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Sex pheromones are deemed to play a significant role in sexual communication of most insects. Although many sex pheromone components in mirid bugs have been identified, the roles of odorant receptors in sex pheromone perception in Adelphocoris spp. (Hemiptera: Miridae) remain unknown so far. Here, AlinOR33, a candidate sex pheromone receptor in Adelphocoris lineolatus was functionally characterized. Phylogenetic analysis showed that AlinOR33 clustered with the sex pheromone receptor AlucOR4 fromApolygus lucorum. Quantitative real-time PCR measurement revealed that the expression of AlinOR33 increased gradually from nymph to adult stage and reached its peak in the antennae of 3-day-old mated male bugs. The subsequent in situ hybridization demonstrated that AlinOR33 was mainly expressed in sensilla trichoid on the antennae of A. lineolatus. In the two-electrode voltage clamp recordings, AlinOR33/AlinOrco was specifically tuned to four sex pheromone components including butyl butyrate, hexyl hexanoate, trans-2-hexenyl butyrate and hexyl butyrate, and especially most sensitive to the major component trans-2-hexenyl butyrate. After dsAlinOR33 injection, the electroantennogram responses of males to four sex pheromone components were reduced significantly (∼50%). Compared to control bugs, dsAlinOR33-injected male bugs almost lost behavioral preference for trans-2-hexenyl butyrate. Furthermore, the wingbeat frequency of dsAlinOR33-injected male bugs notably declined. Therefore, we conclude that as a candidate sex pheromone receptor, AlinOR33 plays essential roles in the sexual behavior of A. lineolatus.

Pheromone-binding proteins (PBPs) have been extensively investigated in lepidopteran moths, but their evolution and function in hemipteran species remain unclear. Our previous study demonstrated that an odorant-binding protein, OBP4, of the mirid bug Adelphocoris lineolatus functions as a candidate hemipteran PBP but clustered with lepidopteran antennae-binding proteins (ABPs) rather than in the PBP/general odorant-binding protein (GOBP) clade. In this study, we hypothesized that origin and function of PBPs in hemipteran bugs may differ from those of lepidopteran moths. To test this hypothesis, we first constructed a phylogenetic tree using insect OBPs from sister hemipteran and holometabolous lineages, and the results indicated that neither OBP4 nor other types of candidate PBPs of mirid bugs clustered with the lepidopteran PBP/GOBP clade. Then, a fluorescence competitive binding assay was employed to determine binding affinities of recombinant OBP4 protein to host plant volatiles, with functional groups different from A. lineolatus sex pheromone components. The results revealed that OBP4 highly bound the female adult attractant 3-hexanone and 15 other mirid bug biologically active plant volatiles. Finally, we examined cellular expression profiles of OBP4 in putative antennal sensilla that are related to female A. lineolatus host plant location. The fluorescence in situ hybridization and immunocytochemical labeling assay showed that the OBP4 gene was highly expressed in the multiporous olfactory sensilla medium-long sensilla basiconica rather than in the short sensilla basiconica or uniporous sensilla chaetica. These results, together with those of our previous studies, indicate that OBP4 not only functions in recognition of bug-produced sex pheromones in males, but is probably involved in detection of host plant volatiles in both A. lineolatus sexes. Our findings support the hypothesis that the origin and function of PBPs in hemipteran bugs differ from those of well-known PBPs in lepidopteran moths, which provides a novel perspective on evolutionary mechanisms of sex pheromone communication across insect orders.

The sex pheromone composition of alfalfa plant bugs, Adelphocoris lineolatus (Goeze), from Central Europe was investigated to test the hypothesis that insect species across a wide geographical area can vary in pheromone composition. Potential interactions between the pheromone and a known attractant, (E)-cinnamaldehyde, were also assessed. Coupled gas chromatography-electroantennography (GC-EAG) using male antennae and volatile extracts collected from females, previously shown to attract males in field experiments, revealed the presence of three physiologically active compounds. These were identified by coupled GC/mass spectrometry (GC/MS) and peak enhancement as hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal. A ternary blend of these compounds in a 5.4:9.0:1.0 ratio attracted male A. lineolatus in field trials in Hungary. Omission of either (E)-2-hexenyl-butyrate or (E)-4-oxo-2-hexenal from the ternary blend or substitution of (E)-4-oxo-2-hexenal by (E)-2-hexenal resulted in loss of activity. These results indicate that this Central European population is similar in pheromone composition to that previously reported for an East Asian population. Interestingly, another EAG-active compound, 1-hexanol, was also present in female extract. When 1-hexanol was tested in combination with the ternary pheromone blend, male catches were reduced. This compound showed a dose-response effect with small doses showing a strong behavioral effect, suggesting that 1-hexanol may act as a sex pheromone antagonist in A. lineolatus. Furthermore, when (E)-cinnamaldehyde was field tested in combination with the sex pheromone, there was no increase in male catch, but the combination attracted both males and females. Prospects for practical application are discussed.

