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Olfaction underpins many insect behaviors, such as foraging, host location, mating, and predator avoidance. In the first step of insect olfaction, odorant-binding proteins (OBPs) bind hydrophobic odorants and transport them to odorant receptors. Methyl eugenol (ME) is a powerful attractant for mature males of the oriental fruit fly Bactrocera dorsalis (Hendel), one of the most destructive fruit pests. The underlying molecular mechanism is unclear, but there is in vitro evidence that BdorOBP56f-2 is involved in ME perception. We used microscale thermophoresis to confirm that BdorOBP56f-2 directly binds ME with strong affinity in vitro. We then used CRISPR/Cas9 to knock out the BdorOBP56f-2 gene, allowing us to establish a homozygous mutant B. dorsalis line. The electroantennogram response and behavioral attraction to ME were significantly reduced in the mutant, providing in vivo evidence that BdorOBP56f-2 is necessary for efficient ME perception. Our results offer insight into the molecular mechanism of ME perception in B. dorsalis and provide a theoretical basis for the functional analysis of other OBPs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mosquitoes rely heavily on their olfactory systems for host seeking, selection of oviposition sites, and avoiding predators and other environmental dangers. Of these behaviors, the preferential selection of a human blood-meal host drives the vectorial capacity of anthropophilic female Anopheles coluzzii mosquitoes. Olfactory receptor neurons (ORNs) are dispersed across several appendages on the head and express an obligate odorant receptor co-receptor (Orco) coupled with a "tuning" odorant receptor (OR) to form heteromeric, odor-gated ion channels in the membrane of these neurons. To examine the mechanistic and functional contributions of Orco/OR complexes to the chemosensory processes of An. coluzzii, we utilized CRISPR/Cas9 gene editing to create a line of homozygous, Orco-knockout, mutant mosquitoes. As expected, orco -/- ORNs across both adult and larval stages of An. coluzzii display significantly lower background activity and lack nearly all odor-evoked responses. In addition, blood-meal-seeking, adult female, orco -/- mutant mosquitoes exhibit severely reduced attraction to human- and non-human-derived odors while gravid females are significantly less responsive to established oviposition attractants. These results reinforce observations in other insects that Orco is crucial in maintaining the activity of ORNs. In that light, it significantly influences a range of olfactory-driven behaviors central to the anthropophilic host preference that is critical to the vectorial capacity of An. coluzzii as a primary vector for human malaria., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Various insect species including moths have shown significant foraging preference to acetic acid. However, the olfactory reception and behavioral outputs of acetic acid in moths remain unsolved. The female adults of Oriental armyworm, Mythimna separata, exhibit high preference to acetic acid enriched sweet vinegar solutions, making them good targets for exploration of acid reception and performance. We first proved that acetic acid is an essential component which elicited electrophysiological responses from volatiles of the sweet vinegar solution. Successive single sensillum recording tests showed that at least 4 types (as1, as2, as3, and as4) of sensilla were involved in acetic acid reception in the antennae. The low dosages of acetic acid elicited upwind flight and close search, and pre-contact proboscis extension responses of the fasted females, indicating it serves as a food related olfactory cue. In vivo optical imaging data showed that low dosages of acetic acid activated one ordinary glomerulus (DC3), and high dosages evoked additional two glomeruli (DC1 and AC1) in the antennal lobe. A systematic survey on olfaction related receptors in three related transcriptomes has yielded 67 olfactory receptors (ORs) and 19 ionotropic receptors (IRs). Among, MsepIR8a, MsepIR64a, MsepIR75q1, and MsepIR75q2 were chosen as putative acid receptors by blasting against known acid IRs in Drosophila and comparing essential amino acid residues which related to acid sensing. Later in situ hybridization revealed that MsepIr8a was co-expressed with each of the other 3 Irs, suggesting its putative co-receptor role. This study reveals olfactory reception of acetic acid as an attractant in M. separata, and it provides the solid basis for later deorphanization of relevant receptors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

