Enhancement of Bacillus thuringiensis toxicity by feeding Spodoptera littoralis larvae with bacteria expressing immune suppressive dsRNA

RNAi interference (RNAi) for insect pest control is often used to silence genes controlling vital functions, thus generating lethal phenotypes. Here, we propose a novel approach, based on the knockout of an immune gene by dsRNA-expressing bacteria as a strategy to enhance the impact of spray applications of the entomopathogen Bacillus thuringiensis ( Bt). The target gene, Sl 102, controls the encapsulation and nodulation responses in the noctuid moth Spodoptera littoralis (Lepidop- tera, Noctuidae). To deliver Sl 102 dsRNA, we have developed a bacterial expression system, using HT115 Escherichia coli. This allows a much cheaper production of dsRNA and its protection against degradation. Transformed bacteria (dsRNA- Bac) administered through artificial diet proved to be more effective than dsRNA synthesized in vitro, both in terms of gene silencing and immunosuppression. This is a likely consequence of reduced dsRNA environmental degradation and of its protected release in the harsh conditions of the gut. The combined oral administration with artificial diet of dsRNA-Bac and of a Bt-based biopesticide (Xentari™) resulted in a remarkable enhancement of Bt killing activity, both on 4th and 5th instar larvae of S. littoralis, either when the two components were simultaneously administered or when gene silencing was obtained before Bt exposure. These results pave the way toward the development of novel Bt spray formulations containing killed dsRNA-Bac, which synergize Bt toxins by suppressing the insect immune response. This strategy will preserve the long-term efficacy of Bt-based products and can, in principle, enhance the ecological services provided by insect natural antagonists

Targeting the Diuretic Hormone Receptor to Control the Cotton Leafworm,<i>Spodoptera littoralis</i>

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Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agriculture for their ability to protect crops from the attack of pathogenic fungi and for plant growth promotion activity. Recently, it has been shown that they may also have complementary properties that enhance plant defense barriers against insects. However, the use of these fungi is somewhat undermined by their variable level of biocontrol activity, which is influenced by environmental conditions. Understanding the source of this variability is essential for its profitable and wide use in plant protection. Here, we focus on the impact of temperature on Trichoderma afroharzianum T22, Trichoderma atroviride P1, and the defense response induced in tomato by insects. The in vitro development of these two strains was differentially influenced by temperature, and the observed pattern was consistent with temperature-dependent levels of resistance induced by them in tomato plants against the aphid, Macrosiphum euphorbiae, and the noctuid moth, Spodoptera littoralis. Tomato plants treated with T. afroharzianum T22 exhibited enhanced resistance toward both insect pests at 25°C, while T. atroviride P1 proved to be more effective at 20°C. The comparison of plant transcriptomic profiles generated by the two Trichoderma species allowed the identification of specific defense genes involved in the observed response, and a selected group was used to assess, by real-time quantitative reverse transcription PCR (qRT-PCR), the differential gene expression in Trichoderma-treated tomato plants subjected to the two temperature regimens that significantly affected fungal biological performance. These results will help pave the way toward a rational selection of the most suitable Trichoderma isolates for field applications, in order to best face the challenges imposed by local environmental conditions and by extreme climatic shifts due to global warming

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel&nbsp;role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems

Variability in genes regulating vitamin D metabolism is associated with vitamin D levels in type 2 diabetes

Mortality rate is increased in type 2 diabetes (T2D). Low vitamin D levels are associated with increased mortality risk in T2D. In the general population, genetic variants affecting vitamin D metabolism (DHCR7 rs12785878, CYP2R1 rs10741657, GC rs4588) have been associated with serum vitamin D. We studied the association of these variants with serum vitamin D in 2163 patients with T2D from the "Sapienza University Mortality and Morbidity Event Rate (SUMMER) study in diabetes". Measurements of serum vitamin D were centralised. Genotypes were obtained by Eco™ Real-Time PCR. Data were adjusted for gender, age, BMI, HbA1c, T2D therapy and sampling season. DHCR7 rs12785878 (p = 1 x 10-4) and GC rs4588 (p = 1 x 10-6) but not CYP2R1 rs10741657 (p = 0.31) were significantly associated with vitamin D levels. One unit of a weighted genotype risk score (GRS) was strongly associated with vitamin D levels (p = 1.1 x 10-11) and insufficiency (&lt;30 ng/ml) (OR, 95%CI = 1.28, 1.16-1.41, p = 1.1 x 10-7). In conclusion, DHCR7 rs12785878 and GC rs4588, but not CYP2R1 rs10741657, are significantly associated with vitamin D levels. When the 3 variants were considered together as GRS, a strong association with vitamin D levels and vitamin D insufficiency was observed, thus providing robust evidence that genes involved in vitamin D metabolism modulate serum vitamin D in T2D

Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agriculture for their ability to protect crops from the attack of pathogenic fungi and for plant growth promotion activity. Recently, it has been shown that they may also have complementary properties that enhance plant defense barriers against insects. However, the use of these fungi is somewhat undermined by their variable level of biocontrol activity, which is influenced by environmental conditions. Understanding the source of this variability is essential for its profitable and wide use in plant protection. Here, we focus on the impact of temperature on Trichoderma afroharzianum T22, Trichoderma atroviride P1, and the defense response induced in tomato by insects. The in vitro development of these two strains was differentially influenced by temperature, and the observed pattern was consistent with temperature-dependent levels of resistance induced by them in tomato plants against the aphid, Macrosiphum euphorbiae, and the noctuid moth, Spodoptera littoralis. Tomato plants treated with T. afroharzianum T22 exhibited enhanced resistance toward both insect pests at 25°C, while T. atroviride P1 proved to be more effective at 20°C. The comparison of plant transcriptomic profiles generated by the two Trichoderma species allowed the identification of specific defense genes involved in the observed response, and a selected group was used to assess, by real-time quantitative reverse transcription PCR (qRT-PCR), the differential gene expression in Trichoderma-treated tomato plants subjected to the two temperature regimens that significantly affected fungal biological performance. These results will help pave the way toward a rational selection of the most suitable Trichoderma isolates for field applications, in order to best face the challenges imposed by local environmental conditions and by extreme climatic shifts due to global warming

Off-label long acting injectable antipsychotics in real-world clinical practice: a cross-sectional analysis of prescriptive patterns from the STAR Network DEPOT study

Introduction Information on the off-label use of Long-Acting Injectable (LAI) antipsychotics in the real world is lacking. In this study, we aimed to identify the sociodemographic and clinical features of patients treated with on- vs off-label LAIs and predictors of off-label First- or Second-Generation Antipsychotic (FGA vs. SGA) LAI choice in everyday clinical practice. Method In a naturalistic national cohort of 449 patients who initiated LAI treatment in the STAR Network Depot Study, two groups were identified based on off- or on-label prescriptions. A multivariate logistic regression analysis was used to test several clinically relevant variables and identify those associated with the choice of FGA vs SGA prescription in the off-label group. Results SGA LAIs were more commonly prescribed in everyday practice, without significant differences in their on- and off-label use. Approximately 1 in 4 patients received an off-label prescription. In the off-label group, the most frequent diagnoses were bipolar disorder (67.5%) or any personality disorder (23.7%). FGA vs SGA LAI choice was significantly associated with BPRS thought disorder (OR = 1.22, CI95% 1.04 to 1.43, p = 0.015) and hostility/suspiciousness (OR = 0.83, CI95% 0.71 to 0.97, p = 0.017) dimensions. The likelihood of receiving an SGA LAI grew steadily with the increase of the BPRS thought disturbance score. Conversely, a preference towards prescribing an FGA was observed with higher scores at the BPRS hostility/suspiciousness subscale. Conclusion Our study is the first to identify predictors of FGA vs SGA choice in patients treated with off-label LAI antipsychotics. Demographic characteristics, i.e. age, sex, and substance/alcohol use co-morbidities did not appear to influence the choice towards FGAs or SGAs. Despite a lack of evidence, clinicians tend to favour FGA over SGA LAIs in bipolar or personality disorder patients with relevant hostility. Further research is needed to evaluate treatment adherence and clinical effectiveness of these prescriptive patterns

The Role of Attitudes Toward Medication and Treatment Adherence in the Clinical Response to LAIs: Findings From the STAR Network Depot Study

