Females smell differently: characteristics and significance of the most common olfactory sensilla of female silkmoths.

In the silkmoth Bombyx mori, the role of male sensilla trichodea in pheromone detection is well established. Here we study the corresponding female sensilla, which contain two olfactory sensory neurons (OSNs) and come in two lengths, each representing a single physiological type. Only OSNs in medium trichoids respond to the scent of mulberry, the silkworm's exclusive host plant, and are more sensitive in mated females, suggesting a role in oviposition. In long trichoids, one OSN is tuned to (+)-linalool and the other to benzaldehyde and isovaleric acid, both odours emitted by silkworm faeces. While the significance of (+)-linalool detection remains unclear, isovaleric acid repels mated females and may therefore play a role in avoiding crowded oviposition sites. When we examined the underlying molecular components of neurons in female trichoids, we found non-canonical co-expression of Ir8a, the co-receptor for acid responses, and ORco, the co-receptor of odorant receptors, in long trichoids, and the unexpected expression of a specific odorant receptor in both trichoid sensillum types. In addition to elucidating the function of female trichoids, our results suggest that some accepted organizational principles of the insect olfactory system may not apply to the predominant sensilla on the antenna of female B. mori.

Odorant detection in a locust exhibits unusually low redundancy.

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.

Unique neural coding of crucial versus irrelevant plant odors in a hawkmoth.

The sense of smell is pivotal for nocturnal moths to locate feeding and oviposition sites. However, these crucial resources are often rare and their bouquets are intermingled with volatiles emanating from surrounding 'background' plants. Here, we asked if the olfactory system of female hawkmoths, Manduca sexta, could differentiate between crucial and background cues. To answer this question, we collected nocturnal headspaces of numerous plants in a natural habitat of M. sexta. We analyzed the chemical composition of these headspaces and used them as stimuli in physiological experiments at the antenna and in the brain. The intense odors of floral nectar sources evoked strong responses in virgin and mated female moths, most likely enabling the localization of profitable flowers at a distance. Bouquets of larval host plants and most background plants, in contrast, were subtle, thus potentially complicating host identification. However, despite being subtle, antennal responses and brain activation patterns evoked by the smell of larval host plants were clearly different from those evoked by other plants. Interestingly, this difference was even more pronounced in the antennal lobe of mated females, revealing a status-dependent tuning of their olfactory system towards oviposition sites. Our study suggests that female moths possess unique neural coding strategies to find not only conspicuous floral cues but also inconspicuous bouquets of larval host plants within a complex olfactory landscape.

The olfactory coreceptor IR8a governs larval feces-mediated competition avoidance in a hawkmoth.

Finding a suitable oviposition site is a challenging task for a gravid female moth. At the same time, it is of paramount importance considering the limited capability of most caterpillars to relocate to alternative host plants. The hawkmoth, Manduca sexta (Sphingidae), oviposits on solanaceous plants. Larvae hatching on a plant that is already attacked by conspecific caterpillars can face food competition, as well as an increased exposure to predators and induced plant defenses. Here, we show that feces from conspecific caterpillars are sufficient to deter a female M. sexta from ovipositing on a plant and that this deterrence is based on the feces-emitted carboxylic acids 3-methylpentanoic acid and hexanoic acid. Using a combination of genome editing (CRISPR-Cas9), electrophysiological recordings, calcium imaging, and behavioral analyses, we demonstrate that ionotropic receptor 8a (IR8a) is essential for acid-mediated feces avoidance in ovipositing hawkmoths.

Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

Acetoin is a key odor for resource location in the giant robber crab Birgus latro.

The terrestrial and omnivorous robber crab Birgus latro inhabits islands of the Indian Ocean and the Pacific Ocean. The animals live solitarily but occasionally gather at freshly opened coconuts or fructiferous arenga palms. By analyzing volatiles of coconuts and arenga fruit, we identified five compounds, including acetoin, which are present in both food sources. In a behavioral screen performed in the crabs' habitat, a beach on Christmas Island, we found that of 15 tested fruit compounds, acetoin was the only volatile eliciting significant attraction. Hence, acetoin might play a key role in governing the crabs' aggregation behavior at both food sources.

Spatial Representation of Feeding and Oviposition Odors in the Brain of a Hawkmoth.

