CRISPR/Cas9 in locusts: Successful establishment of an olfactory deficiency line by targeting the mutagenesis of an odorant receptor co-receptor (Orco)

Locusts are important agricultural pests worldwide and regarded as study models for entomology. However, the absence of targeted gene manipulation systems for locusts has restricted their applications for research. Herein, we report the successful use of the CRISPR/Cas9 system to induce a targeted heritable mutagenesis of the migratory locust, Locusta migratoria. The target sequence of gRNA was designed to disrupt the gene encoding the odorant receptor co-receptor (Orco) and examine the roles of the odorant receptor pathway in the locust. Microinjection of the mixture of Cas9-mRNA and Orco-gRNA into the locust eggs resulted in efficient target-gene editing at a rate of 71.7% in G0 animals and achieved a germline efficiency of up to 88.1% in G1 animals. By a crossing strategy, we successfully established stable Orco mutant lines. EAGs and SSRs indicated that the fourth-instar nymphs of the Orco mutants showed severely impaired electrophysiological responses to multiple odors. The Orco mutant locusts lost an attraction response to aggregation pheromones under the crowding conditions. The locomotor activity and body coloration of the Orco mutant locusts did not significantly differ from those of the two other genotypes. This study provides an easy and effective approach by using the CRISPR/Cas9 system for generating loss-of-function mutants for functional genetic studies of locusts and for managing insect pests.     

Egg hatching of two locusts,Schistocerca gregaria and Locusta migratoria,in response to light and temperature cycles

The present study showed that the eggs of the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria, responded to photoperiod by hatching when placed on sand in the laboratory. S. gregaria mainly hatched during the dark phase and L. migratoria during the light phase. The importance of light as a hatching cue depended on the magnitude of the temperature change during the thermoperiod; photoperiod played a more important role in the control of hatching time in both species when the magnitude of the temperature change was small. In addition, the eggs of the two species that were covered with sand did not respond to photoperiod and hatched during both the light and dark phases, indicating that light did not penetrate through the sand. Because locust eggs are normally laid as egg pods and a foam plug is deposited between the egg mass and the ground surface, we tested a possibility that naturally deposited eggs perceived light through the foam plug. The eggs that were deposited and left undisturbed in the sand hatched during the light and dark phases at similar frequencies. These results suggest that the eggs of both locust species responded to light and controlled their hatching timing accordingly but would not use light as a hatching cue in the field. The evolutionary significance of the ability of eggs to respond to light in these locusts was discussed.     

Transport of hydrocarbons by haemolymph lipophorin in Locusta migratoria

Injection of [1-¹⁴C]acetate into the locust, Locusta migratoria, results in the incorporation of radioactivity into the hydrocarbon fraction which is subsequently released into the haemolymph in association with the lipophorin molecule. The specific capacity of the L. migratoria lipophorin to accept hydrocarbons from the oenocytes, which are believed to be the site of hydrocarbon synthesis, was demonstrated in vitro. Lipophorins from L. migratoria and Periplaneta americana displayed no specificity in their ability to accept hydrocarbons from the oenocytes of the other species. Therefore, it was concluded that the different hydrocarbon compositions of lipophorins from the two species were due to differences in the nature of the oenocytes. When lipophorin containing ¹⁴C-labelled hydrocarbon was injected into the haemocoele of L. migratoria, the labelled hydrocarbon soon appeared at the cuticular surface.The current study together with previous data support the proposal that insect lipophorin serves as a true carrier molecule for the transport of hydrocarbon from the site of synthesis (oenocyte) to the site of deposition (cuticle), in addition to its function of transporting diacylglycerol and cholesterol from the fat body and midgut.     

The primary structure of an endocuticular protein from two locust species,Locusta migratoria and Schistocerca gregaria,determined by a combination of mass spectrometry and automatic Edman degradation

The complete primary structures of two variants of a protein, Abd-5, isolated from the endocuticles of the migratory locust Locusta migratoria and the desert locust Schistocerca gregaria, have been determined. The proteins from the two species are N-terminally blocked with pyroglutamic acid. Their sequences differed only in two positions. Comparison of the sequences to those of other cuticular proteins shows that moderate homologies exist to 11 other cuticular proteins from insects representing four different orders. Amino acid residues in certain positions appear to be strictly conserved.     