BackgroundColor vision and phototactic behavior based on opsins are important for the fitness of insects because of their roles in foraging and mate choice. Related topics, including the duplication and loss of opsin genes, have been well investigated in insect orders such as Coleoptera, Lepidoptera, Hymenoptera, Odonata and Orthoptera, and the findings have been used to develop pest management strategies involving light trapping. Mirid bugs of Hemiptera, which are pests that cause heavy economic losses, show capacity for color discrimination and phototaxis. However, the opsins in mirid bugs remain uncharacterized. Herein, we examined five species to investigate the evolution of opsins in the family Miridae.ResultsUsing RNA-seq, we identified several contigs showing high identity with opsins, including four contigs inApolygus lucorumand three contigs each inAdelphocoris suturalis,Adelphocoris fasciaticollis,Adelphocoris lineolatusandNesidiocoris tenuis. Phylogenetic analyses indicated that one of these genes clustered with ultraviolet-sensitive (UV) opsins and that the others clustered with long-wavelength (LW) opsins, suggesting that duplication of LW opsins and loss of blue light-sensitive (B) opsins occurred in mirid bugs. The existence of introns in the LW opsins of mirid bugs suggested that the duplication events were DNA based. Both LW1 and LW2 opsins of mirid bugs were found to be under strong purifying selection. The LW1 opsins were significantly more highly expressed than the LW2 and UV opsins.ConclusionsWe identified the opsins of mirid bugs using five selected mirid species as a representative sample. Phylogenetic analyses clustered one of the genes with UV opsins and the others with LW opsins, suggesting the occurrence of LW opsin duplication and B opsin loss during the evolution of mirid bugs. Intron detection suggested that the identified duplication event was DNA based. The evidence of strong purifying selection and the relatively high expression levels suggested that these opsins exhibit fundamental functions in mirid bugs.

Plant volatiles such as floral scent compounds play a crucial role in mediating insect host locating, mate search, and oviposition sites selection. The alfalfa plant bug, Adelphocoris lineolatus (Goeze), is a seriously polyphagous herbivore of alfalfa and cotton that has an obvious preference for flowering host plants. In this study, we focused on the role of an odorant receptor AlinOR59 in the perception of plant volatiles in A. lineolatus. In situ hybridization showed that AlinOR59 was coexpressed with the coreceptor AlinORco in the ORNs cell located in the long curved sensilla trichodea on antennae of both genders. The Xenopus oocytes expression coupled with two-electrode voltage clamp recordings demonstrated that AlinOR59 responded to 15 plant volatiles. In electroantennogram assays, all of the above 15 compounds could excite electrophysiological responses in the antennae of adult bugs. Furthermore, an important floral scent compound, methyl salicylate, was utilized to evaluate the behavioral responses of A. lineolatus. It was found that adult bugs of both genders were significantly attracted to methyl salicylate. Taken together, our findings suggest that AlinOR59 plays a crucial role in the perception of floral scents in A. lineolatus and could be used as a potential target to design novel olfactory regulators for the management of bugs.

This paper presents analysis of the effects of the alfalfa crop age, sowing method, phenological development phases, and green mass mowing on the composition and density of insects and spiders on the aboveground organs of alfalfa grown in crop rotation without irrigation, pest control measures, and fertilizers. The main inhabitants of alfalfa crops comprised over 50 species, with prevalence of the chewing herbivores: alfalfa weevil Hypera postica, alfalfa seed chalcid Bruchophagus roddi, nodule weevils Sitona lineatus and S. inops; the sucking herbivores: flower thrips Odontothrips sp., gorse shield bug Piezodorus lituratus, lucerne bug Adelphocoris lineolatus, plant bug Lygus pratensis, cowpea aphid Aphis craccivora, and pea aphid Acyrthosiphon pisum; the predators: spiders, predatory bug Orius niger, predatory thrips Aeolothrips sp., and variegated ladybug Hippodamia variegata. The seasonal dynamics of the composition and density of dominant groups was more pronounced in the first-year crops than in 2–4 year old ones. In field crop rotations without the use of insecticides, plots with periodic green mass mowing during the budding and flowering phases of alfalfa seed crops should alternate with unmown plots, so that plots with blossoming plants should be present in the field throughout the summer. This measure would promote the development and maintenance of highly diverse insect complexes, attract predators and pollinators, provide additional feeding for parasitic Hymeno­ptera, and reduce the number and harmfulness of herbivores as the result of greater trophic competition between them as well as of the impact of entomophages.

Several plant bug species (Miridae) are important pests of crops and vegetables, thus monitoring them is of essential importance for effective pest control. During the current, preliminary study synthetic plant volatile combinations were tested in field conditions in Hungary in alfalfa fields. Beside semiochemical baited traps, sweep-netting was also performed. In the experiments three plant bug species were found in higher numbers: Adelphocoris lineolatus, Lygus rugulipennis and L. pratensis. As a novel, interesting finding L. pratensis was attracted to phenylacetaldehyde baited traps. For all species, both males and females were trapped in all combinations. Sweep-netting and semiochemical baited traps showed different efficacy in case of the three species, as sweep-netting catches were highly biased for A. lineolatus, which indicates the higher efficacy of this method as compared to the tested semiochemical-baited traps. On the other hand, semiochemical baited showed better performance for L. rugulipennis and L. pratensis. For these species none of the tested combinations performed better than phenylacetaldehyde baited traps. The potential implication of results in view of monitoring are discussed.

