Alto AR1 (E+). We observed an 80% endophyte infection rate for (E+) and 0% for (E-). Moreover, 222 individual spiders corresponding to 10 families were identified in both perennial ryegrasses, including 209 individuals for (E-) and 13 for (E+). The most abundant spider family was Lycosidae, representing 71.17% of the total spiders. In addition, 65 insects were collected, corresponding to 6 families, with Carabidae being the most abundant. Furthermore, the Simpson index indicated the dominance of the family Lycosidae. Overall, spider and insect abundance and diversity were reduced in (E+), suggesting a negative effect of the endophyte on predator populations.

Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology.

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Domestication of Plants of Ugni molinae Turcz (Myrtaceae) Interferes in the Biology of Chilesia rudis (Lepidoptera: Erebidae) Larvae.

In terms of the domestication process in murtilla, studies have found changes in the concentration of phenolic compounds, with reduction of chemical defense of plants, depending on the change in the feeding behavior of insects. Thus, we hypothesized that the domestication of Ugni molinae decreases the content of phenolic compounds and modifies the feeding preference of Chilesia rudis larvae. Leaves of three parental ecotypes and four cultivated ecotypes were used in preference experiments to evaluate the mass gain and leaves consumption of larvae. Phenolic extracts from leaves of U. molinae were analyzed by HPLC. Identified compounds were incorporated in an artificial diet to assess their effect on mass gain, consumption, and survival of the larvae. The presence of phenolic compounds in bodies and feces was also evaluated. In terms of choice assays, larvae preferred parental ecotypes. Regarding compounds, vanillin was the most varied between the ecotypes in leaves. However, plant domestication did not show a reduction in phenolic compound concentration of the ecotypes studied. Furthermore, there was no clear relation between phenolic compounds and the performance of C. rudis larvae. Whether this was because of sequestration of some compounds by larvae is unknown. Finally, results of this study could also suggest that studied phenolic compounds have no role in the C. rudis larvae resistance in this stage of murtilla domestication process.

Antifeedant Effects and Repellent Activity of Loline Alkaloids from Endophyte-Infected Tall Fescue against Horn Flies, Haematobia irritans (Diptera: Muscidae).

Haematobia irritans is an obligate bloodsucking ectoparasite of cattle and is the global major pest of livestock production. Currently, H. irritans management is largely dependent upon broad-spectrum pesticides, which lately has led to the development of insecticide resistance. Thus, alternative control methods are necessary. Endophyte-infected grasses have been studied as an alternative due to their capability to biosynthesize alkaloids associated with anti-insect activities. Thus, the main aim of this study was to evaluate the antifeedant and repellent activity of lolines obtained from endophyte-infected tall fescue against H. irritans adults in laboratory conditions. The alkaloid extract (ALKE) was obtained by acid-base extraction. N-formyl loline (NFL) and N-acetyl loline (NAL) were isolated by preparative thin layer chromatography (pTLC) and column chromatography (CC), and the loline was prepared by acid hydrolysis of a NFL/NAL mixture. Loline identification was performed by gas chromatography coupled to mass spectrometry (GC/MS). Feeding behavior was evaluated by a non-choice test, and olfactory response was evaluated using a Y-tube olfactometer. Accordingly, all samples showed antifeedant activities. NFL was the most antifeedant compound at 0.5 µg/µL and 1.0 µg/µL, and it was statistically equal to NAL but different to loline; however, NAL was not statistically different to loline. NFL and NAL at 0.25 µg/µL were more active than loline. All samples except loline exhibited spatial repellency in the olfactometer. Thus, the little or non-adverse effects for cattle and beneficial activities of those lolines make them suitable candidates for horn fly management.

