Transcriptome Analysis Provides Insights into Water Immersion Promoting the Decocooning of Osmia excavata Alfken.

The timing of decocooning and nesting during the flowering period are crucial for the reproduction and pollination activities of Osmia excavata. In order to improve the pollination efficiency of O. excavata, it is crucial to find a way to break the cocoon quickly. Our results showed that the decocooning rates at 6, 12, 24, 36, 48, and 72 h after 30 min of water immersion (WI) were 28.67%, 37.33%, 37.33%, 41.33%, 44.33%, and 53.00%, respectively. The decocooning rate fold of 6 h was 14.33 compared with the control group. Transcriptome sequencing resulted in 273 differentially expressed genes (DEGs) being identified between the WI and control groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that muscle-related functions play important roles in O. excavata decocooning in response to WI. Cluster analysis also showed that DEGs in cardiac muscle contraction and adrenergic signaling in cardiomyocytes were up-regulated in response to WI-promoted decocooning. In conclusion, the rate of decocooning can be improved by WI in a short time. During WI-promoted decocooning, muscle-related pathways play an important role. Therefore, the application of this technology will improve the pollination effect of O. excavata.

Virulence of the fungal pathogen, Aspergillus oryzae XJ-1 to adult locusts (Orthoptera: Acrididae) in both laboratory and field trials.

BACKGROUND: Locusts and grasshoppers are pests of many agricultural crops, and their frequent outbreaks worldwide threaten food security. Microbial control agents are currently used to suppress the early (nymphal) stages of pests, but they are often less effective against adults, which are primarily responsible for locust plagues. The fungal pathogen Aspergillus oryzae XJ-1 has high pathogenicity in locust nymphs. To assess its potential for controlling locust adults, we evaluated the virulence of A. oryzae XJ-1 (i.e., locust Aspergillus, LAsp) in locust adults using laboratory, field-cage experiments, and a field trial. RESULTS: The lethal concentration of LAsp in adult Locusta migratoria was 3.58 ± 0.09 × 105 conidia mL-1 15 days after inoculation in the laboratory. A field-cage experiment showed that the mortalities of adult L. migratoria were 92.0 ± 4.6% and 90.1 ± 3.2% 15 days after inoculation with 3 × 105 and 3 × 103 conidia m-2 of LAsp, respectively. A large-scale field trial of 666.6 ha was conducted, in which a LAsp water suspension was applied at a concentration of 2 × 108 conidia mL-1 in 15 L ha-1 by aerial spraying via drones. The densities of mixed populations of L. migratoria and Epacromius spp. were significantly reduced by 85.4 ± 7.9%-94.9 ± 5.1%. In addition, the infection rates of surviving locusts collected from the treated plots were 79.6% and 78.3% on the 17th and 31st day after treatment, respectively. CONCLUSION: These results indicate that A. oryzae XJ-1 is highly virulent in adult locusts and that it has high potential for the control of locusts. © 2023 Society of Chemical Industry.

Single Sensillum Recordings for Locust Palp Sensilla Basiconica.

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust's food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs' membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation.

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported AlocSWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. AlocSWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT-PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

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.

Differential expression of two novel odorant receptors in the locust (Locusta migratoria).

BACKGROUND: Olfaction in animals is important for host localization, mating and reproduction in heterogeneous chemical environments. Studying the molecular basis of olfactory receptor neurons (ORNs) systems can elucidate the evolution of olfaction and associated behaviours. Odorant receptors (ORs) in insects have been identified, particularly in the holometabolous model Drosophila, and some of them have been functionally studied. However, ORs in the locust-a hemimetabolous model insect and the most important insect crop pest-have not yet been identified, hindering our understanding of locust olfaction. Here, we report for the first time four putative ORs in Locusta migratoria: LmigOR1, LmigOR2, LmigOR3 and LmigOR4. RESULTS: These four putative OR genes encoded proteins with amino acids of 478, 436, 413 and 403 respectively. Sequence identity among them ranged from 19.7% to 35.4%. All ORs were tissue-specifically expressed in olfactory organs, without sex-biased characteristics. However, LmigOR1, LmigOR3 and LmigOR4 were only expressed in the antenna, while LmigOR2 could also be detected in mouthparts. In situ hybridization demonstrated that the LmigOR1antisense probe labelled olfactory receptor neurons (ORNs) in almost all segments of the antenna, but only a few segments housed ORNs expressing LmigOR2. The number of neurons labelled by LmigOR1 antisense probes in each antennal segment was much greater (>10 neurons/segment) than that labelled by LmigOR2 probes (generally 1-3 neurons/segment). Furthermore, some of the labelled neurons could be attributed to the basiconic sensilla, but LmigOR1 and LmigOR2 were expressed in different subtypes. CONCLUSIONS: Our results strongly suggested that these newly discovered genes encode locust ORs and the differential expression patterns of LmigOR1 and LmigOR2 implied distinct functions. These results may offer insights into locust olfaction and contribute to the understanding of the evolution of insect chemoreception.

[Cloning and expression of endoglucanase of marine cold-adapted bacteria Pseudoalteromonas sp. MB-1].

The cold-adapted gram-negative rod bacterium MB-1 which could secret cellulase was screened from mud of the bottom of the Huanghai. According to the sequence of 16S rDNA, this bacterium screened was identified as one species of Pseudoalteromonas and was named as Pseudoalteromonas sp. MB-1. The gene celA encoding cold-adapted endogluanase was cloned and then jointed to pGEX-4T-1 to construct expression plasmid pGEX-celA which was expressed in E. coli BL21. Analysis to the supernatant of E. coli sonicate revealed that the concentration of GST-CelA was about 78.5 mg/L. Properties of the fusion enzyme of GST-CelA including the optimum temperature at 35 degrees C and the optimum pH about 7.2, showed that this fusion enzyme still belonged to cold-adapted enzyme and neutral enzyme. The result lays solid base for the fundamental theory and application research on cold-adapted cellulase from Pseudoalteromonas sp. MB-1.