Odorant-binding proteins (OBPs) can bind and transport hydrophobic odorants across the sensillum lymph to the olfactory receptors (ORs) and play crucial roles in insect chemosensory systems. Although the ligand spectra of classical OBPs have been extensively characterized, little is known about OBPs in the Plus-C subgroup. Here, we focus on AlinOBP14, a Plus-C OBP from the hemipteran mirid bug pest Adelphocoris lineolatus (Goeze). Quantitative real-time PCR experiments suggest that AlinOBP14 is ubiquitously expressed at different developmental stages but is highly expressed in the adult head, the non-chemosensory organ. Fluorescence-based competitive binding assays show that β-ionone, nerolidol, farnesol and insect juvenile hormone III (JHIII) strongly bind to AlinOBP14. No significant internal binding pocket is predicted by homology modeling. Instead, the long N-terminal and C-terminal regions and parts of several α-helixes form a cupped cavity to accommodate ligands. Molecular docking reveals that the four potential ligands have distinct binding orientations, implying different roles of the N-terminal extension in ligand recognition. This hypothesis is further confirmed via a ligand binding assay in which the recombinant N-terminal mutant AlinOBP14 displays comparable binding affinities for β-ionone and trans, trans-farnesol but decreased binding affinities for nerolidol and JHIII. Thus, our current study is the first to characterize the ligand binding spectra of a Plus-C OBP in hemipteran insect species and reveals that N-terminal extensions could be required for its recognition of putative ligands.

Mirid bugs are a group of important insect pests that cause large annual losses in agricultural production. Many studies have focused on the isolation and identification of sex pheromones in mirid bugs, and the components and biological activity of the sex pheromones have also been studied as a way to control these pests. However, few studies have focused on the mechanisms of pheromone perception. In this study, we identified the odorant receptor repertoire in three mirid bug species, Apolygus lucorum, Adelphocoris lineolatus, and Adelphocoris suturalis using antennal transcriptome sequencing and bioinformatics analysis. The candidate pheromone receptor (PR) genes were then identified by comparative transcriptomic and expression pattern analysis. Importantly, in vitro functional studies have shown that the candidate PRs have robust responses to the main mirid bug sex pheromone components (E)-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB). Our study uncovered the mechanism of pheromone peripheral coding in these three species and elucidated the mechanism by which mirid bugs can specifically recognize a mate. Moreover, the results of our study will provide a theoretical basis for screening effective sex attractants or mating disturbance agents at the molecular and neural levels for enhanced control of these destructive pests.

The alfalfa plant bug Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), a serious pest of several agricultural crops, is extremely attracted by flowering plants to perform season host switching. Generally, chemoreception of A. lineolatus plays a crucial role in detecting food sources, locating mates and oviposition sites. In addition, odorant carrier proteins are believed to participate in the initial chemical communication and perception in insects. In this work, we identified 31 putative odorant carrier protein genes from the antennal transcriptomes of A. lineolatus, including 17 new odorant binding proteins (OBPs), 10 new chemosensory proteins (CSPs) and four Niemann-Pick type C2 proteins (NPC2s). Phylogenetic analysis demonstrated that the OBP and CSP genes from four mirid bug species (A. lineolatus, Adelphocoris suturalis, Apolygus lucorum and Lygus lineolaris) had a relatively close evolutionary relationship. Regular patterns and key conserved motifs of OBPs and CSPs in Hemiptera insects are also identified by using Multiple EM for Motif Elicitation (MEME) tool. Tissue expression profiles analysis showed that nine of the 17 OBPs, 10 CSPs and two of the four NPC2s were primarily or uniquely expressed in the adult antennae suggesting their putative roles in chemoreception. Our study provides the first insight into the roles of odorant carrier proteins in chemoreception of A. lineolatus, which will help to facilitate biological functions of odorant carrier proteins and develop novel insect behavioral regulation strategy in future.