Non-protein amino acids of Acacia species and their effect on the feeding of the acridids Anacridium melanorhodon and Locusta migratoria

The leaves of Acacia species have been found to contain homoarginine, pipecolic acid and 4-hydroxy-pipecolic acid. The nymphs of the tree locust Anacridium melanorhodon, which feed on the leaves of Acacia species, were not inhibited from feeding on palatable media containing concentrations of these amino acids equivalent to, or greater than, those found in the leaves. The graminivorous Locusta migratoria was more sensitive to these compounds, inhibitory effects being observed at concentrations comparable to those found in the leaves. The inhibitory effects of mixtures of homoarginine and pipecolic acid were additive in A. melanorhodon but not in L. migratoria. Three of the non-protein amino acids found in the seeds of Acacia species, 2,3-diaminopropionic acid, 2-amino-3-acetylaminopropionic acid and 2-amino-3-oxalylaminopropionic acid, were more effective inhibitors of feeding in Anacridium than were the leaf amino acids.     

Adaptive difference in daily timing of hatch in two locust species,Schistocerca gregaria and Locusta migratoria: The effects of thermocycles and phase polyphenism

This study examined the effects of temperature and phase polyphenism on egg hatching time in the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria. The two species exhibited differences and similarities in hatching behavior when exposed to different temperature conditions. In 12-h thermocycles of various temperatures, the S. gregaria eggs hatched during the cryoperiod (low temperature period), whereas L. migratoria eggs hatched during the thermoperiod (high temperature period). The eggs of both species hatched during the species-specific period of the thermoperiod in response to a temperature difference as small as 1 °C. Furthermore, the locusts adjusted hatching time to a new thermal environment that occurred shortly before the expected hatching time. In both species, the hatching of the eggs was synchronized to a specific time of the day, and two hatching peaks separated by approximately 1 day were observed at a constant temperature after the eggs were transferred from thermocycles 3 days before hatching. Eggs laid by gregarious females hatched earlier than those laid by solitarious females in S. gregaria but this difference was not observed in L. migratoria.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Mitochondrial genomes reveal the global phylogeography and dispersal routes of the migratory locust

The migratory locust, Locusta migratoria, is the most widely distributed grasshopper species in the world. However, its global genetic structure and phylogeographic relationships have not been investigated. In this study, we explored the worldwide genetic structure and phylogeography of the locust populations based on the sequence information of 65 complete mitochondrial genomes and three mitochondrial genes of 263 individuals from 53 sampling sites. Although this locust can migrate over long distances, our results revealed high genetic differentiation among the geographic populations. The populations can be divided into two different lineages: the Northern lineage, which includes individuals from the temperate regions of the Eurasian continent, and the Southern lineage, which includes individuals from Africa, southern Europe, the Arabian region, India, southern China, South‐east Asia and Australia. An analysis of population genetic diversity indicated that the locust species originated from Africa. Ancestral populations likely separated into Northern and Southern lineages 895 000 years ago by vicariance events associated with Pleistocene glaciations. These two lineages evolved in allopatry and occupied their current distributions in the world via distinct southern and northern dispersal routes. Genetic differences, caused by the long‐term independent diversification of the two lineages, along with other factors, such as geographic barriers and temperature limitations, may play important roles in maintaining the present phylogeographic patterns. Our phylogeographic evidence challenged the long‐held view of multiple subspecies in the locust species and tentatively divided it into two subspecies, L. m. migratoria and L. m. migratorioides.     

Pharmacological characterization of dopamine-sensitive adenylate cyclase in the salivary glands of Locusta migratoria L.

The locust (Locusta migratoria) salivary gland adenylate cyclase was stimulated by both dopamine and serotonin (6 and 5-fold stimulation respectively). Since their effects were additive, it is concluded that these neurotransmitters are acting on two discrete receptor types.The agonist and antagonist specificity profiles were very similar to those of the D₁ receptor coupled with an adenylate cyclase in vertebrates. They were clearly different from those of the D₂ receptor of vertebrates. No evidence for octopamine sensitive adenylate cyclase was found in the salivary glands of L. migratoria.In addition to previous electrophysiological and histological studies (House and Ginsborg, 1979) these results suggest that dopamine has a neurotransmitter function in the salivary glands of locusts and that its function is mediated by cyclic AMP.     

