RESUMO
BACKGROUND: Accurate detection of pheromones is crucial for chemical communication and reproduction in insects. In holometabolous flies and moths, the sensory neuron membrane protein 1 (SNMP1) is essential for detecting long-chain aliphatic pheromones by olfactory neurons. However, its function in hemimetabolous insects and its role for detecting pheromones of a different chemical nature remain elusive. Therefore, we investigated the relevance of SNMP1 for pheromone detection in a hemimetabolous insect pest of considerable economic importance, the desert locust Schistocerca gregaria, which moreover employs the aromatic pheromone phenylacetonitrile (PAN) to govern reproductive behaviors. RESULTS: Employing CRISPR/Cas-mediated gene editing, a mutant locust line lacking functional SNMP1 was established. In electroantennography experiments and single sensillum recordings, we found significantly decreased electrical responses to PAN in SNMP1-deficient (SNMP1-/-) locusts. Moreover, calcium imaging in the antennal lobe of the brain revealed a substantially reduced activation of projection neurons in SNMP1-/- individuals upon exposure to PAN, indicating that the diminished antennal responsiveness to PAN in mutants affects pheromone-evoked neuronal activity in the brain. Furthermore, in behavioral experiments, PAN-induced effects on pairing and mate choice were altered in SNMP1-/- locusts. CONCLUSIONS: Our findings emphasize the importance of SNMP1 for chemical communication in a hemimetabolous insect pest. Moreover, they show that SNMP1 plays a crucial role in pheromone detection that goes beyond long-chain aliphatic substances and includes aromatic compounds controlling reproductive behaviors.
Assuntos
Gafanhotos , Proteínas de Membrana , Animais , Gafanhotos/fisiologia , Gafanhotos/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Feromônios/farmacologia , Comportamento Sexual Animal/fisiologia , Comportamento Sexual Animal/efeitos dos fármacos , Feminino , Corte , Acetonitrilas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
The representation of odors in the locust antennal lobe with its >2,000 glomeruli has long remained a perplexing puzzle. We employed the CRISPR-Cas9 system to generate transgenic locusts expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon functional imaging, we mapped the spatial activation patterns representing a wide range of ecologically relevant odors across all six developmental stages. Our findings reveal a functionally ring-shaped organization of the antennal lobe composed of specific glomerular clusters. This configuration establishes an odor-specific chemotopic representation by encoding different chemical classes and ecologically distinct odors in the form of glomerular rings. The ring-shaped glomerular arrangement, which we confirm by selective targeting of OR70a-expressing sensory neurons, occurs throughout development, and the odor-coding pattern within the glomerular population is consistent across developmental stages. Mechanistically, this unconventional spatial olfactory code reflects the locust-specific and multiplexed glomerular innervation pattern of the antennal lobe.
Assuntos
Antenas de Artrópodes , Odorantes , Neurônios Receptores Olfatórios , Animais , Neurônios Receptores Olfatórios/metabolismo , Antenas de Artrópodes/fisiologia , Olfato/fisiologia , Gafanhotos/fisiologia , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Condutos Olfatórios/fisiologia , Receptores Odorantes/metabolismo , Receptores Odorantes/genética , Locusta migratoria/fisiologia , Cálcio/metabolismoRESUMO
Olfactory coding, from insects to humans, is canonically considered to involve considerable across-fiber coding already at the peripheral level, thereby allowing recognition of vast numbers of odor compounds. We show that the migratory locust has evolved an alternative strategy built on highly specific odorant receptors feeding into a complex primary processing center in the brain. By collecting odors from food and different life stages of the locust, we identified 205 ecologically relevant odorants, which we used to deorphanize 48 locust olfactory receptors via ectopic expression in Drosophila. Contrary to the often broadly tuned olfactory receptors of other insects, almost all locust receptors were found to be narrowly tuned to one or very few ligands. Knocking out a single receptor using CRISPR abolished physiological and behavioral responses to the corresponding ligand. We conclude that the locust olfactory system, with most olfactory receptors being narrowly tuned, differs from the so-far described olfactory systems.