The alfalfa plant bug Adelphocoris lineolatus, an economically important pest, has representative behavioral characteristics with host plants transfer. Olfactory system is essential for insects to perceive ever-changing chemical signals in the external environment, and chemosensory genes play crucial roles in signals reception and transduction. In this work, we compared the differences in chemosensory genes expression before and after host plants transfer by constructing 12 antennal transcriptomes of male and female bugs, respectively. The results showed that the expression levels of most chemosensory genes in A. lineolatus changed to adapt to the transformation of the hosts plant. More remarkable, female bugs had more up-regulated chemosensory genes than males. Differentially expressed genes (DEGs) analysis revealed three odorant binding proteins (OBPs), three chemosensory proteins (CSPs), eight odorant receptors (ORs) and one ionotropic receptor (IR) showed significant differences when the host plant transferred. There were complex characteristics of up- and down- regulated genes in male and female adults, among which OBP19 showed higher expression in females exposing to the new host plant alfalfa, suggesting this OBP may be associated with the localization of the oviposition site. The OR54 and OR82 were up-regulated in both genders, indicating their possible roles in recognizing some alfalfa-specific volatiles. These findings will provide valuable insights in biological functions of chemosensory genes in A. lineolatus and facilitate the development of new targets for novel strategies to control the alfalfa plant bug and other herbivores.

The polyphagous mirid bug Adelphocoris lineolatus relies heavily on olfactory cues to track suitable host plants. Thus, a better understanding of the molecular basis of its olfactory detection could contribute to the development of effective pest management strategies. In the present study, we report the expression profile of the odorant binding protein gene A. lineolatus odorant binding protein 6 (AlinOBP6). Quantitative real-time PCR experiments suggest that AlinOBP6 is female adult antennae-biased. Cellular immunolocalization analyses show that AlinOBP6 is highly expressed in the lymph of both multiporous sensilla basiconica and uniporous sensilla chaetica. A ligand binding analysis showed that recombinant AlinOBP6 not only bound tightly to host plant volatile compounds but also to nonvolatile compounds. Homology modelling and molecular docking analyses confirmed these unusual ligand binding profiles and revealed that the amino acid residues involved in the recognition of volatile and nonvolatile compounds are distinct. The results of our study are the first to suggest that an antenna- and female-biased OBP in an hemipteran insect is expressed in both olfactory and gustatory sensilla as a mechanism to respond to volatile and nonvolatile host compounds. These findings warrant further research into the molecular mechanisms of chemosensation for mirid bugs in responsive to host plant location.

Determining the spatio-temporal distribution and association of pests and natural enemies would be useful for implementing biological control of pests and could also be used in site-specific pest management. In this study, the spatio-temporal distribution and association of aphids, plant bugs, and natural enemies were assessed in alfalfa fields using geo-statistics and spatial analysis by distance indices (SADIE). Additionally, the effect of alfalfa hay-harvesting on the spatial and temporal distribution of these insects was investigated for the first time. Geostatistical analysis indicated that the degree of dependence (DD) was &ge, 75% for 11 out of 39, 9 out of 35, 3 out of 12, 10 out of 29, and 2 out of 20 datasets for pea aphid Acyrthosiphon pisum, spotted alfalfa aphid Therioaphis maculata, cowpea aphid Aphis craccivora, alfalfa plant bug Adelphocoris lineolatus, and tarnished plant bug Lygus rugulipennis, respectively. The results also indicated that DD was &ge, 75% in 7 out of 45, 18 out of 45, and 3 out of 20 datasets for Coccinella septempunctata, Hippodamia variegata, and Pterostichus melanarius, respectively. Harvesting decreased the aggregation of the ladybirds, which resulted in a decrease in the index of aggregation. The geo-statistics results were confirmed by SADIE in 75% of datasets. These results can be used in biological control and site-specific management of aphids and plant bugs in alfalfa fields.

Adelphocoris lineolatus (Goeze, 1778 ) Cimex lineolatus Goeze, 1778: 267. NEW DATA. — Valdieri, Valasco, 1450 m, 44.204067°N, 7.256410°E (WGS84), 31.VIII.2013, MN, MD leg., 1 specimen, MZUF. — Valdieri, Valasco, 1450 m, 44.204067°N, 7.256410°E (WGS84), 09.IX.2013, MN, MD leg., 1 specimen, MZUF. — Entracque, Fus Carbonaro, 900 m, 44.24899°N, 7.38166°E (WGS84), 02.IX.2014, MN, MD leg., 1 specimen, MZUF. — Entracque, Fus Carbonaro, 900 m, 44.24899°N, 7.38166°E (WGS84), 28.VII.2015, LB & MN leg., 7 specimens, MZUF, MNHN. — Valdieri, Sant’Anna di Valdieri, Gesso della Valletta stream, 980 m, 44.2433°N, 7.3228°E (WGS84), 26-29.VII.2015, MN & LB leg., 1 specimen, MZUF. CHOROTYPE. — Holarctic., Published as part of Norbiato, Margherita, Dioli, Paride & Cianferoni, Fabio, 2019, Annotated checklist of the Hemiptera Heteroptera of the Site of Community Importance and Special Area of Conservation " Alpi Marittime " (NW Italy), pp. 269-312 in Zoosystema 41 (15) on page 278, DOI: 10.5252/zoosystema2019v41a15, http://zenodo.org/record/3722483, {"references":["GOEZE J. A. E. 1778. - Entomologische Beytrage zu des Ritter Linne zwolften Ausgabe des Natursystems. Zweyter Theil. Weidmanns Erben und Reich, Leipzig, 352 p. https: // doi. org / 10.5962 / bhl. title. 45974"]}