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

Characterization and PCR multiplexing of polymorphic microsatellite loci for the locust Locusta migratoria

Because of the scarcity of polymorphic genetic markers available in locust species, only a few population genetics studies have been carried out on this taxon. We isolated and characterized 11 polymorphic microsatellite loci in the pest locust Locusta migratoria capito, and described experimental conditions for polymerase chain reaction (PCR) multiplexing and simultaneously genotyping these loci. The number of alleles per locus ranged from six to 25, and the expected heterozygosity ranged from 0.431 to 0.957. Results of cross‐taxon amplification tests are reported in six other Locusta migratoria subspecies, six species of the Oedipodinae subfamily and two other pest locust species.     

The current state of natural foci of swarming locusts (Orthoptera,Acrididae) in Turkmenistan

Climatic changes and anthropogenic impact on natural ecosystems result in the growth of locust populations. The general characteristic of the spatial distribution of the Moroccan (Dociostaurus maroccanus Thunb.), Italian (Calliptamus italicus L.), and Asian migratory (Locusta migratoria migratoria L.) locusts in Turkmenistan are given.     

Dynamics of mortality of the migratory locust under synchronous infection with entomopathogenic fungi (<Emphasis Type="Italic">Beauveria bassiana,Metarhizium anisopliae</Emphasis>) and bacteria <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

As a result of search for species and strains of entomopathogenic fungi and bacteria virulent to migratory locust (Locusta migratoria migratoria L.), combinations were found which cause high mortality of insect in a short time interval. Four or five days after the L. migratoria had been infected with Beauveria bassiana (Bals.) Vuill and Metarhizium anisopliae (Metsch.) Sorokin a sharp increase in nymphas’ mortality was observed, reaching 95–100% on the 13th to 17th day after inoculation. The mortality of L. migratoria after infection with Pseudomonas sp. bacteria was approximately 30–50% on the 3rd to 7th day of the experiment. Later deaths of the locusts were not observed. When we made synchronous inoculation with fungi and bacteria, the rate of nymphas’ mortality was higher in comparison with monoinfections, and LT₅₀ was about three days. Microbiological analysis of the dead insects showed that both pathogens could coexist in the locust. To determine the antagonism between Pseudomonas and fungi on a synthetic nutrient medium, the blocking method was used. We showed that the fungi do not affect the development of the bacterium, and the Pseudomonas has an insignificant effect on the fungi growth.     

The Leucophaea maderae hindgut preparation: A rapid and sensitive bioassay tool for the isolation of insect myotropins of other insect species

The isolated hindgut preparation of the cockroach, Leucophaea maderae has provided an effective bioassay tool for the isolation of certain structural types of insect myotropic peptides. Initially, the preparation was used to monitor excitatory and inhibitory activities of numerous HPLC fractions in a study that resulted in the structural characterization of 12 Leucophaea neuropeptides. Subsequently, the preparation was used as the bioassay for the isolation and structural characterization of myotropic neuropeptides of the house cricket, Acheta domesticus, and the locust, Locusta migratoria. Five novel myotropic peptides from the cricket were structurally characterized, and 32 separate myotropic compounds were isolated from nervous tissue of the locust. At present, 8 of the locust peptides have been structurally characterized. Isolation studies using this bioassay have been responsible for the discovery of 25 unique neuropeptides, 4 new peptide families, and the initial demonstration of the natural analog phenomenon in insects.     

Yolk formation in Locusta migratoria and Schistocerca gregaria: Related ligands and oocyte receptors

During vitellogenesis the transport of yolk precursor proteins, the vitellogenins (VTG), from the hemolymph into the oocyte is achieved by receptor-mediated endocytosis. Recently the receptor for the VTG of Locusta migratoria has been isolated. Now a new protocol has been developed for the purification of the VTG receptor of this locust from ovarian membranes. By CHAPS solubilization of the membranes followed by ion exchange and immunoaffinity chromatography, a 100-fold purification of the VTG receptor was achieved. The amino acid composition of the receptor protein has been determined. However, first attempts to sequence the receptor failed due to the N-terminal blocking of the molecule. With the same methods the VTG receptor of another locust, Schistocerca gregaria, has been isolated, purified, and characterized. This receptor has an apparent M_r of 186 kDa under nonreducing conditions. It recognizes L. migratoria VTG and vice versa. However, in cross-competition experiments in which the Schistocerca VTG competed with Locusta VTG for binding to the Locusta VTG receptor, the Schistocerca VTG was less efficient. Furthermore, the VTG receptor proteins of S. gregaria and L. migratoria are immunologically related as revealed by Western blotting with anti-Locusta VTG receptor antibodies. It appears that important structural elements required for efficient and specific endocytosis of VTG have been conserved. © 1994 Wiley-Liss, Inc.