Aquibaculum sediminis sp. nov., a halotolerant bacteria isolated from salt lake sediment.

An aerobic, Gram-stain negative bacterium was isolated from sediment samples of Barkol salt lake in Hami City, Xinjiang Uygur Autonomous Region, China, with the number EGI_FJ10229T. The strain is ellipse-shaped, oxidase-negative, catalase-positive, and has white, round, smooth, opaque colonies on marine 2216 E agar plate. Growth occurs at 4.0-37.0 â„ƒ (optimal:30.0 â„ƒ), pH 7.0-9.0 (optimal: pH 8.0) and NaCl concentration of 0-8.0% (optimal: 3.0%). Phylogenetic analysis based on 16S rRNA gene and genome sequences indicated that the isolated strain should be assigned to the genus Aquibaculum and was most closely related to Aquibaculum arenosum CAU 1616 T. Average nucleotide identity (ANI) and Average amino-acid identity (AAI) values between the type species of the genus Aquibaculum and other related type species were lower than the threshold values recommended for bacterial species. The genomic DNA G + C content of EGI_FJ10229T was 65.41%. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, phosphatidylethanolamine and unidentified phospholipid. The major fatty acids (> 5%) were C19:0 cyclo ω8c (42.0%) and C18:1 ω7c (33.78%). The respiratory quinone identified was Q-10. Differential phenotypic and genotypic characteristics of this strain and species of genus Aquibaculum showed that the strain should be classified as representing a new species belonging to this genus, for which the name Aquibaculum sediminis sp. nov. is proposed. The type strain of the proposed novel species is EGI_FJ10229T (= KCTC 8570 T = GDMCC 1.4598 T).

The irregular developmental duration mainly caused by the broad-complex in Chilo suppressalis.

Chilo suppressalis, a critical rice stem borer pest, poses significant challenges to rice production due to its overlapping generations and irregular developmental duration. These characteristics complicate pest management strategies. According to the dynamic analysis of the overwintering adults of C. suppressalis in fields, it indicates that the phenomenon of irregular development of C. suppressalis exists widely and continuously. This study delves into the potential role of the Broad-Complex (Br-C) gene in the developmental duration of C. suppressalis. Four isoforms of Br-C, named CsBr-C Z1, CsBr-C Z2, CsBr-C Z4, and CsBr-C Z7, were identified. After CsBr-Cs RNAi, the duration of larva development spans extended obviously. And, the average developmental duration of dsCsBr-Cs feeding individuals increased obviously. Meanwhile, the average developmental duration of the dsCsBr-C Z2 feeding group was the longest among all the RNAi groups. After dsCsBr-Cs feeding continuously, individuals pupated at different instars changed obviously: the proportion of individuals pupated at the 5th instar decreased and pupated at the 7th instar or higher increased significantly. Moreover, the pupation rate of dsCsBr-Cs (except dsCsBr-C Z7) were significantly lower than that of dsGFP. The same results were obtained from the mutagenesis in CsBr-C genes mediated by CRISPR/Cas9. The average developmental duration of CsBr-Cs knockout individuals was significantly prolonged. And, the instar of pupation in knockout individuals was also delayed significantly. In conclusion, this work showed that CsBr-Cs played a crucial role in pupal commitment and affected the developmental duration of C. suppressalis significantly.

[Analysis on Spatial Variability of Hydrochemical Characteristics and Control Factors of Jinan Baotu Spring Area].

To understand the karst groundwater hydrochemical characteristics and ion sources of the Jinan Baotu Spring area, this study focused on the three functional zones as an indirect recharge area, direct recharge area, and discharge area. Through water sample collection and testing, the spatial variability of groundwater chemical characteristics in different functional zones and the formation mechanism were analyzed using hydrochemistry parameter statistics, multivariate statistics, self-organizing map, hydrochemistry graphical analysis, ion ratios, and other methods, guided by the theory of groundwater flow system and combined with regional physical geography and hydrogeological conditions. The results showed that： the groundwater of each functional zone was alkaline as is typical in the dissolution of carbonate minerals. Owing to the different groundwater runoff pathways, the variability of water chemistry parameters in different functional areas was obvious. The groundwater of the discharge area was recharged by both the direct recharge area and the indirect recharge area. The hydrochemistry type changed from HCO3-Ca type to HCO3·SO4-Ca type through the indirect recharge area to the discharge area. The presence of a small amount of gypsum dissolution within the aquifer generated Ca2+ and SO42-. The ions in groundwater mainly came from the dissolution and filtration of aquifer rock minerals, and at the same time, they were affected by cation exchange, mineral dissolution equilibrium, and the combined effects of human activities. The groundwater in the Baotu Spring area was greatly influenced by human activities, which to some extent affected the evolution of groundwater hydrochemistry in the spring area.

Reversed oxidative TCA (roTCA) for carbon fixation by an Acidimicrobiia strain from a saline lake.

Acidimicrobiia are widely distributed in nature and suggested to be autotrophic via the Calvin-Benson-Bassham (CBB) cycle. However, direct evidence of chemolithoautotrophy in Acidimicrobiia is lacking. Here, we report a chemolithoautotrophic enrichment from a saline lake, and the subsequent isolation and characterization of a chemolithoautotroph, Salinilacustristhrix flava EGI L10123T, which belongs to a new Acidimicrobiia family. Although strain EGI L10123T is autotrophic, neither its genome nor Acidimicrobiia metagenome-assembled genomes (MAGs) from the enrichment culture encode genes necessary for the CBB cycle. Instead, genomic, transcriptomic, enzymatic, and stable-isotope probing data hinted at the activity of the reversed oxidative TCA (roTCA) coupled with the oxidation of sulfide as the electron donor. Phylogenetic analysis and ancestral character reconstructions of Acidimicrobiia suggested that the essential CBB gene rbcL was acquired through multiple horizontal gene transfer events from diverse microbial taxa. In contrast, genes responsible for sulfide- or hydrogen-dependent roTCA carbon fixation were already present in the last common ancestor of extant Acidimicrobiia. These findings imply the possibility of roTCA carbon fixation in Acidimicrobiia and the ecological importance of Acidimicrobiia. Further research in the future is necessary to confirm whether these characteristics are truly widespread across the clade.

Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Comparison of the efficacy of steroid-free versus classic steroid-containing regimens in primary membranous nephropathy.

Objective: To compare the efficacy of a steroid-free regimen with steroid-based treatment in managing primary membranous nephropathy (PMN) and investigate the potential benefits of steroid-free regimens in PMN therapy. Methods: This was a single-centre prospective cohort study. A total of 81 patients were divided into two groups according to their medication regimen: a rituximab (RTX)/tacrolimus (TAC) group (low-dose RTX combined with low-dose TAC group, without steroids, n = 31) and a prednisone (P)/TAC group (P combined with TAC group, n = 61). The changes in 24-h urine protein quantification, levels of blood albumin, blood creatinine, total cholesterol, triglyceride and fasting blood glucose as well as anti-phospholipase A2 receptor antibody titres were observed in both groups before treatment and after 1, 3, 6 and 12 months of treatment. Clinical remission (complete and partial remission), serological remission and recurrence were assessed in both groups after treatment, and the occurrence of adverse reactions was observed. Results: 1) Before treatment, there was no significant difference in baseline values between the two groups (p > 0.05). 2) After 12 months of treatment, the 24-h proteinuria and total cholesterol levels in the RTX/TAC group were significantly lower than those in the P/TAC group (p < 0.05). 3) After 6 months of treatment, the clinical remission rate of the RTX/TAC group was significantly higher than that of the P/TAC group (p < 0.05). After 12 months of treatment, the clinical remission rate of the RTX/TAC group was significantly higher than that of the P/TAC group (p < 0.05). (4) After 3, 6 and 12 months of treatment, serological remission rates in the RTX/TAC group were significantly higher than those in the P/TAC group (p < 0.05). During treatment, the anti-PLA2R antibody titres in the RTX/TAC group remained lower than those in the P/TAC group (p < 0.05). Conclusion: The low-dose RTX combined with low-dose TAC steroid-free regimen induces serological remission in patients with PMN earlier than the classic regimen of P combined with TAC, and there was no significant difference in adverse effects between the two groups. Besides, the long-term clinical remission effect of low-dose RTX combined with low-dose TAC is better than that of P combined with TAC.

Palmitic acid combined with γ-interferon inhibits gastric cancer progression by modulating tumor-associated macrophages' polarization via the TLR4 pathway.

BACKGROUND: Tumor-associated macrophages (TAMs) constitute the main infiltrating immune cells in the solid tumor microenvironment. Amounting studies have analyzed the antitumor effect on immune response induced by Toll-like receptor (TLR) agonists, such as lipopolysaccharide (LPS), γ-interferon (γ-IFN), and palmitic Acid (PA). However, their combined treatment for gastric cancer (GC) has not been illuminated. METHODS: We investigated the relevance of macrophage polarization and the effect of PA and γ-IFN in GC in vitro and in vivo. M1 and M2 macrophage-associated markers were measured by real-time quantitative PCR and flow cytometry, and the activation level of the TLR4 signaling pathways was evaluated by western blot analysis. The effect of PA and γ-IFN on the proliferation, migration, and invasion of GC cells (GCCs) was evaluated by Cell-Counting Kit-8, transwell assays, and wound-healing assays. In vivo animal models were used to verify the effect of PA and γ-IFN on tumor progression, and the M1 and M2 macrophage markers, CD8 + T lymphocytes, regulatory T cells (Treg) cells, and the myeloid-derived suppressor cells (MDSCs) in tumor tissues were analyzed by flow cytometry and immunohistochemical (IHC). RESULTS: The results showed that this combination strategy enhanced M1-like macrophages and diminished M2-like macrophages through the TLR4 signaling pathway in vitro. In addition, the combination strategy impairs the proliferative and migratory activity of GCC in vitro and in vivo. While, the antitumor effect was abolished using the TAK-424 (a specific TLR-4 signaling pathway inhibitor) in vitro. CONCLUSIONS: The combined treatment of PA and γ-IFN inhibited GC progression by modulating macrophages polarization via the TLR4 pathway.

Changes and significance of Treg and Th17 in adult patients with primary membranous nephropathy.

OBJECTIVE: To investigate the changes in Treg and Th17 cells and explore the significance of Treg/Th17 balance in adult primary membranous nephropathy (PMN) patients. MATERIALS AND METHODS: A total of 60 PMN patients and 50 healthy adults from June 2013 to October 2016 were enrolled in this study. The levels of Treg, Th17, and related cytokines were assessed. Pearson correlation was used for conducting correlation analysis. RESULTS: There was a significant increase in Th17 frequencies and IL-17 (Th17-related cytokines) in the peripheral blood mononuclear cells (PBMCs), as well as a significant decrease in Treg frequencies and IL-10 (Treg-related cytokines). The IL-17 concentrations in the peripheral blood of PMN patients were positively correlated with urinary protein, while IL-10 levels were negatively correlated with urinary protein. Protein expression of Treg transcription factor (Foxp3) was significantly low in the renal tissues of PMN patients, while the expression of IL-17 was much higher. Th17/Treg imbalance was reversed to normal after effective treatment with tacrolimus in 15 PMN patients. CONCLUSION: These results suggested the existence of Treg/Th17 imbalance in PMN patients, showing the importance of Treg/Th17 imbalance in PMN pathogenesis.

Two Splice Isoforms of Leptinotarsa Ecdysis Triggering Hormone Receptor Have Distinct Roles in Larva-Pupa Transition.

Insect ecdysis triggering hormone (ETH) receptors (ETHRs) are rhodopsin-like G protein-coupled receptors. Upon binding its ligand ETH, ETHR initiates a precisely programed ecdysis behavior series and physiological events. In Drosophila melanogaster, the ethr gene produces two functionally distinct splicing isoforms, ethra and ethrb. ETH/ETHRA activates eclosion hormone (EH), kinin, crustacean cardioactive peptide (CCAP), and bursicon (burs and pburs) neurons, among others, in a rigid order, to elicit the behavioral sequences and physiological actions for ecdysis at all developmental stages, whereas ETH/ETHRB is required at both pupal and adult ecdysis. However, the role of ETHRB in regulation of molting has not been clarified in any non-drosophila insects. In the present paper, we found that 20-hydroxyecdysone (20E) signaling triggers the expression of both ethra and ethrb in a Coleopteran insect pest, the Colorado potato beetle Leptinotarsa decemlineata. RNA interference (RNAi) was performed using double-stranded RNAs (dsRNAs) targeting the common (dsethr) or isoform-specific (dsethra, dsethrb) regions of ethr. RNAi of dsethr, dsethra, or dsethrb by the final-instar larvae arrested larva development. The arrest was not rescued by feeding 20E. All the ethra depleted larvae stopped development at prepupae stage; the body cavity was expanded by a large amount of liquid. Comparably, more than 80% of the ethrb RNAi larvae developmentally halted at the prepupae stage. The remaining Ldethrb hypomorphs became pupae, with blackened wings and highly-expressed burs, pburs and four melanin biosynthesis genes. Therefore, ETHRA and ETHRB play isoform-specific roles in regulation of ecdysis during larva-pupa transition in L. decemlineata.

Involvement of Leptinotarsa hormone receptor 38 in the larval-pupal transition.

In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata, the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP, LdE75, LdE74, LdE93, LdBroad and LdHR3, whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata.

Epigenetic therapies in acute myeloid leukemia: the role of hypomethylating agents, histone deacetylase inhibitors and the combination of hypomethylating agents with histone deacetylase inhibitors.

Epigenetic regulation includes changes of DNA methylation and modifications of histone proteins and is essential for normal physiologic functions, especially for controlling gene expression. Epigenetic dysregulation plays a key role in disease pathogenesis and progression of some malignancies, including acute myeloid leukemia (AML). Epigenetic therapies, including hypomethylating agents (HMAs) and histone deacetylase (HDAC) inhibitors, were developed to reprogram the epigenetic abnormalities in AML. However, the molecular mechanisms and therapeutic effects of the two agents alone or their combination remain unknown. An overview of these epigenetic therapies is given here. A literature search was conducted through PubMed database, looking for important biological or clinical studies related to the epigenetic regimens in the treatment of AML until October 15th, 2019. Various types of articles, including original research and reviews, were assessed, identified, and eventually summarized as a collection of data pertaining the mechanisms and clinical effects of HMAs and HDAC inhibitors in AML patients. We provided here an overview of the current understanding of the mechanisms and clinical therapeutic effects involved in the treatment with HMAs and HDAC inhibitors alone, the combination of epigenetic therapies with intensive chemotherapy, and the combination of both types of epigenetic therapies. Relevant clinical trials were also discussed. Generally speaking, the large number of studies and their varied outcomes demonstrate that effects of epigenetic therapies are heterogeneous, and that HMAs combination regimens probably contribute to significant response rates. However, more research is needed to explore therapeutic effects of HDAC inhibitors and various combinations of HMAs and HDAC inhibitors.

Ecdysone receptor isoforms play distinct roles in larval-pupal-adult transition in Leptinotarsa decemlineata.

A heterodimer of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle, mediates 20-hydroxyecdysone (20E) signaling to modulate many aspects in insect life, such as molting and metamorphosis, reproduction, diapause and innate immunity. In the present paper, we intended to determine the isoform-specific roles of EcR during larval-pupal-adult transition in the Colorado potato beetle. Double-stranded RNAs (dsRNAs) were prepared using the common (dsEcR) or isoform-specific (dsEcRA, dsEcRB1) regions of EcR as templates. Ingestion of either dsEcR or dsEcRA, rather than dsEcRB1, by the penultimate (3rd) and final (4th) instar larvae caused failure of larval-pupal and pupal-adult ecdysis. The RNA interference (RNAi) larvae remained as prepupae, or became deformed pupae and adults. Determination of messenger RNA (mRNA) levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1. Moreover, silencing the two EcR transcripts, LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad, and lowered 20E titer. In contrast, the expression levels of HR3, HR4, E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae, but not in LdEcRB1 depleted specimens. Dietary supplement with 20E did not restore the expression of five 20E signaling genes (USP, HR3, HR4, E74 and E75), and only partially alleviated the pupation defects in dsEcR- or dsEcRA-fed beetles. These data suggest that EcR plays isoform-specific roles in the regulation of ecdysteroidogenesis and the transduction of 20E signal in L. decemlineata.

A new O-cinnamoyl threonine derivative from gene adpA overexpression strain Streptomyces sp. HS-NF-1222A.

A new O-cinnamoyl threonine derivative, O-(2-(3-methyloxiranyl) cinnamoyl) threonine (1), was isolated from the gene adpA overexpression strain Streptomyces sp. HS-NF-1222A. The structure of 1 was determined based on HRESIMS and extensive NMR analysis.

Silencing Taiman impairs larval development in Leptinotarsa decemlineata.

An exploration of novel control strategies for Leptinotarsa decemlineata is becoming more pressing given rapid evolution of insecticide resistance and rise of production loss of potato. Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) is a promising alternative for management. An important first step is to uncover possible RNA-interference (RNAi)-target genes effective against both young and old larvae. Taiman (Tai) is a basic-helix-loop-helix/Per-Arnt-Sim transcription factor that is involved in the mediation of both juvenile hormone (JH) and 20-hydroxyecdysone (20E) signaling. In the present paper, we found that continuous ingestion of dsTai for three days by third (penultimate)-instar larvae caused approximately 20% larval mortality and 80% pupation failure. The larval lethality resulted from failed cuticle and tracheae shedding, which subsequently reduced foliage consumption and nutrient absorption, and depleted lipid stores. In contrast, pupation failure derived from disturbed JH and 20E signals, and disordered nutrient homeostasis including, among others, inhibition of trehalose metabolism and reduction of chitin content. Knockdown of LdTai caused similar larval lethality and pupation impairment in second and fourth (final) larval instars. Therefore, LdTai is among the most attractive candidate genes for RNAi to control L. decemlineata larvae.

Importance of Taiman in Larval-Pupal Transition in Leptinotarsa decemlineata.

Insect Taiman (Tai) binds to methoprene-tolerant to form a heterodimeric complex, mediating juvenile hormone (JH) signaling to regulate larval development and to prevent premature metamorphosis. Tai also acts as a steroid receptor coactivator of 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and Ultraspiracle (USP), to control the differentiation of early germline cells and the migration of specific follicle cells and border cells in ovaries in several insect species. In holometabolous insects, however, whether Tai functions as the coactivator of EcR/USP to transduce 20E message during larval-pupal transition is unknown. In the present paper, we found that the LdTai mRNA levels were positively correlated with circulating JH and 20E titers in Leptinotarsa decemlineata; and ingestion of either JH or 20E stimulated the transcription of LdTai. Moreover, RNA interference (RNAi)-aided knockdown of LdTai at the fourth (final) instar stage repressed both JH and 20E signals, inhibited larval growth and shortened larval developing period. The knockdown caused 100% larval lethality due to failure of larval-pupal ecdysis. Under the apolysed larval cuticle, the LdTai RNAi prepupae possessed pupal thorax. In contrast, the process of tracheal ecdysis was uncompleted. Neither JH nor 20E rescued the aforementioned defectives in LdTai RNAi larvae. It appears that Tai mediates both JH and 20E signaling. Our results uncover a link between JH and 20E pathways during metamorphosis in L. decemlineata.

Hormonal signaling cascades required for phototaxis switch in wandering Leptinotarsa decemlineata larvae.

Many animals exploit several niches sequentially during their life cycles, a fitness referred to as ontogenetic niche shift (ONS). To successfully accomplish ONS, transition between development stages is often coupled with changes in one or more primitive, instinctive behaviors. Yet, the underlining molecular mechanisms remain elusive. We show here that Leptinotarsa decemlineata larvae finish their ONS at the wandering stage by leaving the plant and pupating in soil. At middle wandering phase, larvae also switch their phototactic behavior, from photophilic at foraging period to photophobic. We find that enhancement of juvenile hormone (JH) signal delays the phototactic switch, and vise verse. Moreover, RNA interference (RNAi)-aided knockdown of LdPTTH (prothoracicotropic hormone gene) or LdTorso (PTTH receptor gene) impairs avoidance response to light, a phenotype nonrescuable by 20-hydroxyecdysone. Consequently, the RNAi beetles pupate at the soil surface or in shallow layer of soil, with most of them failing to construct pupation chambers. Furthermore, a combination of depletion of LdPTTH/LdTorso and disturbance of JH signal causes no additive effects on light avoidance response and pupation site selection. Finally, we establish that TrpA1 (transient receptor potential (TRP) cation channel) is necessary for light avoidance behavior, acting downstream of PTTH. We conclude that JH/PTTH cascade concomitantly regulates metamorphosis and the phototaxis switch, to drive ONS of the wandering beetles from plant into soil to start the immobile pupal stage.

RNA interference against the putative insulin receptor substrate gene chico affects metamorphosis in Leptinotarsa decemlineata.

It is noted that insect insulin/insulin-like growth factor/target of rapamycin signaling is critical for the regulation of metamorphosis in holometabolous insects. However, the molecular mechanism remains undetermined. Our previous findings reveal that RNA interference (RNAi)-mediated knockdown of an insulin gene (LdILP2) in Leptinotarsa decemlineata disturbs both 20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling, and impairs pupation. In the present paper, we further observed that the expression of the insulin receptor substrate gene chico (Ldchico) and the phosphoinositide-3-kinase gene pi3k (Ldpi3k92E) was repressed in LdILP2 depleted larvae. Moreover, RNAi of Ldchico or Ldpi3k92E decreased food consumption, affected absorption and metabolism of amino acids and sugars, and reduced expression of several 20E (LdEcR, LdHR3 and LdE75) and JH (LdJHAMT, LdKr-h1 and LdHairy) signaling genes. As a result, larval development was postponed and larval growth was inhibited. Intriguingly, knockdown of Ldchico, rather than Ldpi3k92E, impaired larval-pupal and pupal-adult ecdysis, and specifically repressed transcription of another 20E signaling gene LdUSP. Ingestion of 20E rescued the expression of LdEcR, LdHR3 and LdE75, whereas 20E feeding restored neither the decreased LdUSP mRNA level, nor the reduced pupation and adult emergence rates in Ldchico RNAi larvae. Therefore, Chico is critical for the regulation of larval-pupal-adult transition by a PI3K-independent pathway, perhaps through activation of USP in L. decemlineata.

Involvement of methoprene-tolerant (Met) in the determination of the final body size in Leptinotarsa decemlineata (Say) larvae.

In the tobacco hornworm Manduca sexta, juvenile hormone (JH) is critical for the control of species-specific size. However, whether the basic helix-loop-helix/Per-Arnt-Sim domain receptor methoprene-tolerant (Met) is involved remains unconfirmed. In the present paper, we found that RNA interference (RNAi)-aided knockdown of Met gene (LdMet) lowered the larval and pupal fresh weights and shortened the larval development period in the Colorado potato beetle Leptinotarsa decemlineata. Dietary introduction of JH into the LdMet RNAi larvae rescued neither the decreased weights nor the reduced development phase, even though JH ingestion by control larvae extended developmental time and caused large pupae. Moreover, the transcript levels of five genes involved in prothoracicotropic hormone and cap 'n' collar isoform C/Kelch-like ECH associated protein 1 pathways were upregulated in the LdMet silenced larvae. Ecdysteroidogenesis was thereby activated; 20-hydroxyecdysone (20E) titer was increased; and 20E signaling pathway was elicited in the LdMet RNAi larvae. Therefore, JH, acting through its receptor Met, inhibits PTTH production and release before the attainment of critical weight. Once the critical weight is reached, JH production and release are averted; and the hemolymph JH is removed. The elimination of JH allows the brain to release PTTH. PTTH subsequently stimulates ecdysteroid biosynthesis and release to start larval-pupal transition in L. decemlineata.

Leptinotarsa hormone receptor 4 (HR4) tunes ecdysteroidogenesis and mediates 20-hydroxyecdysone signaling during larval-pupal metamorphosis.

Hormone receptor 4 (HR4) is involved in the regulation of 20-hydroxyecdysone (20E) biosynthesis and the mediation of 20E signaling during larval-pupal transition in a holometabolan Drosophila melanogaster, whereas it acts as a repressor in 20E-responsive transcriptional cascade in a hemimetabolan, Blattella germanica. Here we characterized two HR4 splicing variants, LdHR4X1 and LdHR4X2, in a coleopteran Leptinotarsa decemlineata. LdHR4X1 was highly expressed in the prothoracic gland and epidermis while LdHR4X2 was abundantly transcribed in the nervous system. In vivo results showed that both prothoracicotropic hormone and 20E pathways transcriptionally regulated LdHR4, in an isoform-dependent pattern. RNA interference of LdHR4 at the final (fourth) larval instar, in contrast to the second- and third-instar periods, enhanced the expression of two ecdysteroidogenesis genes, increased 20E titer, upregulated transcription of five 20E-response genes, and reduced the mRNA level of Fushi tarazu-factor 1 (FTZ-F1). As a result, the fourth-instar LdHR4 RNAi larvae exhibited accelerated development and reduced body weight. Moreover, knockdown of LdHR4 at the fourth instar resulted in larval lethality and impaired pupation. Feeding of pyriproxyfen (a mimic of juvenile hormone) or silencing of a juvenile hormone degrading enzyme gene restored the normal course of ecdysteroidogenesis, duration of larval development, and body weight in fourth-instar LdHR4 RNAi larvae. The treatment partially suppressed the larval mortality but not the failure to pupate. The dual role of HR4 during larval-pupal metamorphosis appears to be evolutionarily conserved among holometabolans.

Novaluron ingestion causes larval lethality and inhibits chitin content in Leptinotarsa decemlineata fourth-instar larvae.

To accomplish consistent, long-term, integrated management (IPM) of the Colorado potato beetle, Leptinotarsa decemlineata (Say), research assessing the potential of novel, IPM-compatible insecticides is essential. Novaluron is a potent benzoylurea insecticide. In the present paper, we found that novaluron ingestion by the fourth-instar larvae inhibited foliage consumption, reduced larval fresh weight, and delayed development period, in a dose dependent manner. Most of the resulting larvae fail to pupate, and died at prepupae stage, with larvicidal activity comparable with those of cyhalothrin and spinosad but lower than those of fipronil and abamectin. Moreover, many surviving pupae that fed novaluron failed to emerge as adults, in a dose dependent pattern. Furthermore, feeding of novaluron significantly decreased chitin contents in body carcass (without midgut) and integument specimen, whereas the chitin concentration in the midgut peritrophic matrix was not affected. Furthermore, uridine diphosphate-N-acetylglucosamine-pyrophosphorylase gene (LdUAP1) and chitin synthase Aa (LdChSAa), which were mainly responsible for chitin biosynthesis in ectodermally-derived tissues, were surpressed and activated respectively after novaluron ingestion. Therefore, novaluron is an effective benzoylurea insecticide to L. decemlineata fourth-instar larvae. It inhibited chitin biosynthesis in ectodermally-derived tissues, disrupted ecdysis, impaired pupation and adult emergence, and led to death in juvenile life stages.