Heliothis Virescens Research Articles

Species-specific evolution of lepidopteran TspC5 tetraspanins associated with dominant resistance to Bacillus thuringiensis toxin Cry1Ac

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have proven to be highly effective in managing some key pests. However, the evolution of resistance by the target pests threatens the sustainability of Bt crops. The L31S mutation in a tetraspanin encoded by HarmTspC5 (previously known as HaTSPAN1) has been shown to confer dominant resistance to the Bt protein Cry1Ac in Helicoverpa armigera, a globally damaging lepidopteran pest. However, the broader implications of the L31S mutation in the tetraspanins of other lepidopteran species remain unclear. The evolutionary analyses in this study indicate that TspC5s have evolved in a species-specific manner among the lepidopteran insects. To investigate the role of TspC5s in conferring dominant resistance to Cry1Ac, we used the piggyBac-based transformation system to generate four transgenic H. armigera strains that express exogenous TspC5 variants from three phylogenetically close species (Helicoverpa zea, Helicoverpa assulta and Heliothis virescens) and one phylogenetically distant species (Plutella xylostella). In comparison with the background SCD strain of H. armigera, the transgenic strains expressing HzeaTspC5-L31S, HassTspC5-L31S, or HvirTspC5-L31S exhibited significant resistance to Cry1Ac (10.0-, 21.4-, and 81.1-fold, respectively), whereas the strain expressing PxylTspC5-L27S remained susceptible. Furthermore, the Cry1Ac resistant phenotypes followed an autosomal dominant inheritance pattern and were closely linked to the introduced mutant TspC5s. These findings reveal the conserved role of TspC5s from Helicoverpa and Heliothis species in mediating the dominant resistance to Cry1Ac, and they provide crucial insights for assessing resistance risks related to mutant tetraspanins and devising adaptive resistance management strategies for these major lepidopteran pests.

Expressional respones of hsp70 genes against abiotic and entomopathgenic stresses in four different noctuid larval species (Lepidoptera: Noctidae)

Heat shock proteins (Hsps) are stress response proteins. In a previous study, host larval Hsp70s were identified as the structural proteins of virions of Heliothis virescens ascovirus 3h (HvAV-3h), an insect virus that mainly infects noctuid larvae. To investigate the response of hsp70s of healthy Mythimna separata, Spodoptera exigua, Spodoptera frugiperda, and Spodoptera litura larvae to various abiotic or entomopathogenic stresses, quantitative PCR was used to detect larval hsp70s expression patterns. Results showed distinct expression patterns of hsp70s in response to different abiotic stresses. Notably, Mshsp70 expression pattern resembled Slhsp70 under most treatments. In healthy larvae, no tissue tropism was observed concerning the relative expression of Mshsp70, Sfhsp70, and Slhsp70. After infection with HvAV-3h, the expression of hsp70s in all dissected tissues of all tested larval species increased. Significant differences were found in the fat bodies of M. separata, S. exigua, and S. litura as well as in the hemolymph of S. exigua and S. litura. Subsequent silencing of Slhsp70, resulted in a significant decrease in DNA replication levels of HvAV-3h in S. litura larvae at 24 and 72 h post RNA interference, indicating that Slhsp70 is necessary for DNA replication in HvAV-3h. These data can provide references for the studying on the stress response of noctuid larvae to different environmental factors.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

The evolution of glandularity as a defense against herbivores in the tarweed clade.

Glandular trichomes are implicated in direct and indirect defense of plants. However, the degree to which glandular and non-glandular trichomes have evolved as a consequence of herbivory remains unclear, because their heritability, their association with herbivore resistance, their trade-offs with one another, and their association with other functions are rarely quantified. We conducted a phylogenetic comparison of trichomes and herbivore resistance against the generalist caterpillar, Heliothis virescens, among tarweed species (Asteraceae: Madiinae) and a genetic correlation study comparing those same traits among maternal half-sibs of three tarweed species. Within a tarweed species, we found no evidence that herbivore growth rate decreased on tarweed individuals or maternal sib groups with more glandularity or denser trichomes. However, tarweed species with more glandularity and fewer non-glandular trichomes resulted in slower-growing herbivores. Likewise, a trade-off between glandular and non-glandular trichomes was apparent among tarweed species, but not among individuals or sib groups within a species. Our results suggest that this key herbivore does not select for trichomes as a direct defense in tarweed species. However, trichomes differed substantially among species and likely affect herbivore pressure on those species. Our results demonstrate that trade-offs among plant traits, as well as inference on the function of those traits, can depend on scale.

