The excretion of NaCl and KCl loads in mosquitoes. 1. Control data.

The handling of Na(+) and K(+) loads was investigated in isolated Malpighian tubules and in whole mosquitoes of Aedes aegypti. Isolated Malpighian tubules bathed in Na(+)-rich Ringer solution secreted Na(+)-rich fluid, and tubules bathed in K(+)-rich Ringer solution secreted K(+)-rich fluid. Upon Na(+) loading the hemolymph, the mosquito removed 77% the injected Na(+) within the next 30 min. The rapid onset and magnitude of this diuresis and the excretion of more Na(+) than can be accounted for by tubular secretion in vitro is consistent with the release of the calcitonin-like diuretic hormone in the mosquito to remove the Na(+) load from the hemolymph. Downstream, K(+) was reabsorbed with water in the hindgut, which concentrated Na(+) in excreted urine hyperosmotic to the hemolymph. Upon K(+) loading the hemolymph, the mosquito took 2 h to remove 100% of the injected K(+) from the hemolymph. The excretion of K(+)-rich isosmotic urine was limited to clearing the injected K(+) from the hemolymph with a minimum of Cl(-) and water. As a result, 43.3% of the injected Cl(-) and 48.1% of the injected water were conserved. The cation retained in the hemolymph with Cl(-) was probably N-methyl-d-glucamine, which replaced Na(+) in the hemolymph injection of the K(+) load. Since the tubular secretion of K(+) accounts for the removal of the K(+) load from the hemolymph, the reabsorption of K(+), Na(+), Cl(-), and water must be inhibited in the hindgut. The agents mediating this inhibition are unknown.

Cloning, expression and purification of subunit H of vacuolar H⁺-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae).

The vacuolar (H+)-ATPase (V-ATPase) of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends) technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.

A new endophytic Penicillium oxalicum with aphicidal activity and its infection mechanism.

BACKGROUND: Aphid infestation adversely affects the yield and quality of crops. Rapid reproduction and insecticidal resistance have made controlling aphids in the field challenging. Therefore, the present study investigated the insecticidal property of Penicillium oxalicum (QLhf-1) and its mechanism of action against aphids, Hyalopterus arundimis Fabricius. RESULTS: Bioassay revealed that the control efficacy of the spores against aphids (86.30% and 89.05% on the third day and fifth day after infection, respectively) were higher than other components, such as the mycelium. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that QLhf-1 invaded the aphid cuticle through spores and used the aphid tissues as a nutrient source for growth and reproduction, causing stiffness and atrophy and a final death. Three extracellular enzymes, lipase, protease, and chitinase had a synergistic effect with spores, and they acted together to complete the infection process by degrading the aphid body wall and accelerating the infection process. CONCLUSION: The newly discovered endophytic penicillin strain P. oxalicum 'QLhf-1' can effectively kill aphids. The results provided strong evidence for the biological control of aphids, and lay a foundation for the development and utilization of QLhf-1. © 2024 Society of Chemical Industry.

Discovery, Identification, and Insecticidal Activity of an Aspergillus flavus Strain Isolated from a Saline-Alkali Soil Sample.

Aphids are one of the most destructive pests in agricultural production. In addition, aphids are able to easily develop resistance to chemical insecticides due to their rapid reproduction and short generation periods. To explore an effective and environmentally friendly aphid control strategy, we isolated and examined a fungus with aphid-parasitizing activity. The strain (YJNfs21.11) was identified as Aspergillus flavus by ITS, 28S, and BenA gene sequence analysis. Scanning electron microscopy and transmission electron microscopy revealed that the infection hyphae of 'YJNfs21.11' colonized and penetrated the aphid epidermal layer and subsequently colonized the body cavity. Field experiments showed that 'YJNfs21.11' and its fermentation products exerted considerable control on aphids, with a corrected efficacy of 96.87%. The lipase, protease, and chitinase secreted by fungi help aphid cuticle degradation, thus assisting spores in completing the infection process. Additionally, changes were observed in the mobility and physical signs of aphids, with death occurring within 60 h of infection. Our results demonstrate that A. flavus 'YJNfs21.11' exhibits considerable control on Aphis gossypii Glover and Hyalopterus arundimis Fabricius, making it a suitable biological control agent.