Female hawkmoths, Manduca sexta, use olfactory cues to locate nectar sources and oviposition sites. We investigated if the behavioral significance of odorants is represented already in the antennal lobe, the first olfactory neuropil of the insect's brain. Using in vivo calcium imaging, we first established a functional map of the dorsal surface of the antennal lobe by stimulating the moths with 80 ecologically relevant and chemically diverse monomolecular odorants. We were able to address 23 olfactory glomeruli, functional subunits of the antennal lobe, in each individual female. Next, we studied the relevance of the same odorants with two-choice experiments (odorant versus solvent) in a wind tunnel. Depending on odorant identity, naive moths made attempts to feed or to oviposit at the scented targets. A correlation of wind tunnel results with glomerular activation patterns revealed that feeding and oviposition behaviors are encoded in the moth's antennal lobe by the activation of distinct groups of glomeruli.

Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori.

The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth's olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.

Feeding-induced rearrangement of green leaf volatiles reduces moth oviposition.

The ability to decrypt volatile plant signals is essential if herbivorous insects are to optimize their choice of host plants for their offspring. Green leaf volatiles (GLVs) constitute a widespread group of defensive plant volatiles that convey a herbivory-specific message via their isomeric composition: feeding of the tobacco hornworm Manduca sexta converts (Z)-3- to (E)-2-GLVs thereby attracting predatory insects. Here we show that this isomer-coded message is monitored by ovipositing M. sexta females. We detected the isomeric shift in the host plant Datura wrightii and performed functional imaging in the primary olfactory center of M. sexta females with GLV structural isomers. We identified two isomer-specific regions responding to either (Z)-3- or (E)-2-hexenyl acetate. Field experiments demonstrated that ovipositing Manduca moths preferred (Z)-3-perfumed D. wrightii over (E)-2-perfumed plants. These results show that (E)-2-GLVs and/or specific (Z)-3/(E)-2-ratios provide information regarding host plant attack by conspecifics that ovipositing hawkmoths use for host plant selection. DOI:http://dx.doi.org/10.7554/eLife.00421.001.

Olfactory coding in five moth species from two families.

The aim of the present study was to determine what impact phylogeny and life history might have on the coding of odours in the brain. Using three species of hawk moths (Sphingidae) and two species of owlet moths (Noctuidae), we visualized neural activity patterns in the antennal lobe, the first olfactory neuropil in insects, evoked by a set of ecologically relevant plant volatiles. Our results suggest that even between the two phylogenetically distant moth families, basic olfactory coding features are similar. But we also found different coding strategies in the moths' antennal lobe; namely, more specific patterns for chemically similar odorants in the two noctuid species than in the three sphingid species tested. This difference demonstrates the impact of the phylogenetic distance between species from different families despite some parallel life history traits found in both families. Furthermore, pronounced differences in larval and adult diet among the sphingids did not translate into differences in the olfactory code; instead, the three species had almost identical coding patterns.

Odour maps in the brain of butterflies with divergent host-plant preferences.

Butterflies are believed to use mainly visual cues when searching for food and oviposition sites despite that their olfactory system is morphologically similar to their nocturnal relatives, the moths. The olfactory ability in butterflies has, however, not been thoroughly investigated. Therefore, we performed the first study of odour representation in the primary olfactory centre, the antennal lobes, of butterflies. Host plant range is highly variable within the butterfly family Nymphalidae, with extreme specialists and wide generalists found even among closely related species. Here we measured odour evoked Ca(2+) activity in the antennal lobes of two nymphalid species with diverging host plant preferences, the specialist Aglais urticae and the generalist Polygonia c-album. The butterflies responded with stimulus-specific combinations of activated glomeruli to single plant-related compounds and to extracts of host and non-host plants. In general, responses were similar between the species. However, the specialist A. urticae responded more specifically to its preferred host plant, stinging nettle, than P. c-album. In addition, we found a species-specific difference both in correlation between responses to two common green leaf volatiles and the sensitivity to these compounds. Our results indicate that these butterflies have the ability to detect and to discriminate between different plant-related odorants.

Impact of intraoperative behavior on surgical site infections.

BACKGROUND: The aim of this study was to identify intraoperative risk factors for surgical site infections (SSIs), which are accessible to interventions. We evaluated the effect of extensive intraoperative antiseptic measures and the impact of the behavior of members of the surgical team on SSIs. METHODS: Standard versus extensive antiseptic measures were randomly assigned in 1,032 surgical patients. The adherence to principles of asepsis by members of the surgical team was assessed prospectively. RESULTS: The rate of SSI was 14% with standard antiseptic measures and 15% with extensive measures (P = .581). Multivariate analysis identified following independent risk factors: lapses in discipline (odds ratio [OR] 2.02, confidence interval [CI] 1.05-3.88), intestinal anastomosis (OR 6.74, CI 3.42-13.30), duration of operation more than 3 hours (OR 3.34, CI 1.82-6.14), and body mass index >30 kg/m2 (OR 1.98, CI 1.22-3.20). CONCLUSION: Extensive measures of antisepsis did not reduce the incidence of SSI. A lapse to adhere to principles of asepsis was identified as an independent risk factor for the development of SSI (ClinicalTrials.gov number, NCT00555815).