Volatiles induction in response to mechanical damage is reduced by domestication in murtilla / La inducción de volátiles en respuesta al daño mecánico es reducida por la domesticación en murtilla

Volatiles compounds are involved in defensive induction against insects, playing an important role in insect-plant interaction being induced by response to mechanical damage. However, they could decrease according to the domestication degree in cultivated plants. Currently, it has been established that secondary metabolites are reduced due to the domestication process in murtilla. Hence, the follow question emerges: Are volatile organic compounds induced by mechanical damage reduced in cultivated murtilla plants in relation to wild plants? Two cultivated ecotypes and their respective wild counterparts were sampled. Volatiles compounds were obtained using Porapak-Q columns and analyzed by gas chromatography. Results showed that compounds as 2-hexanone, α-pinene, 2-thujene, 3-thujene and 1,8- cineole were more abundant in wild plants exposed to a mechanical damage than cultivated plants. Hence, these compounds have been associated to induced defense, these results suggest that domestication reduced the induction of defensive volatiles in cultivated murtilla in response to mechanical damage.

Los compuestos volátiles están implicados en la defensa inducida contra insectos, desempeñando un papel importante en esta interacción. Sin embargo, estos compuestos podrían disminuir según el grado de domesticación. Actualmente, se ha reportado que algunos metabolitos secundarios son reducidos en plantas de murtilla domesticadas. Por lo tanto, surge la siguiente pregunta de investigación: ¿Los compuestos orgánicos volátiles inducidos por el daño mecánico son reducidos en plantas cultivadas de murtilla en comparación con plantas silvestres? Para dos ecotipos cultivados y sus respectivas contrapartes silvestres, los compuestos volátiles fueron capturados usando columnas de Porapak-Q y las muestras analizadas por cromatografía gaseosa. Los resultados mostraron que compuestos tales como 2- hexanona, α-pineno, 2-tujeno, 3-tujeno y 1,8-cineol fueron más abundantes en plantas silvestres expuestas a daño mecánico que en cultivadas. Debido a que estos compuestos se han asociado a defensa inducida, estos resultados sugieren que la domesticación reduce la inducción de volátiles en plantas cultivadas sometidas a daño mecánico.

Structural investigation of selective binding dynamics for the pheromone-binding protein 1 of the grapevine moth, Lobesia botrana.

The European grapevine moth, Lobesia botrana (Denis & Schiffermüller), is a serious pest in vineyards in North and South America. Mating disruption techniques have been used to control and monitor L. botrana on the basis of its sexual communication. This needs a well-tuned olfactory system, in which it is believed that pheromone-binding proteins (PBPs) are key players that transport pheromones in the antennae of moths. In this study, the selectivity of a PBP, named as LbotPBP1, was tested by fluorescence binding assays against 11 sex pheromone components and 6 host plant volatiles. In addition, its binding mechanism was predicted on the basis of structural analyses by molecular docking and complex and steered molecular dynamics (SMD). Our results indicate that LbotPBP1 binds selectively to sex pheromone components over certain host plant volatiles, according to both in vitro and in silico tests. Thus, chain length (14 carbon atoms) and functional groups (i.e., alcohol and ester) appear to be key features for stable binding. Likewise, residues such as Phe12, Phe36, and Phe118 could participate in unspecific binding processes, whilst Ser9, Ser56, and Trp114 could participate in the specific recognition and stabilization of sex pheromones instead of host plant volatiles. Moreover, our SMD approach supported 11-dodecenyl acetate as the best ligand for LbotPBP1. Overall, the dynamics simulations, contact frequency analysis and SMD shed light on the binding mechanism of LbotPBP1 and could overcome the imprecision of molecular docking, supporting the in vitro binding assays. Finally, the role of LbotPBP1 in the chemical ecology of L. botrana is discussed.