Ultrastructural effects of Celangulin V on midgut cells of the oriental armyworm, Mythimna separata walker (Lepidoptera: Noctuidae).

The ultrastructural effects of ingested Celangulin V (CA-V), an insecticidal component extracted from the root bark of Chinese bittersweet (Celastrus angulatus Maxim), on the midgut epithelial cells of the oriental armyworm larva (Mythimna separata Walker) were studied. Transmission electron microscopy showed that CA-V could induce a severe, time-dependent cytotoxicity of the midgut epithelial cells, which had large cytoplasmic spaces, disrupted microvilli and swollen mitochondria. The cisternae of the rough endoplasmic reticulum were excessively dilated, vesiculated and then fragmented. The nuclei were pycnotic and contained one or two divided nucleoli and agglomerated chromatin. Numerous lysosome-like vacuoles and secretion granules were observed. Finally, the CA-V resulted in cell death by necrosis with plasma membrane lysis and the cytoplasm content leakage into the migdut lumen.

Preparation of monoclonal antibody against celangulin V and immunolocalization of receptor in the oriental armyworm, Mythimna separata walker (Lepidoptera: Noctuidae).

The botanical insecticide celangulin V (CA-V) is an insect digestive poison acting on midgut tissue of the target insect larvae. With the aim of localizing the receptor enacted by CA-V, monoclonal antibodies (MAbs) specific to the compound were developed. A hapten was synthesized by introducing a succinoyl into the CA-V structure and conjugated with three carrier proteins. From mice immunized with one conjugate, three MAbs were obtained with a potential capacity of detecting protein-bound residue forms of CA-V in the biological tissues. The oriental armyworm larvae ingested CA-V were examined by the technique of immuno-electron-microscopy (IEM) using the anti-CA-V MAb as the primary antibody and goat anti-mouse/IgG labeled with colloidal gold as the secondary antibody. Electron micrographs of the armyworm midgut tissues showed that the CA-V was associated with the midgut epithelia of the insects. These results demonstrated the existence of a receptor enacted by CA-V on the midgut cells of the oriental armyworm larvae.

Validation of the Target Protein of Insecticidal Dihydroagarofuran Sesquiterpene Polyesters.

A series of insecticidal dihydroagarofuran sesquiterpene polyesters were isolated from the root bark of Chinese bittersweet (Celastrus angulatus Max). A previous study indicated that these compounds affect the digestive system of insects, and aminopeptidase N3 and V-ATPase have been identified as the most putative target proteins by affinity chromatography. In this study, the correlation between the affinity of the compounds to subunit H and the insecticidal activity or inhibitory effect on the activity of V-ATPase was analyzed to validate the target protein. Results indicated that the subunit H of V-ATPase was the target protein of the insecticidal compounds. In addition, the possible mechanism of action of the compounds was discussed. The results provide new ideas for developing pesticides acting on V-ATPase of insects.

Effects of Periplocoside P from Periploca sepium on the Midgut Transmembrane Potential of Mythimna separata Larvae.

Periplocoside P (PSP) isolated from the root bark of Periploca sepium contains a pregnane glycoside skeleton and possesses high insecticidal properties. Preliminary studies indicated that PSP disrupts epithelial functions in the midgut of lepidopteran larvae. In the present study, we examined the effects of PSP on the apical and basolateral membrane voltages, Va and Vbl, respectively, of cells from (1) midguts isolated from the larvae of the oriental armyworm Mythimna separata that were in vitro incubated with toxins and (2) midguts isolated from M. separata larvae force-fed with PSP. We compared the effects of PSP with the effects of the Bacillus thuringiensis toxin Cry1Ab and inactive periplocoside E (PSE) on the midgut epithelial cells. The results showed that Va rapidly decreased in the presence of PSP in a time- and dose-dependent manner, similar to the effects of Cry1Ab. By contrast, PSE did not affect the Va and Vbl. Additionally, PSP did not influence the Vbl. Given these results, we speculate that PSP may modulate transport mechanisms at the apical membrane of the midgut epithelial cells by inhibiting the V-type H+ ATPase.

Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography.

Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.