Chemosensory membrane proteins, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs) and sensory neurone membrane proteins (SNMPs), are supposed to be crucial macromolecules in the insect olfactory signal transduction pathway. The alfalfa plant bug Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) is highly attracted to high-nitrogen or flowering plants and destroys many important agricultural crops. We assembled the antennal transcriptome of A. lineolatus using Illumina sequencing technology and identified a total of 108 transcripts encoding chemosensory membrane proteins (88 ORs, 12 IRs, four GRs and four SNMPs), amongst which 90 candidates appeared to be full length. Subsequently, both semiquantitative reverse transcription PCR and quantitative real-time PCR experiments were performed to investigate their tissue- and sex-biased expression profiles. The results showed that nearly all of the 108 candidate chemosensory membrane protein genes were largely expressed in adult antennae, and some genes additionally displayed significant differences in the expression levels between sexes. The results of our phylogenetic analysis and the detailed tissue- and sex-biased expression characteristics given here provide an important foundation for further understanding of the complex chemoreception system of the alfalfa plant bug and other Hemiptera species, which also could help us use chemosensory membrane proteins as targets to manipulate insect olfactory behaviour and broaden the applications of available tools for insect pest control.

Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. Here, we injected AlinOBP4-siRNA into the conjunctivum between prothorax and mesothorax of the lucerne plant bug, Adelphocoris lineolatus and evaluated the silencing of AlinOBP4 by reverse transcription polymerase chain reaction (RT-PCR) analysis, quantitative real-time PCR (qPCR) test and electroantennogram (EAG) assay. The combination of RT-PCR and qPCR analyses revealed that the levels of messenger RNA transcript were significantly reduced ∼95% in AlinOBP4-siRNA-treated A. lineolatus males and ∼75% in RNAi-treated females within 48 hours. It was found that there are different EAG responses between male and female bugs when the AlinOBP4 gene was silenced by RNAi. The EAGs of A. lineolatus to two plant volatiles, tridecanal and hexyl alcohol, were reduced 9.09% and 79.45% in RNAi-treated males, 62.08% and 62.08% in RNAi-treated females compared to the controls, separately. Antennae of RNAi-treated bugs showed significantly lower electrophysiological responses to four sex pheromone analogs, butyl butanoate, 1-hexyl butyrate, (E)-2-hexenyl butyrate and hexyl hexanoate. The EAG recordings were reduced 35.43%, 35.24%, 39.96% and 78.47% in RNAi-treated males and 64.52%, 18.13%, 36.88% and 49.52% in RNAi-treated females, respectively. The results suggested that AlinOBP4 might play dual-roles in the identification of plant volatiles and sex pheromones. It was suspected that AlinOBP4 may have different functions in odor perception between male and female A. lineolatus.

Pheromone binding proteins (PBPs) are well studied in lepidopteran moths and are considered to be crucial in detection of sex pheromones as well as some green leaf volatiles. In contrast, evidence that PBPs interact with sex pheromones of hemipteran species is not available. The mirid plant bug, Adelphocoris lineolatus (Goeze), is a notorious hemipteran pest that uses two butyrate esters, trans-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB), and one hexenoic aldehyde trans-4-oxo-2-hexenal (E4O2H), as sex pheromones. In the present study, we report on an odorant binding protein, AlinOBP4, with particular focus on its potential physiological roles in the detection of A. lineolatus sex pheromone components. Phylogenetic analyses indicated that AlinOBP4 and two mirid orthologs clustered in a general phylogenetic clade with the lepidopteran ABX OBPs, the fly LUSH and the OBP83a/b subfamily. Cellular localization by fluorescence in situ hybridization and immunolabeling further demonstrated that AlinOBP4 was strongly expressed in the multiporous sensilla trichodea (str) and middle long sensilla basiconica (mlsba) of male A. lineolatus adults, suggesting a key role associated with sex pheromone and odorant detection. A ligand binding assay revealed that recombinant AlinOBP4 protein highly bound not only to the sex pheromone components E4O2H but also to some host plant volatiles. These findings together with the evidence of insect PBPs available in the literature support the view that AlinOBP4 is involved in sex pheromone detection in male A. lineolatus and provide foundational information for further elucidating the molecular mechanisms of chemosensory based mating behavior in hemipteran mirid bugs.