Assuntos
Gafanhotos , Neurônios Receptores Olfatórios , Receptores Odorantes , Animais , Humanos , Odorantes , Olfato/fisiologia , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismo , InsetosRESUMO
Many animals engage in cannibalism to supplement their diets. Among dense populations of migratory locusts, cannibalism is prevalent. We show that under crowded conditions, locusts produce an anticannibalistic pheromone called phenylacetonitrile. Both the degree of cannibalism and the production of phenylacetonitrile are density dependent and covary. We identified the olfactory receptor that detects phenylacetonitrile and used genome editing to make this receptor nonfunctional, thereby abolishing the negative behavioral response. We also inactivated the gene underlying phenylacetonitrile production and show that locusts that lack this compound lose its protection and are more frequently exposed to intraspecific predation. Thus, we reveal an anticannibalistic feature built on a specifically produced odor. The system is very likely to be of major importance in locust population ecology, and our results might therefore provide opportunities in locust management.
Assuntos
Acetonitrilas , Canibalismo , Aglomeração , Gafanhotos , Feromônios , Animais , Acetonitrilas/metabolismo , Gafanhotos/genética , Gafanhotos/metabolismo , Feromônios/genética , Feromônios/metabolismoRESUMO
Insect sexual communication often relies upon sex pheromones. Most insect pheromones, however, contain carbon-carbon double bonds and potentially degrade by oxidation. Here, we show that frequently reported increased levels of Anthropocenic ozone can oxidize all described male-specific pheromones of Drosophila melanogaster, resulting in reduced amounts of pheromones such as cis-Vaccenyl Acetate and (Z)-7-Tricosene. At the same time female acceptance of ozone-exposed males is significantly delayed. Interestingly, groups of ozone-exposed males also exhibit significantly increased levels of male-male courtship behaviour. When repeating similar experiments with nine other drosophilid species, we observe pheromone degradation and/or disrupted sex recognition in eight of them. Our data suggest that Anthropocenic levels of ozone can extensively oxidize double bonds in a variety of insect pheromones, thereby leading to deviations in sexual recognition.
Assuntos
Drosophila melanogaster , Atrativos Sexuais , Animais , Masculino , Feminino , Drosophila melanogaster/metabolismo , Comportamento Sexual Animal , Feromônios/metabolismo , CorteRESUMO
Understanding neuronal representations of odor-evoked activities and their progressive transformation from the sensory level to higher brain centers features one of the major aims in olfactory neuroscience. Here, we investigated how odor information is transformed and represented in higher-order neurons of the lateral horn, one of the higher olfactory centers implicated in determining innate behavior, using Drosophila melanogaster. We focused on a subset of third-order glutamatergic lateral horn neurons (LHNs) and characterized their odor coding properties in relation to their presynaptic partner neurons, the projection neurons (PNs) by two-photon functional imaging. We show that odors evoke reproducible, stereotypic, and odor-specific response patterns in LHNs. Notably, odor-evoked responses in these neurons are valence-specific in a way that their response amplitude is positively correlated with innate odor preferences. We postulate that this valence-specific activity is the result of integrating inputs from multiple olfactory channels through second-order neurons. GRASP and micro-lesioning experiments provide evidence that glutamatergic LHNs obtain their major excitatory input from uniglomerular PNs, while they receive an odor-specific inhibition through inhibitory multiglomerular PNs. In summary, our study indicates that odor representations in glutamatergic LHNs encode hedonic valence and odor identity and primarily retain the odor coding properties of second-order neurons.
Assuntos
Odorantes , Neurônios Receptores Olfatórios , Animais , Drosophila , Drosophila melanogaster/fisiologia , Neurônios Receptores Olfatórios/fisiologia , OlfatoRESUMO
The sensory neuron membrane protein, SNMP1, was initially discovered in moths and is associated with sex pheromone sensitive neurons, suggesting a role in the detection of these semiochemicals. Although DrosophilaSNMP1 has been reported to be involved in detecting of the sex pheromone cis-vaccenyl acetate (cVA), the role of this protein in moths in vivo is still largely unexplored. In this study we developed a SNMP1-/- homozygous mutant line of Helicoverpa armigera using CRISPR/Cas9. Wind-tunnel behavioral experiments showed that HarmSNMP1-/- males could not be attracted by sex pheromones (Z11-16:Ald/Z9-16:Ald = 97/3), while mating behavior obvervations revealed that the SNMP1 mutant males didn't react much to calling females and the rate of copulation was significantly decreased. The electrophysiological results indicated that HarmSNMP1 contributes to the detection of 16-carbon liner sex pheromones, (Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-hexadecenal (Z9-16:Ald), (Z)-11-hexadecanol (Z11-16:OH) and 16-carbon acetate (Z)-11-hexadecenyl acetate (Z11-16:OAc), but is not required for detecting the 14-carbon sex pheromone component (Z)-9-tetradecenal (Z9-14:Ald) an analogue of Z11-16:Ald, (Z)-9-tetradecen-1-yl formate (Z9-14:OFor), which can activate the Z11-16:Ald-responsive neuron. Taken together, our studies indicated that HarmSNMP1 has an important role in the detection of long-chain sex pheromones, but is not essential for detecting shorter chain sex pheromone in vivo.
