Mating, but Not Male Accessory Gland Products, Changes Female Response to Olfactory Cues in Anastrepha Fruit Flies

Copulation and/or ejaculate components can alter female physiological state and female post-mating behavior. The objective of the present study was to determine if copulation and male reproductive accessory gland products (MAGs) modify the behavior of female Anastrepha ludens (Loew) and Anastrepha obliqua (Macquart; Diptera: Tephritidae) in response to two stimuli: male-emitted pheromone and oviposition host volatiles. Olfactometry studies revealed that mated females of both A. ludens and A. obliqua have a stronger response for host volatiles compared to unmated females, which have a stronger response for male pheromone. We also examined olfactory responses of females mated to testectomized males who could transfer MAGs but not sperm. In both species, MAGs alone did not cause the change in the olfactory response observed after copulation, unlike what has been found in Ceratitis capitata (Wiedemann). Females mated to testectomized males responded equally to the male sex pheromone or to host volatiles, thus suggesting that the whole ejaculate is needed to elicit the complete behavioral switch in olfactory response. The function of MAGs is still unknown in these two pests of economic importance. The response for host volatiles by mated females has implications for the development of baits and traps that should preferably attract and target this population.

Identification and Characterization of an Antennae-Specific Glutathione S-Transferase From the Indian Meal Moth

Insect glutathione-S-transferases (GSTs) play essential roles in metabolizing endogenous and exogenous compounds. GSTs that are uniquely expressed in antennae are assumed to function as scavengers of pheromones and host volatiles in the odorant detection system. Based on this assumption, antennae-specific GSTs have been identified and functionally characterized in increasing number of insect species. In the present study, 17 putative GSTs were identified from the antennal transcriptomic dataset of the Indian meal moth, Plodia interpunctella, a severe stored-grain pest worldwide. Among the GSTs, only PiGSTd1 is antennae-specific according to both Fragments Per Kilobase Million (FPKM) and quantitative real-time PCR (qRT-PCR) analysis. Sequence analysis revealed that PiGSTd1 has a similar identity as many delta GSTs from other moths. Enzyme kinetic assays using 1-chloro-2,4-dinitrobenzene (CDNB) as substrates showed that the recombinant PiGSTd1 gave a Km of 0.2292 ± 0.01805 mM and a Vmax of 14.02 ± 0.2545 μmol·mg−1·min−1 under the optimal catalytic conditions (35°C and pH = 7.5). Further analysis revealed that the recombinant PiGSTd1 could efficiently degrade the sex pheromone component Z9-12:Ac (75.63 ± 5.52%), as well as aldehyde volatiles, including hexanal (89.10 ± 2.21%), heptanal (63.19 ± 5.36%), (E)-2-octenal (73.58 ± 3.92%), (E)-2-nonenal (75.81 ± 1.90%), and (E)-2-decenal (61.13 ± 5.24%). Taken together, our findings suggest that PiGSTd1 may play essential roles in degrading and inactivating a variety of odorants, especially sex pheromones and host volatiles of P. interpunctella.

Functional Characterization of Olfactory Proteins Involved in Chemoreception of Galeruca daurica

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) play a fundamental role in insect olfaction. Galeruca daurica (Joannis) is a new pest with outbreak status in the Inner Mongolia grasslands, northern China. In this study, six olfactory protein genes (GdauOBP1, GdauOBP6, GdauOBP10, GdauOBP15, GdauCSP4, and GdauCSP5) were cloned by RACE and expressed by constructing a prokaryotic expression system. Their binding affinities to 13 compounds from host volatiles (Allium mongolicum) were determined by fluorescence-binding assay. In order to further explore the olfactory functions of GdauOBP15 and GdauCSP5, RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted. Ligand-binding assays showed that the binding properties of the six recombinant proteins to the tested volatiles were different. GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 could bind several tested ligands of host plants. It was suspected that GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 were related to the host location in G. daurica. We also found that there were different EAG responses between males and females when the GdauOBP15 and GdauCSP5 genes were silenced by RNAi. The EAG response of G. daurica females to 2-hexenal was significantly decreased in dsRNA-OBP15-injected treatment compared to the control, and the dsRNA-CSP5-treated females significantly reduced EAG response to eight tested host volatiles (1,3-dithiane, 2-hexenal, methyl benzoate, dimethyl trisulfide, myrcene, hexanal, 1,3,5-cycloheptatriene, and p-xylene). However, the EAG response had no significant difference in males. Both GdauOBP15 and GdauCSP5 may have different functions between males and females in G. daurica and may play more important roles in females searching for host plants.