The phytophagous mirid bugs of Apolygus lucorum, Lygus pratensis as well as three Adelphocoris spp., including Adelphocoris lineolatus, A. suturalis, and A. fasciaticollis are major pests of multiple agricultural crops in China, which have distinct geographical distribution and occurrence ranges. Like many insect species, these bugs heavily rely on olfactory cues to search preferred host plants, thereby investigation on functional co-evolution and divergence of olfactory genes seems to be necessary and is of great interest. In the odorant detection pathway, olfactory receptor co-receptor (Orco) plays critical role in the perception of odors. In this study, we identified the full-length cDNA sequences encoding three putative Orcos (AsutOrco, AfasOrco, and LpraOrco) in bug species of A. suturalis, A. fasciaticollis, and L. pratensis based on homology cloning method. Next, sequence alignment, membrane topology and gene structure analysis showed that these three Orco orthologs together with previously reported AlinOrco and AlucOrco shared high amino acid identities and similar topology structure, but had different gene structure especially at the length and insertion sites of introns. Furthermore, the evolutional estimation on the ratios of non-synonymous to synonymous (Ka/Ks) revealed that Orco genes were under strong purifying selection, but the degrees of variation were significant different between genera. The results of quantitative real-time PCR experiments showed that these five Orco genes had a similar antennae-biased tissue expression pattern. Taking these data together, it is thought that Orco genes in the mirid species could share conserved olfaction roles but had different evolution rates. These findings would lay a foundation to further investigate the molecular mechanisms of evolutionary interactions between mirid bugs and their host plants, which might in turn contribute to the development of pest management strategy for mirid bugs.

* Adelphocoris lineolatus (Goeze, 1778) Kotłowy Stream, 15 Aug 2006, 1 ex., Published as part of TASZAKOWSK, Artur & PASI��SKA, Agnieszka, 2017, New data on the occurrence of terrestrial true bugs (Hemiptera: Heteroptera) in Pieniny Mountains, pp. 1-13 in Fragmenta Faunistica 60 (1) on page 5, DOI: 10.3161/00159301FF2017.60.1.001, http://zenodo.org/record/6251980

In northern China, due to the large-scale adoption of transgenic Bacillus thuringiensis Berliner (Bt) cotton, the number of Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) has increased quickly, causing significant loss in cotton and alfalfa production. One of the environmentally safe strategies without use of pesticides is the application of insect pheromone for mating disruption. In our study, we aim to identify the active components in sexually mature virgin female A. lineolatus, and activity and optimal ratio of these components. By using gas chromatography–mass spectrometry (GC-MS) and gas chromatography–electroantennographic detection (GC-EAD), we identified three active compounds: hexyl butyrate (HB), (E)-2-hexenyl butyrate (E2HB), and (E)-4-oxo-2-hexenal (4-OHE). We examined the release rate of septum and tube dispensers in a wind tunnel. In a field experiment, deletion of 4-OHE or E2HB resulted in significant suppression of male trap catches, indicating that E2HB or 4-OHE may be the active pheromone components. Traps baited with a blend of 4-OHE and E2HB at 2:5, 3:4, and 4:3 caught significantly more males, suggesting that the optimal ratio of 4-OHE and E2HB could be in the range of 1:1 to 1:2. The addition of a large amount of HB could strongly reduce the attractive activity of both virgin females and artificial lures. We also compared the attractiveness of septum lures and tube lures in field experiments. The septum lures attracted males in the first 3 days. The tube lures caught more males after 3 days and the attractive effects existed up to 5 weeks, suggesting them as a tool for long-term monitoring and control of plant bugs.

In China, Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) is an important pest of alfalfa, cotton, and other crops, while Peristenus spretus (Chenvan Achterberg) (Hymenoptera: Braconidae) is the dominant nymphal parasitoid of this mirid bug. In the present study, the toxicity of 17 common insecticides to A. lineolatus was evaluated, and the susceptibility of P. spretus to the insecticides with high toxicity to A. lineolatus was tested under laboratory conditions. Of the 17 insecticides tested, 12 (beta cypermethrin, deltamethrin, carbosulfan, acetamiprid, emamectin benzoate, imidacloprid, phoxim, chlorpyrifos, acephate, profenophos, hexaflumuron, and abamectin) had a highly toxic effect on second-instar nymphs of A. lineolatus, with LC(50) values ranging from 0.58 to 14.85 mg a.i. (active ingredient) liter(-1). Adults of P. spretus were most sensitive to chlorpyrifos, with LC(50) values of 0.03 mg a.i. liter(-1), followed by phoxim, acetamiprid, profenophos, carbosulfan, acephate, deltamethrin, emamectin benzoate, imidacloprid, beta-cypermethrin, and abamectin, with LC(50) values ranging from 0.06 to 3.09, whereas hexaflumuron exhibited the least toxicity to the parasitoid, with LC(50) values500 mg a.i. liter(-1). A risk quotient analysis indicated that beta-cypermethrin, emamectin benzoate, abamectin, and hexaflumuron when applied against A. lineolatus were the least toxic to P. spretus.

In Manitoba, Canada, commercial soybeans and two types of field beans, navy and pinto, were sampled for plant bugs. At the centre and margins of 36 fields, sweep net and tap tray samples were taken weekly. In all three crops, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae) comprised >78% and Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) Lygus Hahn, nymphs were 46% of the catch in trays and 23% in sweeps, but total Lygus catch in trays was >2% of that in sweeps. Catch/sample effort was similar at field centres and margins for both Lygus adults and nymphs. Lygus lineolaris reproduced and developed in all three crops. In field beans, Lygus adults were first collected in late vegetative and early pod set stages and late instar nymphs and adults were present from pod elongation until maturity – results consistent with immigration of first summer generation reproductive adults and development of the second generation in the crops. There was evidence of late season immigration of Lygus into all crops. There was no loss of seed quantity attributable to mirids in any of the crops; in field beans there was no evidence that mirids reduced seed quality.

