The fatty acid elongase gene LmELO7 is required for hydrocarbon biosynthesis and cuticle permeability in the migratory locust, Locusta migratoria.

Insect cuticular lipids are a complex cocktail of highly diverse cuticular hydrocarbons (CHCs), which form a hydrophobic surface coat to maintain water balance and to prevent desiccation and penetration of exogenous substances. Fatty acid elongases (ELOs) are key enzymes that participate in a common CHC synthesis pathway in insects. However, the importance of ELOs for CHC synthesis and function remains understudied. Using transcriptomic data, we have identified seven ELO genes (LmELO1-7) in the migratory locust Locusta migratoria. We determined their tissue-specific and temporal expression profiles in fifth instar nymphs. As we are interested in cuticle barrier formation, we performed RNA interference against LmELO7, which is mainly expressed in the integument. Suppression of LmELO7 significantly decreased its expression and caused lethality during or shortly after molting. CHC quantification by GC-MS analysis indicated that suppression of LmELO7 resulted in a decrease in total CHC amounts. By consequence, CHC deficiency reduced desiccation resistance and enhanced cuticle permeability in LmELO7-suppressed L. migratoria. Interestingly, LmELO7 expression is induced at low air humidity. Our results indicate that LmELO7 plays a vital role in the production of CHCs and, hence, cuticle permeability. Induction of LmELO7 expression in drought conditions suggests a key role of this gene in regulating desiccation resistance. This work is expected to help developing new strategies for insect pest management based on CHC function.

Looming detection by identified visual interneurons during larval development of the locust Locusta migratoria.

Insect larvae clearly react to visual stimuli, but the ability of any visual neuron in a newly hatched insect to respond selectively to particular stimuli has not been directly tested. We characterised a pair of neurons in locust larvae that have been extensively studied in adults, where they are known to respond selectively to objects approaching on a collision course: the lobula giant motion detector (LGMD) and its postsynaptic partner, the descending contralateral motion detector (DCMD). Our physiological recordings of DCMD axon spikes reveal that at the time of hatching, the neurons already respond selectively to objects approaching the locust and they discriminate between stimulus approach speeds with differences in spike frequency. For a particular approaching stimulus, both the number and peak frequency of spikes increase with instar. In contrast, the number of spikes in responses to receding stimuli decreases with instar, so performance in discriminating approaching from receding stimuli improves as the locust goes through successive moults. In all instars, visual movement over one part of the visual field suppresses a response to movement over another part. Electron microscopy demonstrates that the anatomical substrate for the selective response to approaching stimuli is present in all larval instars: small neuronal processes carrying information from the eye make synapses both onto LGMD dendrites and with each other, providing pathways for lateral inhibition that shape selectivity for approaching objects.

Symmorphosis and the insect respiratory system: a comparison between flight and hopping muscle.

Weibel and Taylor's theory of symmorphosis predicts that the structural components of the respiratory system are quantitatively adjusted to satisfy, but not exceed, an animal's maximum requirement for oxygen. We tested this in the respiratory system of the adult migratory locust Locusta migratoria by comparing the aerobic capacity of hopping and flight muscle with the morphology of the oxygen cascade. Maximum oxygen uptake by flight muscle during tethered flight is 967±76 µmol h(-1) g(-1) (body mass specific, ±95% confidence interval CI), whereas the hopping muscles consume a maximum of 158±8 µmol h(-1) g(-1) during jumping. The 6.1-fold difference in aerobic capacity between the two muscles is matched by a 6.4-fold difference in tracheole lumen volume, which is 3.5×10(8)±1.2×10(8) µm(3) g(-1) in flight muscle and 5.5×10(7)±1.8×10(7) µm(3) g(-1) in the hopping muscles, a 6.4-fold difference in tracheole inner cuticle surface area, which is 3.2×10(9)±1.1×10(9) µm(2) g(-1) in flight muscle and 5.0×10(8)±1.7×10(8) µm(2) g(-1) in the hopping muscles, and a 6.8-fold difference in tracheole radial diffusing capacity, which is 113±47 µmol kPa(-1) h(-1) g(-1) in flight muscle and 16.7±6.5 µmol kPa(-1) h(-1) g(-1) in the hopping muscles. However, there is little congruence between the 6.1-fold difference in aerobic capacity and the 19.8-fold difference in mitochondrial volume, which is 3.2×10(10)±3.9×10(9) µm(3) g(-1) in flight muscle and only 1.6×10(9)±1.4×10(8) µm(3) g(-1) in the hopping muscles. Therefore, symmorphosis is upheld in the design of the tracheal system, but not in relation to the amount of mitochondria, which might be due to other factors operating at the molecular level.