Evidence for a role of SNMP2 and antennal support cells in sensillum lymph clearance processes of moth pheromone-responsive sensilla

In insect antenna, following the activation of olfactory sensory neurons, odorant molecules are inactivated by enzymes in the sensillum lymph. How the inactivation products are cleared from the sensillum lymph is presently unknown. Here we studied the role of support cells (SCs) and the so-called sensory neuron membrane protein 2 (SNMP2), a member of the CD36 family of lipid transporters abundantly expressed in SCs, in sensillum lymph clearance processes in the moths Heliothis virescens and Bombyx mori. In these species, the sex pheromone components are inactivated to long-chain fatty acids. To approach a role of SNMP2 in the removal of such inactivation products, we analyzed the uptake of a fluorescent long-chain fatty acid analog into a newly generated HvirSNMP2-expressing cell line. We found an increased uptake of the analog into SNMP2-cells compared to control cells, which could be blocked by the CD36 protein inhibitor, SSO. Furthermore, analyses of sensilla from antenna treated with the fatty acid analog indicated that SNMP2-expressing SCs are able to take up fatty acids from the sensillum lymph. In addition, sensilla from SSO-pretreated antenna of B. mori showed reduced removal of the fluorescent analog from the sensillum lymph. Finally, we revealed that SSO pretreatment of male silkmoth antenna significantly prolonged the duration of the female pheromone-induced wing-fluttering behavior, possibly as a result of impaired lymph clearance processes. Together our findings in H. virescens and B. mori support a pivotal role of olfactory SCs in sensillum lymph maintenance processes and suggest an integral role of SNMP2 in the removal of lipophilic “waste products” such as fatty acids resulting from sex pheromone inactivation.

Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affect pathogenicity against different host larvae species

ABSTRACT The pathogenicity of a virus to a specific host species is an inerratic and describable ability of a virus to cause infection but is generally shaped by a variety of abiotic and biotic factors. In this investigation, the variations in pathogenicity of Heliothis virescens ascovirus 3h (HvAV-3h) to five noctuid pests were assessed based on mass spectrometry analysis on the virion compositions. Twenty-nine common HvAV-3h proteins were shared across all hosts, and different flexible proteins were identified in the virions of each specific host. Different host proteins were identified as HvAV-3h virion-associated proteins, including different detoxification enzyme proteins. Furthermore, a relatively fixed relationship between viral replication and changes in host detoxification enzyme activity caused by deficiencies in various viral structural proteins was found in the host larvae using a correlation matrix analysis: the host larval carboxylesterase and cytochrome P450 monooxygenases generally had highly similar responses to the viruses blocked by different structural proteins’ antisera and their effects on viral DNA replication. Different interaction patterns for the virion structural proteins were found in different host larvae-produced virions, and the interactions between Spodoptera litura glutathione S-transferases and viral structural proteins were confirmed. The different host responses after viral infection could be the reason for the changes in viral pathogenicity, while the virus responses gradually adapted to the different hosts and there were flexible changes in the virion structures. IMPORTANCE Different pathogenic processes of a virus in different hosts are related to the host individual differences, which makes the virus undergoes different survival pressures. Here, we found that the virions of an insect virus, Heliothis virescens ascovirus 3h (HvAV-3h), had different protein composition when they were purified from different host larval species. These “adaptive changes” of the virions were analyzed in detail in this study, which mainly included the differences of the protein composition of virions and the differences in affinity between virions and different host proteins. The results of this study revealed the flexible changes of viruses to help themselves adapt to different hosts. Also, these interesting findings can provide new insights to improve our understanding of virus adaptability and virulence differentiation caused by the adaptation process.