There is growing evidence in the literature suggesting that caste differentiation in the stingless bee, Melipona scutellaris, and other bees in the genus Melipona, is triggered by environmental signals, particularly a primer pheromone. With the proper amount of food and a chemical stimulus, 25% of females emerge as queens, in agreement with a long-standing "two loci/two alleles model" proposed in the 1950s. We surmised that these larvae must be equipped with an olfactory system for reception of these chemical signals. Here we describe for the first time the diversity of antennal sensilla in adults and the morphology of larvae of M. scutellaris. Having found evidence for putative olfactory sensilla in larvae, we next asked whether olfactory proteins were expressed in larvae. Since the molecular basis of M. scutellaris is still unknown, we cloned olfactory genes encoding chemosensory proteins (CSP) and odorant-binding proteins (OBPs) using M. scutellaris cDNA template and primers designed on the basis CSPs and OBPs previously reported from the European honeybee, Apis mellifera. We cloned two CSP and two OBP genes and then attempted to express the proteins encoded by these genes. With a recombinant OBP, MscuOBP8, and a combinatorial single-chain variable fragment antibody library, we generated anti-MscuOBP8 monoclonal antibody. By immunohistochemistry we demonstrated that the anti-MscuOBP8 binds specifically to the MscuOBP8. Next, we found evidence that MscuOBP8 is expressed in M. scutellaris larvae and it is located in the mandibular region, thus further supporting the hypothesis of olfactory function in immature stages. Lastly, molecular modeling suggests that MscuOBP8 may function as a carrier of primer pheromones or other ligands.

In the last decade, it has been demonstrated that brain functional asymmetry occurs not only in vertebrates but also in invertebrates. However, the mechanisms underlying functional asymmetry remain unclear. In the present study, we trained honeybees of the same parentage and age, on the proboscis extension reflex (PER) paradigm with only one antenna in use. The comparisons of gene expression between the left and right hemispheres were carried out using high throughput sequencing. Our research revealed that gene expression in the honeybee brain is also asymmetric, with more genes having higher expression in the right hemisphere than the left hemisphere. Our studies show that during olfactory learning, the left hemisphere is more responsible for long term memory and the right hemisphere is more responsible for the learning and short term memory.

The oriental fruit fly (Bactrocera dorsalis) is a species of tephritid fruit fly, endemic to Southeast Asia but also introduced to many regions of the US, and it is one of the major pest species with a broad host range of cultivated and wild fruits. Although males of B. dorsalis respond strongly to methyl eugenol and this is used for monitoring and estimating populations, the molecular mechanism of the oriental fruit fly olfaction has not been elucidated yet. Therefore, in this project, using next generation sequencing technologies, we sequenced the transcriptome of the antennae of male and female adults of B. dorsalis. We identified a total of 20 candidate odorant binding proteins (OBPs), 5 candidate chemosensory proteins (CSPs), 35 candidate odorant receptors (ORs), 12 candidate ionotropic receptors (IRs) and 4 candidate sensory neuron membrane proteins (SNMPs). The sex-specific expression of these genes was determined and a subset of 9 OR genes was further characterized by qPCR with male and female antenna, head, thorax, abdomen, leg and wing samples. In the male antennae, 595 genes showed a higher expression, while 128 genes demonstrated a higher expression in the female antennae. Interestingly, 2 ORs (BdorOR13 and BdorOR14) were highly and specifically expressed in the antennae of males, and 4 ORs (BdorOR13, BdorOR16, BdorOR18 and BdorOR35) clustered with DmOR677, suggesting pheromone reception. We believe this study with these antennae-enriched OBPs, CSPs, ORs, IRs and SNMPs can play an important role in the detection of pheromones and general odorants, and so in turn our data improve our current understanding of insect olfaction at the molecular level and provide important information for disrupting the behavior of the oriental fruit fly using chemical communication methods.

We studied the involvement of the α8 subunit of nicotinic acetylcholine receptors (nAChRs) in olfactory learning and memory in Apis mellifera. We have previously shown, by injecting different nicotinic antagonists into the bee brain, that pharmacologically different subtypes of nAChRs are important for honeybee memory -α-bungarotoxin-sensitive receptors are necessary for memory consolidation and mecamylamine-sensitive receptors are involved in retrieval processes. Here, we took advantage of the honeybee genome sequencing and the development of a small interfering RNA (siRNA) tool to focus on the role of the α8 subunit, which has been shown to be expressed in brain areas important for olfactory learning, such as the antennal lobes and mushroom bodies. We first demonstrated the efficacy of the siRNA tool by showing a decrease of the α8 protein level at 6 h after brain injection of α8 siRNA. We then tested the general role of this subunit in olfactory conditioning, using brain systemic or localized siRNA injections in the antennal lobes or the calyces and vertical lobes of the mushroom bodies. These injections were performed at either 6 h before the learning acquisition or 6 h before the memory test. The most prominent result was that 6-h pre-test injection of siRNA in the mushroom body vertical lobes impaired memory retrieval at 24 and 48 h post-training. This indicated the importance of cholinergic extrinsic neurons and nAChRs containing the α8 subunit for this process., (© 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)