Assuntos
Proteínas de Insetos/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Receptores de Feromônios/genética , Atrativos Sexuais/metabolismo , Animais , Sequência de Bases , Proteínas de Insetos/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Mariposas , Proteínas do Tecido Nervoso/metabolismo , Receptores de Feromônios/metabolismoRESUMO
Trehalase is an indispensable component of insect hemolymph that plays important role in energy metabolism and stress resistance. In this study, we cloned and expressed the gene encoding soluble trehalase (HaTreh-1) of Helicoverpa armigera (cotton bollworm) and characterized the enzyme. HaTreh-1 had a full-length open reading frame encoding a protein of 571 amino acids. Sequence comparison indicated that HaTreh-1 was similar to some known insect trehalases. Two essential active sites (D321 and E519) and three essential residues (R168, R221, and R286) were conserved in HaTreh-1. The recombinant trehalase was expressed in Escherichia coli and purified by nickel exchange chromatography. Molecular weight of the recombinant protein was about 71 kDa, and the optimum HaTreh-1 enzyme activity is at 55°C with pH 6.0. Enzymatic assays showed a Km value of 72.8 mmol/liter and a Vmax value of 0.608 mmol/(liter·min). Inhibition assays in vitro indicated that castanospermine, a polyhydroxylated alkaloid, was an effective competitive inhibitor of trehalase with a Ki value of 6.7 µmol/liter. The inhibitor action of castanospermine was linked to its modification effect on trehalase structure. The circular dichroism spectrum showed that the percentage of α-helix increased under the presence of castanospermine. Results of our study will aid in developing effective trehalase inhibitors for controlling H. armigera in the future.
Assuntos
Mariposas/enzimologia , Trealase/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Escherichia coli , Indolizinas , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Masculino , Mariposas/genética , Conformação Proteica , Análise de Sequência de DNA , Trealase/antagonistas & inibidores , Trealase/genéticaRESUMO
In order to acquire enough nutrients and energy for further development, larvae need to invest a large portion of their sensory equipments to identify food sources. Yet, the molecular basis of odor-driven behavior in larvae has been poorly investigated. Information on olfactory genes, particularly odorant binding proteins (OBPs) and chemosensory proteins (CSPs) which are involved in the initial steps of olfaction is very scarce. In this study, we have identified 26 OBP and 21 CSP genes from the transcriptomes of Helicoverpa armigera larval antennae and mouthparts. A comparison with the 34 OBP and 18 CSP genes of the adult antenna, revealed four novel OBPs and seven novel CSPs. Similarly, 27 OBPs (six novel OBPs) and 20 CSPs (6 novel CSPs) were identified in the transcriptomes of Helicoverpa assulta larval antennae and mouthparts. Tissue-specific profiles of these soluble proteins in H. armigera showed that 6 OBP and 4 CSP genes are larval tissue-specific, 15 OBPs and 13 CSPs are expressed in both larvae and adult, while the rest are adult- specific. Our data provide useful information for functional studies of genes involved in larval foraging.