An Overview of Ovarian Calyx Fluid Proteins of Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae): An Integrated Transcriptomic and Proteomic Approach.

The larval stages of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae), are parasitized by the endophagous parasitoid wasp, Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae). During the injections of eggs, this parasitoid wasp also injects into the host body the secretion of the venom gland and the calyx fluid, which contains a polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian calyx fluid Proteins (OPs). The effects of the OPs on the host immune system have recently been described. In particular, it has been demonstrated that the OPs cause hemocytes to undergo a number of changes, such as cellular oxidative stress, actin cytoskeleton modifications, vacuolization, and the inhibition of hemocyte encapsulation capacity, which results in both a loss of hemocyte functionality and cell death. In this study, by using a combined transcriptomic and proteomic analysis, the main components of T. nigriceps ovarian calyx fluid proteins were identified and their possible role in the parasitic syndrome was discussed. This study provides useful information to support the analysis of the function of ovarian calyx fluid proteins, to better understand T. nigriceps parasitization success and for a more thorough understanding of the components of ovarian calyx fluid proteins and their potential function in combination with other parasitoid factors.

No interaction of fluctuating or constant temperature and virulence of entomopathogenic fungi in two noctuid species

AbstractThe potential interaction of fluctuating temperature and the virulence of entomopathogens has implications for biological control. The objective of this study was to investigate the effect of entomopathogenic fungi (Metarhizium brunneum, Beauveria bassiana) on noctuid pest caterpillars (Spodoptera littoralis, Heliothis virescens) under constant and fluctuating temperature regimes. The results revealed similar virulence of entomopathogenic fungi under fluctuating temperature (20–30 °C or 15–35 °C) compared with constant temperature (25 °C). Experiments with cotton leaves as food confirmed that S. littoralis was less susceptible to entomopathogenic fungi (M. brunneum) than H. virescens. Results of additional experiments with H. virescens larvae, B. bassiana, and artificial diet were comparable to experiments with M. brunneum and cotton leaves, despite that susceptibility to B. bassiana was three orders of magnitude lower than to M. brunneum. The fact that both fungus species showed reduced growth on medium under fluctuating temperatures when compared to constant temperatures did not translate to an interaction of temperature regime and the virulence against noctuid larvae. Our study implies that virulence studies with noctuid larvae under constant temperatures using plant material or artificial diet might be fair models also for environments with fluctuating temperatures.

Heliothis virescens chymotrypsin is translationally controlled by AeaTMOF binding ABC putative receptor.

Heliothis virescens larval chymotrypsin (GenBank accession number AF43709) was cloned, sequenced and its three dimensional (3D) conformation modeled. The enzyme's transcript was first detected 6 days after larval emergence and the transcript level was shown to fall between larval ecdysis periods. Comparisons between the activities of larval gut chymotrypsin and trypsin shows that chymotrypsin activity is only 16% of the total trypsin activity and the pH optimum of the larval chymotrypsin is between pH 9-10, however the enzyme also exhibited a broad activity between pH 4-6. Injections of AeaTMOF and several shorter analogues into 3rd instar larvae followed by Northern blot analyses showed that although the chymotrypsins activities were inhibited by 60%-80% the transcript level of the sequenced chymotrypsin was not reduced and was similar to controls in which the chymotrypsin activity was not inhibited, indicating that AeaTMOF and its analogues exert a translational control. Based on these observations a putative AeaTMOF receptor (ABCC4) homologous to the Ae. aegypti ABC receptor sequence was found in the H. virescens genome. 3D molecular modeling and docking of the AeaTMOF and several of its analogues to the ABCC4 receptor showed that it can bind AeaTMOF and its analogues as was shown before for the Ae. aegypti receptor.