Interaction of visual and odour cues in the mushroom body of the hawkmoth Manduca sexta.

The responses to bimodal stimuli consisting of odour and colour were recorded using calcium-sensitive optical imaging in the mushroom bodies of the hawkmoth Manduca sexta. The results show that the activity in the mushroom bodies is influenced by both olfaction and vision. The interaction between the two modalities depends on the odour and the colour of the visual stimulus. A blue stimulus suppressed the response to a general flower scent (phenylacetaldehyde). By contrast, the response to a green leaf scent (1-octanol) was enhanced by the presence of the blue stimulus. A green colour had no influence on these odours but caused a marked increase in the response to an odour component (benzaldehyde) of the hawkmoth-pollinated Petunia axillaris.

Engrailed-like immunoreactivity in the embryonic ventral nerve cord of the Marbled Crayfish (Marmorkrebs).

The homeobox transcription factor Engrailed is involved in controlling segmentation during arthropod germ band formation but also in establishing individual neuronal identities during later embryogenesis. In Crustacea, most studies analysing the expression of Engrailed so far have focussed on its function as segment polarity gene. In continuation to these previous studies, we analysed the neuronal expression of the Engrailed protein by immunohistochemistry in the embryonic nerve cord of a parthenogenetic crustacean, the Marbled Crayfish (Marmorkrebs). We paid particular attention to the individual identification of Engrailed expressing putative neuroblasts in the crayfish embryos. Engrailed positive cells in the neuroectoderm were counted, measured and mapped from 38 to 65% of embryonic development. That way, several Engrailed positive putative neuroblasts and putative neurons were identified. Our findings are compared with earlier studies on Engrailed expression during germ band formation in Crustacea. Recent data on neurogenesis in an amphipod crustacean have provided compelling evidence for the homology of several identified neuroblasts between this amphipod and insects. The present report may serve as a basis to explore the question if during crustacean neurogenesis additional communalities with insects exist.

Sirolimus and intraoperative hyperthermic peritoneal chemoperfusion with mitomycin-C do not impair healing of bowel anastomoses.

Surgeons will increasingly have to address the development of gastrointestinal disease in transplant patients or deal with extended bowel resection and bowel anastomosis in advanced cancer patients. Immunosuppressants as well as intraoperative hyperthermic peritoneal chemoperfusion (IHPC) may alter intestinal anastomotic healing. We evaluated the effects of the immunosuppressant sirolimus and of IHPC on healing and stability of bowel anastomoses in pigs. Twenty-four pigs were divided into four groups (SIR: sirolimus was administered orally; IHPC: animals received IHPC with mitomycin-C; COMP: combination of sirolimus and IHPC was administered; CON: sham-treated control group). Animals underwent hand-sutured small bowel and left colon anastomoses and were killed on postoperative day 4. Anastomoses were evaluated by morphometric analysis and immunohistochemistry (IHC) and by measuring the bursting pressure (BP). In all experimental groups (SIR, IHPC, COMP), anastomotic BPs remained unaltered and were not statistically different compared with control (CON). In addition, ileum villous height and colonic crypt depth analysis revealed no significant difference in mucosal thickness, and IHC showed no difference among groups in proliferation, as assessed by the number of KI-67- and bromodeoxyuridine-labeled cells. Immunosuppression with sirolimus as well as IHPC with mitomycin-C do not alter healing of intestinal anastomosis in pigs.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver.

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

Granulocyte colony-stimulating factor supports liver regeneration in a small-for-size liver remnant mouse model.

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 microg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Landmark memories are more robust when acquired at the nest site than en route: experiments in desert ants.

Foraging desert ants, Cataglyphis fortis, encounter different sequences of visual landmarks while navigating by path integration. This paper explores the question whether the storage of landmark information depends on the context in which the landmarks are learned during an ant's foraging journey. Two experimental set-ups were designed in which the ants experienced an artificial landmark panorama that was placed either around the nest entrance (nest marks) or along the vector route leading straight towards the feeder (route marks). The two training paradigms resulted in pronounced differences in the storage characteristics of the acquired landmark information: memory traces of nest marks were much more robust against extinction and/or suppression than those of route marks. In functional terms, this result is in accord with the observation that desert ants encounter new route marks during every foraging run but always pass the same landmarks when approaching the nest entrance.