Background: Long-acting injectable (LAI) antipsychotics are efficacious in managing psychotic symptoms in people affected by severe mental disorders, such as schizophrenia and bipolar disorder. The present study aimed to investigate whether attitude toward treatment and treatment adherence represent predictors of symptoms changes over time. Methods: The STAR Network \u201cDepot Study\u201d was a naturalistic, multicenter, observational, prospective study that enrolled people initiating a LAI without restrictions on diagnosis, clinical severity or setting. Participants from 32 Italian centers were assessed at three time points: baseline, 6-month, and 12-month follow-up. Psychopathological symptoms, attitude toward medication and treatment adherence were measured using the Brief Psychiatric Rating Scale (BPRS), the Drug Attitude Inventory (DAI-10) and the Kemp's 7-point scale, respectively. Linear mixed-effects models were used to evaluate whether attitude toward medication and treatment adherence independently predicted symptoms changes over time. Analyses were conducted on the overall sample and then stratified according to the baseline severity (BPRS &lt; 41 or BPRS 65 41). Results: We included 461 participants of which 276 were males. The majority of participants had received a primary diagnosis of a schizophrenia spectrum disorder (71.80%) and initiated a treatment with a second-generation LAI (69.63%). BPRS, DAI-10, and Kemp's scale scores improved over time. Six linear regressions\u2014conducted considering the outcome and predictors at baseline, 6-month, and 12-month follow-up independently\u2014showed that both DAI-10 and Kemp's scale negatively associated with BPRS scores at the three considered time points. Linear mixed-effects models conducted on the overall sample did not show any significant association between attitude toward medication or treatment adherence and changes in psychiatric symptoms over time. However, after stratification according to baseline severity, we found that both DAI-10 and Kemp's scale negatively predicted changes in BPRS scores at 12-month follow-up regardless of baseline severity. The association at 6-month follow-up was confirmed only in the group with moderate or severe symptoms at baseline. Conclusion: Our findings corroborate the importance of improving the quality of relationship between clinicians and patients. Shared decision making and thorough discussions about benefits and side effects may improve the outcome in patients with severe mental disorders