Hybridization in heliothine moths: impacts on reproduction, pheromone communication, and pest management

Heliothine moths (Family Noctuidae : Subfamily Heliothinae) are ubiquitous crop pests with three documented species combinations known to hybridize: Helicoverpa zea x Helicoverpa armigera, H. armigera x Helicoverpa assulta, and Heliothis virescens x Heliothis subflexa. Hybrids can have advantageous traits, such as increased host range, resistance to insecticides, and increased mating success, posing serious threats to agriculture. However, deleterious traits of hybrids, such as developmental abnormalities or sterility, can be exploited for pest management. In this review, the characteristics of F1 hybrids and backcrosses are examined through a historical lens. Topics reviewed include developmental characteristics, sex pheromone synthesis and perception, mating and calling behavior, sperm morphology, gene expression, electrophysiological responses, structures of the central and peripheral nervous systems, hybrid sterility, and applications in pest control. Recommendations for future studies based on existing gaps of knowledge are given, as are proposed pest management strategies.

More to legs than meets the eye: Presence and function of pheromone compounds on heliothine moth legs.

Chemical communication is ubiquitous in nature and chemical signals convey species-specific messages. Despite their specificity, chemical signals may not be limited to only one function. Identifying alternative functions of chemical signals is key to understanding how chemical communication systems evolve. Here, we explored alternative functions of moth sex pheromone compounds. These chemicals are generally produced in, and emitted from, dedicated sex pheromone glands, but some have recently also been found on the insects' legs. We identified and quantified the chemicals in leg extracts of the three heliothine moth species Chloridea (Heliothis) virescens, Chloridea (Heliothis) subflexa and Helicoverpa armigera, compared their chemical profiles and explored the biological function of pheromone compounds on moth legs. Identical pheromone compounds were present on the legs in both sexes of all three species, with no striking interspecies or intersex differences. Surprisingly, we also found pheromone-related acetate esters in leg extracts of species that lack acetate esters in their female sex pheromone. When we assessed gene expression levels in the leg tissue, we found known and putative pheromone-biosynthesis genes expressed, which suggests that moth legs may be additional sites of pheromone production. To determine possible additional roles of the pheromone compounds on legs, we explored whether these may act as oviposition-deterring signals, which does not seem to be the case. However, when we tested whether these chemicals have antimicrobial properties, we found that two pheromone compounds (16:Ald and 16:OH) reduce bacterial growth. Such an additional function of previously identified pheromone compounds likely coincides with additional selection pressures and, thus, should be considered in scenarios on the evolution of these signals.

Spodoptera litura larvae are attracted by HvAV-3 h-infected S. litura larvae-damaged pepper leaves.

Herbivore-induced plant volatiles (HIPVs) are important self-defense outputs of pepper plants to resist insect pests. Ascoviruses are pathogenic to the larvae of most lepidopteran vegetable pests. However, whether Heliothis virescens ascovirus 3 h (HvAV-3 h)-infected Spodoptera litura larvae can change pepper leaf HIPVs is not well understood. S. litura larvae preferred S. litura-infested leaves, and this preference was stronger with longer duration of S. litura infestation. In addition, S. litura larvae significantly chose pepper leaves damaged by HvAV-3 h-infected S. litura over the healthy pepper leaves. Results also showed that S. litura larvae preferred leaves mechanically damaged and treated with oral secretions from HvAV-3 h infected-S. litura larvae in a simulation test. We captured the volatiles emitted by leaves under 6 treatments. Results showed that the volatile profile changed with the different treatments. Testing of volatile blends, prepared to the proportion released showed that the blend from simulated HvAV-3 h-infected S. litura larvae-damaged plants was the most attractive to S. litura larvae. Further, we also found that some of the compounds significantly attracted S. litura larvae at specific concentrations. HvAV-3 h-infected S. litura can alter the release of HIPVs in pepper plants and thus become more attractive to S. litura larvae. We speculate that this may be due to alterations in the concentration of some compounds (such as geranylacetone and prohydrojasmon) affecting the behavior of S. litura larvae. This article is protected by copyright. All rights reserved.