The genus Adelphocoris (Hemiptera: Miridae) is a group of key insect pests in cotton fields in China that includes three dominant species: A. suturalis (Jakovlev), A. lineolatus (Goeze) and A. fasciaticollis (Reuter). Previous field surveys have found that adults of these Adelphocoris species usually move onto specific host plants when the plants enter the flowering stage. To determine the potential trade-offs for this host-plant preference behavior, the performance of these three Adelphocoris spp. on flowering and non-flowering cotton and alfalfa were compared in the laboratory. The results showed that Adelphocoris spp. had significantly higher nymphal developmental and survival rates, along with increased adult longevity and fecundity on flowering cotton and alfalfa than on non-flowering plants of either species. In addition, compared with cotton plants, alfalfa generally promoted better performance of these three Adelphocoris species, especially for A. lineolatus. Simple correlation analysis indicated that female adult longevity was positively correlated to male adult longevity and female fecundity, and female fecundity was positively correlated to nymphal development and survival rates. This study demonstrated a positive correlation between adult preference and offspring/adult performance for all three Adelphocoris species, with no evidence of any trade-offs for this preference for flowering host plants. These findings support the hypothesis that hemimetabolous insects such as mirid bugs have positive adult preference-adult/nymphal performance relationships, which is likely due to the similar feeding habits and nutritional requirements of adults and nymphs.

Odorant-binding proteins (OBPs) are crucial in the olfactory pathway of insects. In the present study, the antenna-enriched OBP AlinOBP13 was investigated because of its potential contribution to the peripheral olfactory perception in the alfalfa plant bug Adelphocoris lineolatus. The results of quantitative reverse transcriptase-PCR showed that the transcript level of AlinOBP13 was higher in the adult stage than in the nymph stages. The transcript levels of AlinOBP13 in the male and female antennae significantly increased after 4 and 8 h of starvation, respectively. Fine ultrastructures of different types of chemosensilla in both female and male antennae were investigated using transmission electron microscopy and immunocytochemical labelling. The results revealed that the anti-AlinOBP13 antiserum strongly and specifically labelled short basiconic sensilla; this antiserum was restricted to the inner lumen and the cavities below the sensillum base of the sensilla. By contrast, multiporous sensilla trichodea, medium long sensilla basiconica, and aporous sensilla chaetica were not labelled. The present study is the first to report an OBP showing specific expression in the short basiconic sensilla of a member of the Hemipteran species. The results of a fluorescence displacement binding assay indicated that recombinant AlinOBP13 showed a more specific binding preference to terpenoids than to sex pheromones and other classes of chemicals. This binding ability was dramatically affected by pH; higher binding affinities were displayed at pH 10.0 than at pH 7.4 and 5.0. In addition, the results of dose-dependent electroantennogram recordings from the antennae showed that both female and male adult bugs responded to the terpenoids tested, suggesting an apparent physiological relevance of AlinOBP13 in A. lineolatus chemoreception. The results of this study suggest that AlinOBP13 functions as a specific carrier of terpenoids and provide insights into the mechanism of A. lineolatus in response to green volatiles.

In agroecosystems, potential species distribution models are extensively applied in pest management strategies, revealing species ecological requirements and demonstrating relationships between species distribution and predictive variables. The Maximum Entropy model was used to predict the potential distribution of five heteropteran key pests in Iran, namely Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), Lygus pratensis (L.), Apodiphus amygdali (Germar) (Hemiptera: Pentatomidae), Nezara viridula (L.), and Nysius cymoides (Spinola) (Hemiptera: Lygaeidae). A total of 663 samples were collected from different parts of Iran. The altitude and climate variable data were included in the analysis. Based on test and training data, the area under the receiver operating characteristic curve values were above 0.80, the binomial omission test with the lowest presence threshold for all species was statistically significant (< 0.01), and the test omission rates were less than 3%. The suitability of areas in Iran for A. amygdale (Germar) (Hemiptera: Pentatomidae), N. cymoides (Spinola) (Hemiptera: Lygaeidae), A. lineolatus (Goeze) (Hemiptera: Miridae), L. pratensis (L.), and N. viridula (L.) (Hemiptera: Pentatomidae), ranked as 78.86%, 68.78%, 43.29%, 20%, and 15.16%, respectively. In general, central parts of Iran including salt lakes, deserts, and sand dune areas with very high temperatures and windy weather were predicted to be less suitable, while other regions, mainly northern parts, were most suitable. These new data could be applied practically for the design of integrated pest management and crop development programs.