Fine structure of the sensilla and immunolocalisation of odorant binding proteins in the cerci of the migratory locust, Locusta migratoria.

Using light and electron microscopy (both scanning and transmission), we observed the presence of sensilla chaetica and hairs on the cerci of the migratory locust, Locusta migratoria L. (Orthoptera: Acrididae). Based on their fine structures, three types of sensilla chaetica were identified: long, medium, and short. Males presented significantly more numbers of medium and short sensilla chaetica than females (p<0.05). The other hairs can also be distinguished as long and short. Sensilla chaetica were mainly located on the distal parts of the cerci, while hairs were mostly found on the proximal parts. Several dendritic branches, enveloped by a dendritic sheath, are present in the lymph cavity of the sensilla chaetica. Long, medium, and short sensilla chaetica contain five, four and three dendrites, respectively. In contrast, no dendritic structure was observed in the cavity of the hairs. By immunocytochemistry experiments only odorant-binding protein 2 from L. migratoria (LmigOBP2) and chemosensory protein class I from the desert locust, Schistocerca gregaria Forsskål (SgreCSPI) strongly stained the outer lymph of sensilla chaetica of the cerci. The other two types of hairs were never labeled. The results indicate that the cerci might be involved in contact chemoreception processes.

Moulting of insect tracheae captured by light and electron-microscopy in the metathoracic femur of a third instar locust Locusta migratoria.

The insect tracheal system is an air-filled branching network of internal tubing that functions to exchange respiratory gases between the tissues and the environment. The light and electron-micrographs presented in this study show tracheae in the process of moulting, captured from the metathoracic hopping femur of a juvenile third instar locust (Locusta migratoria). The images provide evidence for the detachment of the cuticular intima from the tracheal epithelial cells, the presence of moulting fluid between the new and old cuticle layers, and the withdrawal of the shed cuticular lining through larger upstream regions of the tracheal system during moulting. The micrographs also reveal that the cuticular intima of the fine terminal branches of the tracheal system is cast at ecdysis. Therefore, the hypothesis that tracheoles retain their cuticle lining at each moult may not apply to all insect species or developmental stages.

The chemosensilla on tarsi of Locusta migratoria (Orthoptera: Acrididae): distribution, ultrastructure, expression of chemosensory proteins.

The chemosensilla on the tarsi of Locusta migratoria were mapped using light microscopy, as well as scanning and transmission electron microscopy. Only chemosensilla chaetica were found on the tarsi. On the basis of their ultrastructure, these can be grouped into three main subtypes: short, long, and sunken sensilla chaetica. Short sensilla chaetica can be further divided into two groups containing 6 or 7 neurons. Long sensilla chaetica are mainly located on the lateral surface of the tarsi. Short sensilla chaetica were mainly found on the dorsal surface of the tarsi. Sunken sensilla chaetica were only found on the ventral surface, such as the pulvilli and arolium. Immunocytochemical localization of chemosensory protein (CSP) was performed on ultrathin sections of chemosensilla on tarsi. The antiserum against LmigCSP-II intensively labeled all three types of sensilla chaetica. Gold granules were concentrated in the outer sensillum lymph surrounding the dendrite sheath, while the inner sensillum lymph containing dendrite branches was never labeled. Massive labeling with the anti-LmigCSP-II was also found in cuticle of the pulvilli on the ventral surface of tarsi.

Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts.

We have identified, cloned and expressed a new chemosensory protein (CSP) in the desert locust Schistocerca gregaria belonging to a third sub-class of these polypeptides. Polyclonal antibodies stained a band of 14 kDa, as expected, in the extracts of antennae and palps of the adults, but not in the 4th and 5th instars. In the related species Locusta migratoria, instead, the same antibodies cross-reacted only with a band of apparent molecular mass of 35 kDa in the extract of 1st-5th instars, but not in the adults. The recombinant protein binds the fluorescent probe N-phenyl-1-naphthylamine, but none of the compounds so far reported as pheromones for S. gregaria. The expression of the odorant-binding protein (OBP) and of CSPs of sub-classes I and II was also monitored in antennae, tarsi, palpi, wings and other organs of solitary and gregarious locusts in their nymphal and adult stages. OBP was found to be antenna specific, where it is expressed at least from the 3rd instar in both solitary and gregarious locusts. CSPs, instead, appear to be more ubiquitous, with different expression patterns, according to the sub-class. Immunocytochemistry experiments revealed that OBP is present in the sensillum lymph of sensilla trichodea and basiconica, while CSP-I and CSP-III were found in the outer sensillum lymph of sensilla chaetica and in the sub-cuticular space between epidermis and cuticle of the antenna. Sensilla chaetica on other parts of the body showed the same expression of CSP-I as those on the antenna.

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization.

Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects.