Immunological regulation by Toll-1 and Spätzle-4 in larval density-dependent prophylaxis of the oriental armyworm, Mythimna separata.

High population density has been shown to alter insect prophylactic immunity. Toll-Spätzle pathway performs a key function in insect innate immune response. To determine the role of Toll and Spätzle, two main components of Toll-Spätzle pathway, in the density-dependent prophylaxis of Mythimna separata. We identified full-length cDNA encoding the Toll-1 and Spätzle-4 genes in M. separata (designed MsToll-1 and Ms Spätzle-4). Both MsToll-1 and MsSpätzle-4 were expressed throughout all developmental stages. MsToll-1 expression was highly in fat body and brain and MsSpätzle-4 was highly expressed in brain and Malpighian tubule. With increased larval density, MsToll-1 expression was markedly up-regulated. MsSpätzle-4 expression was found to be raised in larvae that were fed in high density (5 and 10 larvae per jar). Co-immunoprecipitation assays demonstrated that MsToll-1 interacted with MsSpätzle-4. Immune-related genes transcriptions were considerably reduced in high-density larvae MsToll-1 (or MsSpätzle-4) was silenced by dsRNA injection. Meanwhile, a discernible reduction in the survival rate of the larvae exposed to Bacillus thuringiensis infection with silence of MsToll-1 (or MsSpätzle-4) was observed. This study implies that prophylactic immunity was influenced by crowded larvae via modulating the Toll-Spätzle pathway in M. separata and allow for a new understanding of into density-dependent prophylaxis in insects.

Oxalate­induced renal pyroptotic injury and crystal formation mediated by NLRP3­GSDMD signaling in vitro and in vivo.

Calcium oxalate kidney stone has become an urgent issue due to its high incidence and recurrence rate. Thus, it is necessary to explore for mechanisms of calcium oxalate stones formation. Previous studies demonstrated that oxalate crystals could induce the activation of nucleotide­binding domain and leucine­rich repeat­containing family pyrin domain­containing 3 (NLRP3) inflammasome and change the renal tubular epithelium adhesion. However, the type and molecular mechanism of NLRP3 inflammasome­mediated calcium oxalate stones formation still need to be further investigated. In the present study, it was confirmed that the NLRP3­gasdermin D (GSDMD) signaling was involved in oxalate­induced cell injury in vitro and in vivo. Inhibition of reactive oxygen species production could effectively prevent the NLRP3 inflammasome formation in oxalate­treated HK­2 cells. NLRP3 gene silence could inhibit the DNA damage and cellular membrane injury of HK­2 cells treated with oxalate. The ultrastructural changes of several organelles and particular structures, similar to typical cell pyroptosis, were observed in oxalate­stimulated HK­2 cells. NLRP3 gene silence could antagonize the oxalate­induced injury and ultrastructure changes. Additionally, NSA (GSDMD inhibitor) could prevent the oxalate­induced injury of membrane integrity in HK­2 cells. Moreover, oxalate crystals were significantly decreased in GSDMD­/­ mice compared with wild­type mice with glyoxylic acid. Together, NLRP3­GSDMD pathway was involved in the oxalate­induced pyroptotic injury in HK­2 cells. GSDMD and its cleavage form GSDMD­N played an important role in the oxalate­induced renal cell injury and oxalate calcium crystals formation in vitro and in vivo. This provided a new target for prevention and treatment of oxalate nephropathy and oxalate calcium stones.

A randomized controlled study of ureteral stent extraction string on patient's quality of life and stent-related complications after percutaneous nephrolithotomy in the prone position.

To demonstrate the Tianjin Institute of Urology (TJIU) technique to place and remove the ureteral stent with extraction string after percutaneous nephrolithotomy (PCNL). Additionally, we aim to compare the pain experienced during stent removal, quality of life during stent retention, and stent-related complications between patients with and without extraction string. 65 patients were included in the final analysis in the string group constructed by the TJIU technique and 66 patients in the conventional double-J ureteral stent (non-string) group. All patients underwent the surgery in a prone position under general anesthesia. They completed the Ureteral Stent Symptom Questionnaire (USSQ) on postoperative days (POD) 7, as well as before their ureteral stent was removed. The visual analogue scale (VAS) pain score (0-10) was completed immediately after the removal of the ureteral stent. Moreover, a specialized person was responsible for recording stent-related complications. All patients completed the USSQ on POD 7, and we did not find a difference in scores in each field. However, there was a significant difference in the "sex" domain before removing the ureteral stent (4.34 vs 3.23; p = 0.01). Notably, the use of extraction string after PCNL could decrease the pain associated with stent removal significantly (mean VAS scores 1.45 vs 2.76; p < 0.01). Extraction string did not increase the incidence of stent-related complications. We concluded that placing a ureteral stent with an extraction string after PCNL reduces the pain of ureteral stent removal without increasing complications such as accidental removal of the stent, febrile urinary tract infection (UTI).

Bursicon mediates antimicrobial peptide gene expression to enhance crowded larval prophylactic immunity in the oriental armyworm, Mythimna separata.

It has been reported that a high population density alters insect prophylactic immunity. Bursicon plays a key role in the prophylactic immunity of newly emerged adults. In this paper, full-length cDNAs encoding the alpha and beta subunits of bursicon in Mythimna separata larvae (Msburs α and Msburs ß) were identified. The cDNAs of Msburs α and Msburs ß contain open reading frames (ORFs) encoding 145- and 139-amino acid residue proteins, respectively. Multiple alignment sequences and phylogenetic analysis indicated that Msbursicons (Msburs α and Msburs ß) are orthologous to bursicons in other lepidopterans. The Msbursicons were expressed throughout all developmental states with higher relative expression during the egg, pupae, and adult stages. Msbursicons (Msburs α and Msburs ß) were highly expressed in the ventral nerve cord and brain relative to other tested tissues. Msbursicon expression of larvae subject to high-density treatment (10 larvae per jar) was significantly increased compared with that of the larvae subject to low-density treatment (1 larva per jar) in the whole fourth and fifth instar stages. The trend in the expression of the antimicrobial peptide (AMP) genes cecropin C and defensin in the test stage was accorded and delayed with increased expression of bursicons. Silencing Msburs α (or Msburs ß) expression by dsRNA injection in larvae subject to high-density treatment significantly decreased the expression levels of the cecropin C and defensin genes. Recombinant Msbursicon homodimers significantly induced the expression of the cecropin C and defensin genes. There was a notable decrease in the survival rate of the Msburs α (or Msburs ß or Mscecropin C or Msdefensin) knockdown larvae infected by Beauveria thuringiensis. Our findings provide the first insights into how larval density mediates AMP gene expression, which subsequently affects the prophylactic immunity of insects under high-density conditions.

Effects of Larval Density on Plutella xylostella Resistance to Granulosis Virus.

It has been reported that some phase-polyphenic insects from high-density conditions are more resistant to pathogens than those from low-density conditions. This phenomenon is termed "density-dependent prophylaxis" (DDP). However, whether non phase-polyphenic insects exhibit DDP has rarely been elucidated. The diamondback moth (DBM), Plutella xylostella, one of the most destructive insect pests affecting cruciferous crops, is non phase-polyphenic. In this study, the resistance of DBM larvae to P. xylostella granulosis virus (Plxy GV) and their immune response to the virus when reared at densities of 1, 2, 5, 10, 15, and 20 larvae per Petri dish were investigated under laboratory conditions. Compared with larvae reared at lower densities, larvae reared at moderate density showed a significantly higher survival rate, but the survival rate significantly decreased with further increases in rearing density. Furthermore, the phenoloxidase, lysozyme and antibacterial activity and total hemocyte count in the hemolymph of the larvae, regardless of whether they were challenged with the virus, from different larval densities corresponded to the observed differences in resistance to Plxy GV. These results demonstrated that P. xylostella larvae exhibited DDP within a certain limited density. This study may help to elucidate the biocontrol effect of different density populations of P. xylostella by granulosis virus and guide improvements in future management strategy.

Immunological regulation by a ß-adrenergic-like octopamine receptor gene in crowded larvae of the oriental Armyworm, Mythmina separata.

Recent reports demonstrate that octopamine plays an important immunological role in crowded larvae of the Oriental Armyworm, Mythmina separata. We identified an octopamine receptor, the ß-adrenergic-like gene (designated MsOctß2R), with a 1191 bp open reading frame that encodes 396 amino acids and contains seven conserved hydrophobic transmembrane domains. Multiple sequence alignments and a phylogenetic analysis indicated that MsOctß2R was orthologous to Octß2R that is present in other lepidopterans. MsOctß2R was expressed throughout all developmental stages with higher relative expression during the fourth instar and adult stages. MsOctß2R was highly expressed in the ventral nerve cord and the fat body relative to other examined tissues. Elevated MsOctß2R expression was observed in larvae that were under higher-density conditions (7 and 10 larvae per jar). Silencing MsOctß2R expression via dsRNA injections in larvae from higher-density conditions significantly decreased phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival rates against Beauveria bassiana infections (54.06%, 9.91%, 36.22%, and 23.53%, respectively) when compared with control larvae. These results suggest that high-density conditions might alter prophylactic immunity in larvae by regulating the MsOctß2R gene in M. separara and provide new insights into density-dependent prophylaxis in insects.

The Role of Tyramine ß-Hydroxylase in Density Dependent Immunityof Oriental Armyworm (Mythmina separata) Larva.

High population density alters insect prophylactic immunity, with density-dependent prophylaxis (DDP) being reported in many polyphonic insects. However, the molecular mechanism for DDP remains unclear. In current study, the role of tyramine ß-hydroxylase (Tßh) in the immune response of M. separata larvae that were subject to different rearing densities conditions was investigated. The tyramine ß-hydroxylase activity of larvae from high density treatments (10 and 30 larvae per jar) was significantly higher than that of the larvae from low density treatments (one, two, and five larvae/jar). A tyramine ß-hydroxylase (designated MsTßh) containing a 1779 bp open reading frame was identified. Multiple sequence alignment and phylogenetic analysis indicated that MsTßh was orthologous to the Tßh that was found in other lepidopterans. Elevated MsTßh expression was observed in larvae under high density (10 larvae per jar). Silencing MsTßh expression by the injection of dsRNA in larvae from the high density treatment produced a 25.1% reduction in octopamine levels, while at the same time, there was a significant decrease in phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival against Beauveria infection 56.6%, 88.5%, 82.0%, and 55.8%, respectively, when compared to control larvae. Our findings provide the first insights into how MsTßh mediates the octopamine level, which in turn modulates the immune response of larvae under different population densities.

Transcriptomic insight into antimicrobial peptide factors involved in the prophylactic immunity of crowded Mythimna separata larvae.

Similar to pathogenic infection, a high population density alters insect prophylactic immunity. Antimicrobial peptides (AMPs) are known to play critical roles in an insect's humoral immune response to microbial infection. We applied RNA sequencing to investigate differential gene expression levels in fat body and hemocyte samples from larvae reared in high- (10 larvae per jar) and low-density (1 larva per jar) conditions; the samples exhibited density-dependent prophylaxis. A number of AMP molecule-related proteins were annotated for the first time from 145,439 assembled unigenes from M. separata larvae. The transcript levels of AMP molecules such as gloverin-, defensin-, cecropin-, lebocin- and attacin-related unigenes were increased with the prophylactic immunity of high-density larvae. The pattern recognition receptor peptidoglycan recognition protein (PGRP), a key protein in the synthesis of AMPs in IMD- and Toll pathway-related unigenes, was also upregulated in the larvae from the high-density group. The resultant transcriptomic database was validated by the transcript levels of four selected AMP genes quantified from the high- and low-density larval groups with quantitative real-time PCR. The antimicrobial activity against gram-positive Staphylococcus aureus and Bacillus subtilis and gram-negative Edwardsiella ictaluri and Vibrio anguillarum in the hemolymph of larvae from the high-density group was significantly higher than that of larvae from the low-density group. Our findings provide the first insight into the role of AMP genes in the mechanisms of density-dependent prophylaxis in M. separata and provide new insight into the control of M. separata with biopesticides.

Altered immunity in crowded Mythimna separata is mediated by octopamine and dopamine.

Similar to pathogenic infection, high population density alters insects' prophylactic immunity. Density-dependent prophylaxis has been reported in many polyphenic insects, but the regulatory mechanism underlying this phenomenon remains unclear. The biogenic monoamines are known to play critical roles in mediating insect immune responses. In the current study, the immune capacity and the levels of three biogenic monoamines were investigated in the polyphenic larvae of Mythimna separata, reared at the densities of 1, 2, 5, 10, and 30 larvae per 650-mL jar. Concomitant with the increased phenoloxidase (PO) activity and total haemocyte count in the larvae at high densities (5, 10, 30 larvae/jar), the octopamine level was also increased. In contrast, the dopamine level was decreased, and the 5-hydroxytryptamine level was not significantly affected. Injection of octopamine induced significant increases in the total haemocyte count and PO activity. Conversely, epinastine, a specific antagonist of octopamine, decreased the total haemocyte count and PO activity. Another octopamine antagonist, phentolamine, inhibited the activity of PO and lysozymes. In addition, injection of dopamine induced a significant increase in PO activity and decreased the total haemocyte count and lysozyme activity. These results suggested that both octopamine and dopamine mediate the increases in total haemocyte count and PO activity in the crowded larvae.

Differing Behavioural Responses of Bemisia tabaci MEAM1 and MED to Cabbage Damaged by Conspecifics and Heterospecifics.

The whitefly Bemisia tabaci is a serious pest with an extensive host range. Previous research has shown that B. tabaci is a species complex with many cryptic species or biotypes and that the two most important species are MEAM1 (Middle East-Minor Asia 1) and MED (Mediterranean genetic group). MEAM1 and MED are known to differ in their preference for cabbage, Brassica oleracea, as a host plant, however, the mechanism underlying this preference is unknown. In the current study, a host choice experiment showed that MED prefers to settle and oviposit on undamaged cabbage plants rather than MED-damaged cabbage plants. However, MEAM1 prefers MED-damaged cabbage plants to undamaged plants and does not exhibit a significant preference for undamaged or MEAM1-damaged cabbage plants. On the basis of gas chromatography-mass spectrometry (GC-MS) analysis, the following volatiles were released in larger quantities from Q-damaged cabbage plants than from undamaged plants: 2-ethyl-1-hexanol, benzenemethanol, (E)-2-decenol, benzaldehyde, nonanal, acetic acid geraniol ester, 4-hydroxy-4-methyl-2-pentanone, decane, and α-longipinene. Only one volatile, 4-hydroxy-4-methyl-2-pentanone, was released in greater quantities from MEAM1-damaged cabbage plants than from undamaged plants. Our results suggest that differences in herbivore-induced host volatile release may help explain the differences between the preference of B. tabaci MEAM1 and MED for cabbage as a host.

Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis).

There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions.

Effects of larval density on flight potential of the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae).

Tethered-flight techniques were used to investigate the flight potential of 1-d-old adult beet webworm, Loxostege sticticalis L. (Lepidoptera: Pyralidae), reared at densities of1, 10, 20, 30, and 40 larvae per 650-ml jar. Larval density had a significant effect on the flight potential, including total flight duration, distance, average velocity, and longest flight duration. Adults reared at a density of 10 larvae per jar, and those reared in isolation displayed the greatest and poorest flight capacity,respectively, relative to the other density treatments. Larval density also significantly affected length of the preoviposition period. Females with longer preoviposition period usually showed greater flight potential, and preoviposition period was positively correlated with flight potential. Body weight, water content, and triglyceride content of the moths were significantly affected by larval density, whereas glycogen content was not. Triglyceride content of adults reared at the different larval densities corresponded to the observed differences inflight potential, whereas water content and body weight were not related to their flight potential. These results suggest that larval density exerts a significant influence on preoviposition period and triglyceride content, which in turn influence flight potential of adult L. sticticalis.