Uso di RNAi per il controllo degli insetti

La presente tesi di dottorato contribuisce allo sviluppo di nuove strategie di controllo degli insetti basate sull'utilizzo di RNA interference (RNAi) per silenziare geni che giocano un ruolo importante nella omeostasi fisiologica e nella riproduzione. Una delle questioni più importanti per l'uso dell’RNAi è la selezione di geni bersaglio il cui silenziamento, idealmente, dovrebbe generare fenotipi letali e mostrare limitati effetti off-target. In questo lavoro abbiamo affrontato questo problema selezionando geni che sono il target di strategie di virulenza adottate dagli imenotteri parassitoidi per sottomettere i propri ospiti, cercando di imitare i meccanismi generati dalla lunga co-evoluzione di queste associazioni antagonistiche. La prima parte del mio lavoro di dottorato è stata dedicata all'analisi funzionale di una proteina codificata da un gene (102) della larva di Heliothis virescens (F.). Questo gene è stato ritenuto un buon candidato per le strategie di silenziamento, in quanto i dati già disponibili, ottenuti nei nostri laboratori, sembrano indicare la sua sotto-regolazione in larve parassitizzate dal braconide endofago Toxoneuron nigriceps (Viereck), attraverso un meccanismo mediato da un RNA non codificante (rc5'ntTnBV) complementare al 5'UTR. Esperimenti condotti in vitro e in vivo hanno dimostrato che il gene 102 ha un ruolo chiave nella risposta immunitaria dell’insetto, infatti esso risulta coinvolto nella localizzazione del processo di melanizzazione della capsula emocitaria intorno agli oggetti estranei ed è essenziale per la formazione della capsula stessa. Questo processo è mediato dalla produzione di fibre amiloidi, a partire da frammenti della P102, che sono raccolte in grandi cisterne del reticolo endoplasmatico rugoso di granulociti e sferulociti. A seguito di stimolo immunitario, le fibre amiloidi sono rilasciate sulla superficie del corpo estraneo, dove formano uno strato che funge da impalcatura molecolare, che promuove la sintesi localizzata della melanina e l’incapsulamento. Questi risultati dimostrano, per la prima volta, che le fibre amiloidi hanno un ruolo funzionale chiave nell'immunità degli insetti, e sono stati già pubblicati in un lavoro frutto della collaborazione tra gruppi di lavoro appartenenti a diversi laboratori (Falabella et al., 2012). Per dimostrare che il trascritto non codificante rc5'ntTnBV è in grado, potenzialmente, di sopprimere attivamente la risposta immunitaria di H. virescens silenziando l'espressione genica 102 attraverso un meccanismo RNAi, abbiamo trattato le larve del nottuide con dsRNA diretto contro questo gene. Microiniezione orali di dsRNA102 in larve mature di H. virescens (ma non iniezioni emoceliche) hanno inibito l'incapsulamento delle sferette (beads) cromatografiche iniettate, provocando, in seguito a stimolo immunitario, la melanizzazione sistemica e quindi la morte. La Real Time PCR quantitativa (qRT-PCR) ha dimostrato che questo fenotipo immunodepresso è associato ad una significativa sotto-regolazione trascrizionale del gene 102. Questi cambiamenti sono perfettamente in linea con i dati funzionali raccolti per la P102, e indicano che il silenziamento di questo gene ha un forte impatto sulla risposta immunitaria di H. virescens. Il fatto che sequenze nucleotidiche correlate al gene 102 siano presenti in diverse specie di insetti, suggerisce l’esistenza di geni omologhi dal punto di vista funzionale. L’analisi delle librerie ESTs disponibili nei database pubblici ha permesso l'identificazione dell’omologo 102 in Spodoptera littoralis (Boisduval) (102Sl). Il ruolo di questo gene nella risposta immunitaria è stato valutato utilizzando il silenziamento mediato da RNAi. Microiniezioni orali di dsRNA 102Sl nelle larve ospiti mature hanno causato una notevole inibizione dell’incapsulamento delle beads, promuovendone solo una limitata melanizzazione superficiale. Tuttavia, diversamente da quanto osservato nelle larve di H. virescens, non si è verificata la melanizzazione sistemica, e larve trattate sono sopravvissute regolarmente. Il fenotipo immunosoppresso è associato a livelli significativi di silenziamento del gene 102Sl, confermandone il ruolo immunitario osservato anche per H. virescens. In realtà, la melanogenesi e la deposizione localizzata della melanina nelle due specie di lepidottero prese in considerazione appaiono controllate da meccanismi diversi. La comprensione di questo fenomeno richiede, pertanto, ulteriori studi. Queste osservazioni hanno stimolato l'idea di perseguire strategie per il controllo di S. littoralis, basato sul silenziamento del gene 102Sl mediato da RNAi. Inoltre, questo gene, così come il gene 102, è anche presumibilmente coinvolto nella formazione delle membrane basali degli epiteli da parte degli emociti (Falabella et al., 2012 e riferimenti ivi citati), e quindi l'interruzione della sua espressione potrebbe potenzialmente influenzare sia l'immunità, sia lo sviluppo dell’insetto. Per verificare questa ipotesi, abbiamo prima silenziato il gene 102 in uova di S. littoralis, per vedere se lo sviluppo embrionale fosse, in qualche modo, influenzato. Il silenziamento è stato eseguito mediante l’immersione delle uova in una soluzione contenente dsRNA102Sl, e questo ha portato ad un significativo aumento della mortalità delle uova e delle larve neonate, associato ad una sotto-regolazione trascrizionale del gene bersaglio. Per poter sfruttare la compromissione della funzione immunitaria indotta dal silenziamento del gene 102Sl, abbiamo valutato il possibile effetto del trattamento con dsRNA102Sl sull'attività insetticida di Bacillus thuringiensis. È stato dimostrato che l’immunosoppressione innescata da questo trattamento ha significativamente migliorato il tasso di mortalità di stadi di S. littoralis , come le larve mature, che sono poco sensibili all'esposizione di Bt. Questo esperimento fornisce la prova pratica che l'approccio proposto, basato sull’RNAi, può migliorare l’attività degli agenti di controllo biologico. Collettivamente, questi dati sperimentali indicano che il gene 102 è un bersaglio promettente per tecnologie di controllo di insetti dannosi basate sull’RNAi

Trade and Infrastructure in the Belt and Road Initiative: A Gravity Analysis Based on Revealed Trade Preferences

This paper aims at investigating whether and how the intensity of trade between a pair of countries changes when they experience improvements in their infrastructural systems. We carry out our analysis considering countries participating in the Belt and Road Initiative (BRI), a project specifically designed to promote infrastructural connectivity and therefore boost trade among the countries involved. Our empirical strategy relies on a particular specification of the gravity model, in which the dependent variable consists in an index of revealed trade preferences, calculated by comparing the actual value of trade flows between two countries with their expected value, proportional to the two countries’ total trade. Such methodology allows us to estimate bilateral trade intensity without resorting to the traditional “size” variables of the gravity model, taking the entire network of multilateral trade into account. We then study the possible impact of an improvement in infrastructure on a ‘gravity-adjusted’ measure of trade preferences, given by the residuals of our first estimations. Our results indicate that bilateral preferences among BRI countries will intensify inasmuch as they succeed in coordinating their infrastructural projects