Identification of a Male-Produced Volatile Pheromone for Phymatodes dimidiatus (Coleoptera: Cerambycidae) and Seasonal Flight Phenology of Four Phymatodes Species Endemic to the North American Intermountain West

Research over the last 15 yr has shown widespread pheromone parsimony within the coleopteran family Cerambycidae, with a number of highly conserved pheromone motifs, often shared within and across subfamilies, tribes, and genera. Our goals were to increase our understanding of the evolution of volatile pheromones within the Cerambycidae, their role in reproductive isolation and to identify pheromones for use in the development of lures for monitoring cerambycids. Over 3 yr, we tested 12 compounds known to be cerambycid pheromones as possible attractants at sites across Idaho. This study focused on species within the cerambycine genus Phymatodes (Tribe: Callidiini). We also collected and analyzed headspace volatiles of captured Phymatodes dimidiatus (Kirby). Our results demonstrate that (R)-2-methylbutan-1-ol is a male-produced volatile pheromone for P. dimidiatus. These results are consistent with prior research suggesting that (R)-2-methylbutan-1-ol and (R)-3-hydroxyhexan-2-one, individually or in a blend of both compounds, commonly serve as pheromones for Phymatodes spp. We captured Phymatodes starting in mid-May, continuing through mid-August. Our data indicate that flight periods of Phymatodes spp. in Idaho overlap. These species may be utilizing various mechanisms to ensure reproductive isolation, such as the production of different volatile pheromones, minor components, and/or proportions of components, utilizing different host species and/or host volatiles, differing daily activity periods, and/or occupying different heights in the tree canopy. Our results contribute to the basic understanding of the chemical and behavioral ecology of the Cerambycidae and can be applied to the development of pheromone lures for monitoring of economically important or endangered species.

Expression Profiles and Binding Properties of the Chemosensory Protein PxylCSP11 from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) is one of the most destructive pests to cruciferous plants worldwide. The oligophagous moth primarily utilizes its host volatiles for foraging and oviposition. Chemosensory proteins (CSPs) are soluble carrier proteins with low molecular weight, which recognize and transport various semiochemicals in insect chemoreception. At present, there is limited information on the recognition of host volatiles by CSPs of P. xylostella. Here, we investigated expression patterns and binding characteristics of PxylCSP11 in P. xylostella. The open reading frame of PxylCSP11 was 369-bp encoding 122 amino acids. PxylCSP11 possessed four conserved cysteines, which was consistent with the typical characteristic of CSPs. PxylCSP11 was highly expressed in antennae, and the expression level of PxylCSP11 in male antennae was higher than that in female antennae. Fluorescence competitive binding assays showed that PxylCSP11 had strong binding abilities to several ligands, including volatiles of cruciferous plants, and (Z)-11-hexadecenyl acetate (Z11-16:Ac), a major sex pheromone of P. xylostella. Our results suggest that PxylCSP11 may play an important role in host recognition and spouse location in P. xylostella.

Characterization of the Expression and Functions of Two Odorant-Binding Proteins of Sitophilus zeamais Motschulsky (Coleoptera: Curculionoidea)

Odorant-binding proteins (OBPs) are important in insect chemical communication. The objective of this research was to identify the functions of two OBPs in Sitophilus zeamais. qRT-PCR and western blot (WB) were performed to investigate the expression profiles at the transcript and protein levels, respectively. Fluorescence competitive binding assays were used to measure the ability of the OBPs to bind to host volatiles, and a Y-tube olfactometer was used to verify the results (attraction/no response) via behavioral experiments. The RNAi was used to verify the function by knocking down the ability of proteins to bind odorants. qRT-PCR showed the highest expression SzeaOBP1 and SzeaOBP28 at the low-instar larva (LL) and eclosion adult (EA) stages, respectively. WB showed that both SzeaOBP1 and SzeaOBP28 were highly expressed in the EA stage. Fluorescence competitive binding assays indicated that SzeaOBP1 exhibited extremely high binding affinity with cetanol. SzeaOBP28 exhibited a pronounced binding affinity for 4-hydroxy-3-methoxybenzaldehyde. The behavioral experiment showed that the adult S. zeamais responded strongly to 4-hydroxy-3-methoxybenzaldehyde and valeraldehyde from Sorghum bicolor. The RNAi knockdown individuals displayed behavioral differences between normal insects and dsRNA (SzeaOBP1)-treated insects. We infer that they both have functions in perception and recognition of host volatiles, whereas SzeaOBP28 may also have other functions.

Electroantennogram, behavioural responses, and field trapping of Trypophloeus klimeschi (Coleoptera: Curculionidae: Scolytinae) to eight host volatiles

Trypophloeus klimeschiEggers (Coleoptera: Curculionidae: Scolytinae) was first discovered in China in 2003, and it exhibits strong species specificity toPopulus albavar.pyramidalisBunge (Salicaceae). To screen plant volatile compounds for monitoring and trappingT. klimeschi, the electroantennogram responses of adultT. klimeschito eight plant volatiles, including nonanal, 2-methylbutanal, decanal, 2-hydroxybenzaldehyde, (Z)-3-hexen-1-ol benzoate, methyl benzoate, methyl salicylate, and geraniol were tested at various concentrations. Behavioural responses of female and male adults to various concentrations of these eight plant volatiles were also determined using a Y-tube olfactometer. We then tested the effectiveness of these compounds as lures for trappingT. klimeschiin the field. Electroantennogram tests showed thatT. klimeschipossesses olfactory sensitivity for eight compounds. Additionally, walkingT. klimeschiexhibited attraction to low concentrations (≤ 1 μg/μL) of all eight compounds in Y-tube olfactometer. Field experiment results indicated that baits composed of each volatile compound alone were more attractive to greater numbers ofT.klimeschithan the control. The methyl benzoate bait was better attracted byT.klimeschithan other tested volatiles. These results suggest that these compounds could be used in attraction of this stem-boring pest. This study could have important implications for the development of an effective semiochemical-based management tool forT. klimeschiin the field.