Olfaction plays an important role in insects' survival and reproduction. Odorant-binding proteins (OBPs) are considered to be one of the crucial proteins in the insect olfactory pathway. In this study, an antenna-specific OBP of the alfalfa plant bug, Adelphocoris lineolatus AlinOBP5, was expressed and purified in vitro. The binding affinities of AlinOBP5 with sex pheromone analogues of the Miridae and cotton volatiles were investigated by fluorescence competitive binding assays. The binding sites of AlinOBP5 were predicted by three-dimensional structure modelling and molecular docking, and site-directed mutagenesis. AlinOBP5 could not effectively bind with sex pheromone analogues of Miridae but showed high binding abilities with specific cotton volatiles, such as cis-nerolidol, ethyl laurate, β-ionone, β-caryophyllene, 2,3-dimethylbenzoic acid and (E)-farnesol. The strongest binding affinity was to cis-nerolidol, suggesting a role of AlinOBP5 in general odorant chemoreception. Based on the relatively strong binding affinity and the reported physiological activity of cis-nerolidol in other insects, we chose cis-nerolidol for further homology modelling and ligand docking. The results of molecular simulation and site-directed mutagenesis indicated that two amino acids, Lys74 and Pro121, in the protein binding pocket are the key amino acids involved in the binding of cis-nerolidol. The Lys74 residue may participate in specific recognition of ligands, and the Pro121 residue plays a crucial role in ligand binding and release by changing the binding pocket environment and stabilizing the conformation of the C-terminus of AlinOBP5.

A trial was conducted in the experimental field of the Institute of Forage Crops, Bulgaria, over the period 2012-2014 with alfalfa wich grew for seed production. It was studied the action of three insecticides (two biological /NeemAzal T/S and Pyrethrum FS EC/ and one synthetic insecticide Nurelle D - standard) to a control of lucerne seed weevil, Tychius flavus Beck. (Coleoptera: Curculionidae); alfalfa plant bug, Adelphocoris lineolatus Goeze (Hemiptera, Heteroptera: Miridae) and pea aphid, Acyrthosiphon pisum Harr. (Hemiptera, Sternorrhyncha: Aphididae) - major pests in alfalfa seed production. The method of sweeping with entomological net was used. Organic products were most effective against A. pisum. Increasing efficacy of NeemAzal reached satisfactory values of 67.8 and 69.1 percent of the seventh and ninth day after the application. Pyrethrum exhibited strong efficacy throughout the reporting period in the range 73.9-82.8% and differences between bioinsecticides were significant. In compared with standard its toxic action after the fifth day was approached the effect of synthetic insecticide with minor differences between them. Bioinsecticides exhibited good control against A. lineolatus as the trend of the better protective effect of Pyrethrum was retained. A dominant influence on the efficacy variation and seed productivity had the type of insecticide followed by factors year of treatment. The productivity of аlfalfa grown for seed production realized after application of Pyrethrum significantly exceeded control and was comparable to that of Nurelle D.

Field experiments were carried out to ascertain whether synthetic floral odour compounds were attractive for two pest bug species. The European tarnished plant bug (Lygus rugulipennis Poppius) has been reported to damage various crops (e.g. strawberry, sugarbeet, alfalfa, cucumber), and the alfalfa plant bug (Adelphocoris lineolatus (Goeze)) is considered as a pest of alfalfa and Bt-cotton. In our field tests, traps baited with phenylacetaldehyde caught significantly more L. rugulipennis than unbaited traps. In addition, A. lineolatus was also attracted to phenylacetaldehyde-baited traps. When testing other, EAG active compounds, (E)-cinnamaldehyde attracted A. lineolatus as well. This compound was also attractive for L. rugulipennis, however, to a lesser extent than phenylacetaldehyde. When the two compounds were presented in combination, no synergistic or inhibitory effect was detected in either species. By attracting both sexes of both species, these new attractants may prove to be useful and provide the basis for further development of new lures for agricultural use.

In northern China, Adelphocoris suturalis, Adelphocoris lineolatus and Adelphocoris fasciaticollis (Hemiptera: Miridae) are common pests of cotton and several other crops. These species have vastly diverse geographic distribution, seasonal dynamics and abundance, the underlying causal factors of which are poorly understood. In this study, the importance of a broad range of plant species as overwintering hosts for each Adelphocoris sp. was compared. Nymphal emergence from a total of 126 plant species was monitored at two distinct locations. The eggs of A. suturalis successfully eclosed from un-plowed cotton field soil and 115 plant species, primarily pastures, weeds and agricultural crops. The eggs of A. lineolatus successfully eclosed from 40 plant species, mainly pastures and weeds. Finally, A. fasciaticollis overwintered on 35 plant species, primarily tree species, weeds and agricultural crops. In conclusion, the most common and widely distributed mirid species, A. suturalis, overwintered on a comparatively broader range of plants compared to the other two species. These observations help to understand the differences in geographical distribution and abundance of the three Adelphocoris species, and constitute the basis for forecasting and pest management protocols for Adelphocoris spp. in China.