An Ascovirus Utilizes Different Types of Host Larval Regulated Cell Death Mechanisms To Produce and Release Vesicles.

Ascoviruses are insect-specific viruses that are thought to utilize the cellular apoptotic processes of host larvae to produce numerous virion-containing vesicles. In this study, we monitored the in vivo infection processes of Heliothis virescens ascovirus 3h (HvAV-3h) to illustrate the regulated cell death (RCD) of host cells. Transmission electron microscopic observations did not reveal any morphological markers of apoptosis in the fat bodies or hemocytes of HvAV-3h-infected Helicoverpa armigera or Spodoptera exigua larvae. However, several hemocytes showed the morphological criteria for necrosis and/or pyroptosis. Further in vitro biochemical tests were performed to confirm the RCD type of host cells after infection with HvAV-3h. Different morphological characteristics were found between the early (prior to 24 hours post-infection, [hpi]) and later (48 to 120 hpi) stages in both HvAV-3h infected larval fat bodies and hemocytes. In the early stages, the virions could only be found in several adipohemocytes, and the fat bodies were cleaving their contained lipid inclusions into small lipid dots. In the later stage, both fat bodies and hemocytes were filled with numerous virions. According to the morphological characteristics of HvAV-3h infected larval fat bodies or hemocytes, the pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, and the systematic pathogenic mode of ascovirus infection was refined in this study. This study details the complete infection process of ascoviruses, which provides insights into the relationship between a pathogenesis of an insect virus and the RCD of different host tissues at different stages of infection. IMPORTANCE Viruses and other pathogens can interrupt host cellular apoptosis to gain benefits, such as sufficient resources and a stable environment that enables them to complete their replication and assembly. It is unusual for viruses to code proteins with homology to caspases, which are commonly recognized as apoptosis regulators. Ascoviruses are insect viruses with special cytopathology, and they have been hypothesized to induce apoptosis in their host larvae via coding a caspase-like protein. This enables them to utilize the process of cellular apoptosis to facilitate vesicle formation and replication. However, our previous studies revealed different trends. The fat bodies and hemocytes of Heliothis virescens ascovirus 3h (HvAV-3h)-infected larvae did not show any morphological markers of apoptosis but did display necrosis and/or pyroptosis morphological characteristics. The pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, which can help us understand the relationship between the pathogenesis of an insect virus and host RCD.

Insight into the regulation of the Nrf2 pathway in response to ascovirus infection in Spodoptera exigua.

Ascoviruses are a type of entomopathogenic microorganism with high biological pest control potential and are expected to contribute to the natural control of lepidopteran pests. However, knowledge of the molecular mechanism underlying the biocidal activity of ascovirus on its host insects remains limited. In this study, the relative enzyme activity of superoxide dismutase and peroxidase, as well as the expression level of Spodoptera exigua peroxidase (SePOD), were found to be significantly increased at 6h post infection with Heliothis virescens ascovirus 3h (HvAV-3h). H2 O2 accumulation and enhanced expression of NADPH Oxidase (SeNOX) were also observed. In addition, Nuclear Factor erythroid 2-Related Factor 2 (SeNrf2) and muscle aponeurosis fibromatosis (SeMaf) were overexpressed following infection with HvAV-3h. Silencing of SeNrf2 decreased the expression of SePOD, whereas the mortality of SeNrf2-silenced larvae and viral genome copy number also increased. Further RNA interference of SeNOX significantly decreased expression of SeNrf2 and SePOD and therefore increased the mortality and viral genome copy number of the ascovirus-infected host. The HvAV-3h activated Nrf2/ARE pathway of S. exigua and reactive oxygen species were found to respond to ascovirus infection by regulating alterations in antioxidant enzyme genes mediated by the host Nrf2/ARE pathway. These findings enhance our knowledge of ascovirus-host interactions and lay the foundation for the application of ascoviruses in biological pest control. © 2022 Society of Chemical Industry.