Differential formononetin content in cultivars and experimental lines of red clover (Trifolium pratense L. ) plants affect the feeding behaviour of Hylastinus obscurus (Coleoptera: Curculionidae) / Contenido diferencial de formononetina en cultivares y líneas experimentales de plantas de trébol rosado (Trifolium pratense L. ) afecta la conducta alimenticia de Hylastinus obscurus (Coleoptera: Curculionidae)

Red clover (Trifolium pratense L.) is a perennial plant widely used as a forage resource for several animals. This plant is the exclusive host of Hylastinus obscurus (Marsham) which causes irreparable damages to the root system affecting their persistence. It has been reported that the presence of the isoflavonoid formononetin in roots of red clover could act as an antifeedant on H. obscurus. There are not studies related to the formononetin content in red clover roots to the antifeedant effect elicited by experimental lines and cultivar of red clover. Six red clover genotypes were investigated in both formononetin content and their respective antifeedant action. The results showed to Sabtoron High and Superqueli-INIA with both the highest formononetin content in red clover roots and antifeedant effect, allowing to suggest that this secondary metabolites could be used as a chemical factor for red clover plants. Moreover, a rapid methodology for searching red clover genotypes with high formononetin content is reported.

El trébol rosado (Trifolium pratense L.) es una planta perenne ampliamente utilizada como fuente de forraje de variados animales. Esta planta es el exclusivo hospedero de Hylastinus obscurus (Marsham) el cual causa irreparables daños al sistema radical afectando seriamente su persistencia. Se ha reportado que la presencia del isoflavonoide formononetina en raíces del trébol rosado podría actuar como antialimentario sobre H. obscurus. Actualmente no existen estudios que relacionen el contenido de formononetina en raíces de trébol rosado con el efecto antialimentario elicitado por líneas experimentales y cultivares de trébol rosado. Seis genotipos de esta leguminosa fueron evaluados en cuanto a su contenido de formononetina y actividad antialimentaria. Los resultados mostraron que los cultivares Sabtoron High y Superqueli-INIA presentaron altos niveles de formononetina en sus raíces y efecto antialimentario sobre H. obscurus, lo que permite sugerir que este metabolito secundario podría ser usado como factor químico para incrementar la persistencia de plantas de trébol rosado. Además, se informa una metodología rápida para la búsqueda de genotipos con altos contenidos de formononetina.

Antifeedant Effects of Essential Oil, Extracts, and Isolated Sesquiterpenes from Pilgerodendron uviferum (D. Don) Florin Heartwood on Red Clover Borer Hylastinus obscurus (Coleoptera: Curculionidae).

The beetle Hylastinus obscurus Marsham (Coleoptera: Curculionidae), endemic to Europe and Northern Africa, is one of the most important red clover pests in Chile. As commercial insecticides are less effective against this pest, plant secondary metabolites have been considered as an alternative for its control. Here, we have investigated the chemical composition of essential oil (EO), petroleum ether extract (PEE), and dichloromethane extract (DCME) from Pilgerodendron uviferum heartwood. Additionally, the effects of EO and extracts on the feeding behavior (% of weight shift) of H. obscurus have been evaluated. The composition of EO, PEE, and DCME were analyzed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The results showed the presence of a similar mixture of sesquiterpenes in the essential oil and in both of the extracts, which accounted for circa 60% of the total mixture. Sesquiterpenes were further isolated using chromatographic methods and were structurally characterized by optical rotation, GC⁻MS, FTIR, and 1D and 2D NMR experiments. The physicochemical properties of the isolated sesquiterpenes, including lipophilicity and vapor pressure, were also determined. The sesquiterpenes were identified as the following: (-)-trans-calamenene (1), cadalene (2), (-)-cubenol (3), (-)-epi-cubenol (4), (-)-torreyol (5), and (-)-15-copaenol (6). The antifeedant activity of EO, extracts, and isolated sesquiterpenes were evaluated using artificial diets in a non-choice test. Relative to the control, the EO, DCME extract, and the isolated sesquiterpenes, namely, (-)-trans-calamenene (1), cadalene (2), and (5) torreyol, were found to be the most effective treatments against H. obscurus. Our study showed that the compounds occurring in P. uviferum heartwood were effective in reducing the adult growth of H. obscurus. The physicochemical properties of the isolated sesquiterpenes might have been associated with antifeedant effects.