Assuntos
Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Receptores Odorantes/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/genética , Anotação de Sequência Molecular , Mariposas/genética , Filogenia , Receptores Odorantes/química , Receptores Odorantes/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Transcriptoma/genéticaRESUMO
Many insect species use multi-component sex pheromones to discriminate among potential mating partners [1-5]. In moths, pheromone blends tend to be dominated by one or two major components, but behavioral responses are frequently optimized by the inclusion of less abundant minor components [6]. An increasing number of studies have shown that female insects use these chemicals to convey their mating availability to males, who can assess the maturity of females and thus decide when to mate [7, 8]. However, little is known about the biological mechanisms that enable males to assess female reproductive status. In this study, we found that females of Helicoverpa armigera avoid nonoptimal mating by inhibiting males with pheromone antagonist cis-11-Hexadecenol (Z11-16:OH). We also show that this antagonist-mediated optimization of mating time ensures maximum fecundity. To further investigate molecular aspects of this phenomenon, we used the CRISPR/Cas9 system to knock out odorant receptor 16 (OR16), the only pheromone receptor tuned to Z11-16:OH. In mutant males, electrophysiological and behavioral responses to Z11-16:OH were abolished. Inability to detect Z11-16:OH prompted the males to mate with immature females, which resulted in significantly reduced viability of eggs. In conclusion, our study demonstrates that the sensitivity of OR16 to Z11-16:OH regulates optimal mating time and thus ensures maximum fecundity. These results may suggest novel strategies to disrupt pest insect mating.
Assuntos
Aldeídos/metabolismo , Mariposas/fisiologia , Receptores Odorantes/metabolismo , Atrativos Sexuais/antagonistas & inibidores , Comportamento Sexual Animal/fisiologia , Animais , Sistemas CRISPR-Cas/genética , Fenômenos Eletrofisiológicos , Feminino , Fertilidade/fisiologia , Técnicas de Silenciamento de Genes , Masculino , Controle Biológico de Vetores/métodos , Receptores Odorantes/genética , Reprodução/fisiologia , Fatores de TempoRESUMO
Male moths efficiently recognize conspecific sex pheromones thanks to their highly accurate and specific olfactory system. The Heliothis/Helicoverpa species are regarded as good models for studying the perception of sex pheromones. In this study, we performed a series of experiments to investigate the peripheral mechanisms of pheromone coding in two-closely related species, Helicoverpa armigera and H. assulta. The morphology and distribution patterns of sensilla trichoidea are similar between the two species when observed at the scanning electron microscope, but their performances are different. In H. armigera, three functional types of sensilla trichoidea (A, B and C) were found to respond to different pheromone components, while in H. assulta only two types of such sensilla (A and C) could be detected. The response profiles of all types of sensilla trichoidea in the two species well matched the specificities of the pheromone receptors (PRs) expressed in the same sensilla, as measured in voltage-clamp experiments. The expressions of PRs in neighboring olfactory sensory neurons (OSNs) within the same trichoid sensillum were further confirmed by in situ hybridization. Our results show how the same pheromone components can code for different messages at the periphery of two Helicoverpa species.
Assuntos
Mariposas/fisiologia , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Receptores de Feromônios/metabolismo , Sensilas/fisiologia , Atrativos Sexuais/metabolismo , Potenciais de Ação/fisiologia , Animais , Feminino , Regulação da Expressão Gênica , Hibridização in Situ Fluorescente , Masculino , Microscopia Eletrônica de Varredura , Mariposas/ultraestrutura , Neurônios Receptores Olfatórios/ultraestrutura , Técnicas de Patch-Clamp , Receptores de Feromônios/genética , Sensilas/ultraestrutura , Atrativos Sexuais/genética , Transdução de Sinais , Especificidade da EspécieRESUMO
Sexual communication in moths offers a simplified scenario to model and investigate insect sensory perception. Both PBPs (pheromone-binding proteins) and PRs (pheromone receptors) are involved in the detection of sex pheromones, but the interplay between them still remains largely unknown. In this study, we have measured the binding affinities of the four recombinant PBPs of Chilo suppressalis (CsupPBPs) to pheromone components and analogs and characterized the six PRs using the Xenopus oocytes expression system. Interestingly, when the responses of PRs were recorded in the presence of PBPs, we measured in several combinations a dramatic increase in signals as well as in sensitivity of such combined systems. Furthermore, the discrimination ability of appropriate combinations of PRs and PBPs was improved compared with the performance of PBPs or PRs alone. Besides further supporting a role of PBPs in the pheromone detection and discrimination, our data shows for the first time that appropriate combinations of PRs and PBPs improved the discrimination ability of PBPs or PRs alone. The variety of responses measured with different pairing of PBPs and PRs indicates the complexity of the olfaction system, which, even for the relatively simple task of detecting sex pheromones, utilises a highly sophisticated combinatorial approach.