Production of Bacillus thuringiensis biopesticide using penicillin fermentation waste matter and application in agriculture

Bacillus thuringiensis (Bt) has been widely used in pest control, but the large scale production of this biopesticide is expensive because of the high cost of the medium and production method. In this study, we attempted to use inexpensive penicillin fermentation waste matter for the growth of Bacillus thuringiensis . The penicillin fermentation waste samples were used under conditions: without pre-treatment, with Na 2 CO 3 treatment, with acetic acid treatment and with drying treatment. The penicillin fermentation waste matter with Na 2 CO 3 treatment gave the highest cell counts. A maximum spore count (24.5 ×10 8 cfu/ml) was obtained by cultivating B. thuringiensis 2387 in medium containing 5% penicillin fermentation waste matter. Insecticidal potency of Bacillus thuringiensis biopesticide(Bt insecticidal concentrate) was about 8 000IU/㎎. Bt insecticidal concentrate showed 77–90% insecticidal efficacy against Pieris rapae, Plutella xylostella and Heliothis virescens in the field trial.

Insecticide compounds selection based on activation of ecdysteroid receptors in Diptera (S2) and Lepidoptera (Sf9) cell lines

Nuclear receptors are important targets in the research for the discovery of brand-new insecticides, and the ecdysteroid receptor is one of the most important targets nowadays. In this study, nine organocalcogenic compounds analogs of dibenzoylhydrazine were tested in a screening system, that presented at least 80% of compatibility in at least one of the three micromolar concentrations tested. We optimized in embryonic cell lines of the order Lepidoptera (SF9) and Diptera (S2), and these cell lines were transfected with a specific reporter plasmid. The nine compounds tested did not present agonist activity in both cell lines tested. In S2 cells the DAF-2 molecule inhibited the ECR-USP luminescence in 29.91%, and the DAF-4 molecule significantly inhibited the nuclear receptor luminescence in 51 to 55% compared to 20-Hydroxyecdysone (control). In SF9 cells, DAF-2 inhibited ECR-USP luminescence in 19.62%, and DAF-4 inhibited the receptor luminescence by 35.14% compared to 20-Hydroxyecdysone (control). In silico analysis of the Drosophila melanogaster receptor with the compound, DAF-2 showed inhibition of amino acids THR467 and MET-508 and with compound DAF-4 inhibition of ARG-511. For the of Heliothis virescens receptor for dendritic compounds, DAF-2 promoted inhibition of amino acids THR-343 and ARG-387 and DAF-4 inhibited ASN-504 and ARG-387

Engineering of <i>Bacillus thuringiensis</i> insecticidal proteins

Bacillus thuringiensis (Bt) has been used as sprayable pesticides for many decades. Bt strains utilized in these products produce multiple insecticidal proteins to complement a narrow insect specificity of each protein. In the late 1990s, genes encoding Bt insecticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry1Ac to control Heliothis virescens (tobacco budworm). Cry1Ab was applied to corn to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry1Ac and Cry1Ab, new Bt proteins are required to overcome the resistance. In order to protect corn furthermore, it is desired to control Diabrotica virgifera (Western corn rootworm), Helicoverpa zea (corn earworm) and Spodoptera frugiperda (fall armyworm). Recently, many new Bt insecticidal proteins have been discovered, but most of them require protein engineering to meet the high activity standard for commercialization. The engineering process for higher activity necessary for Bt crops is called optimization. The seed industry has been optimizing Bt insecticidal proteins to improve their insecticidal activity. In this review, several optimization projects, which have led to substantial activity increases of Bt insecticidal proteins, are described.

Synthesis and biological testing for pesticidal activity of 8-azasteroids

The aim of the present study is the synthesis and testing for pesticidal activities of 2,3-dimethoxy16,16-dimethyl-D-homo-8-azagona-1,3,5(10),13-tetraene-12,17а-one and 2,3-dimethoxy-16,16-dimethyl-dhomo-8-azagona-1,3,5(10),13-tetraene-12-imino-17а-one hydrochloride which could become the basis the basis of plant protection products. The first compound was obtained by condensation of 6,7-dimethoxy-2,3- dihydroisoquinoline with 2-acetyl-5,5-dimethylcyclohexane-1,3-dione. The second substance was synthesized by interaction of the first with ammonium chloride. 2-Acetyl-5,5-dimethylcyclohexane-1,3-dione was prepared by heating dimedone with acetic acid in polyphosphoric acid. 6,7-Dimethoxy-2,3-dihydroisoquinoline was synthesized in two steps. Boiling 2-(3,5-dimethoxyphenyl)ethylamine in formic acid gave the corresponding amide, which was cyclized in the presence of phosphorus oxychloride. The structure of the obtained compounds is confirmed by the data of IR, 1H NMR, UV spectra and elemental analysis. In the IR absorption spectra of 2,3-dimethoxy-16,16-dimethyl-D-homo-8-azagona-1,3,5(10),13-tetraene-12,17a-dione and 2,3-dimethoxy-16,16-dimethyl-D-homo-8-azagona-1,3,5(10),13-tetraen-12-imino-17a-one hydrochloride, enaminodiketone bands are present (1535, 1580, 1615, 1625, 1670 cm-1 ) and enimine ketone (1595, 1650, 3260 cm-1 ) groups, respectively. Their UV absorption spectra recorded in ethanol contain two absorption bands (265.303 and 268.317 nm) corresponding to ππ* transitions of the same molecular fragments. The mass spectra of the two obtained tetracycles contain peaks of molecular ions. 1H NMR spectra correspond to the structures of all obtained compounds The synthesized compounds were tested for certain types of insecticide (against Toxoptera graminum, Musca domestica, Meloidogyne incognita, Heliothis virescens, Diabrotica undecimpunctata howardi, Caenorhabditis elegans), fungicidal (against Drechslera, Erysiphe, Puccinia, Peronospora) and herbicidal (against Amaranthus retroflexus, Brassica rapa, Abutilon theophrasti, Alopecurus myosuroides, Avena fatua, Echinochloa crus galli) activities. Both synthesized compounds showed herbicidal activity against Amaranthus retroflexus, Brassica rapa, Abutilon theophrasti and insecticidal activity against Toxoptera graminum. Hydrochloride 2,3-dimethoxy-16,16-dimethyl-D-homo-8-azagon-1,3,5(10),13- tetraene-12-imino-17a-one showed insecticidal activity against Musca domestica and fungicidal activity against Drechslera.

Mating status affects female choice when females are signalers.

Sexual selection in animals has been mostly studied in species in which males are signalers and females are choosers. However, in many species, females are (also) signalers. In species with non‐signaling females, virgin females are hypothesized to be less choosy than mated females, as virgins must mate to realize fitness and the number of available males is generally limited. Yet, when females signal to attract males, mate limitation can be overcome. We tested how virgin and mated females differ in their calling behavior, mating latency, and in mate choice, using the tobacco budworm Chloridea (Heliothis) virescens as an example for a species in which females are not only choosers but also signalers. We found that virgin females signaled longer than mated females, but virgin and mated signaling females were equally ready to mate, in contrast to non‐signaling females. However, we found that virgin signaling females showed weaker mate preference than mated females, which can be explained by the fact that females increase their fitness with multiple matings. Mated females may thus further increase their fitness by more stringent mate selection. We conclude that signaling is a crucial aspect to consider when studying female mate choice because signaling may affect the number of available mates to choose from.

Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation.

Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups. In combination with phosphoproteomic results and conservation estimations, 30 conserved phosphorylation sites on 17 phosphoproteins were identified from a total of 176 phosphosites on 57 phosphoproteins from Heliothis virescens ascovirus 3h (HvAV-3h), providing potential research targets for investigating the role of these protein in the regulation of viral infection. This study will facilitate genome annotation and comparison of further Ascoviridae members as well as functional genomic investigations.