TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10–7 mol L−1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70–80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL−1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

Silicon fertilisation affects morphological and immune defences of an insect pest and enhances plant compensatory growth

Herbivorous insects have evolved various anti-predator defences, including morphological, behavioural, and immune defences, which can make biocontrol of herbivorous pests challenging. Silicon (Si) accumulation in plants is a potent physical defence against mandibulate insects. However, it remains uncertain how Si affects the anti-predator defences of insect herbivores and plant defences following herbivory. We grew the model grass, Brachypodium distachyon, hydroponically with (+Si) or without (–Si) Si and investigated the plant-mediated effects of Si on the anti-predator defences of the cotton bollworm, Helicoverpa armigera, integrating morphological (i.e. integument resistance and thickness), behavioural, and immune defences. We also examined the effects of Si on plant compensatory growth and leaf trichome production. Larval growth, leaf consumption, and integument resistance were lower when feeding on +Si plants compared to when feeding on –Si plants. Larval integument thickness, defensive behaviours, haemocyte density, and lysozyme-like activity in the haemolymph were unaffected by Si. Larvae fed on +Si plants had higher haemolymph phenoloxidase (PO) and total-PO activities than larvae fed on –Si plants, although this did not enhance the melanisation response of larvae. Furthermore, Si supplies increased plant compensation for herbivory and constitutive trichome production, whereas herbivory induced trichome production only on –Si plants. We provide the first evidence for plant-mediated effects of Si on anti-predator defences of an insect herbivore. We suggest that the lower integument resistance of larvae when feeding on Si-supplemented plants could contribute to their vulnerability to natural enemies and that high PO activity may impose fitness costs (e.g. delayed development).

Comparative Studies of Renin-Null Zebrafish and Mice Provide New Functional Insights

Background: The renin-angiotensin system is highly conserved across vertebrates, including zebrafish, which possess orthologous genes coding for renin-angiotensin system proteins, and specialized mural cells of the kidney arterioles, capable of synthesising and secreting renin. Methods: We generated zebrafish with CRISPR-Cas9-targeted knockout of renin ( ren −/− ) to investigate renin function in a low blood pressure environment. We used single-cell (10×) RNA sequencing analysis to compare the transcriptome profiles of renin lineage cells from mesonephric kidneys of ren −/− with ren +/+ zebrafish and with the metanephric kidneys of Ren1 c−/− and Ren1 c +/+ mice. Results: The ren −/− larvae exhibited delays in larval growth, glomerular fusion and appearance of a swim bladder, but were viable and withstood low salinity during early larval stages. Optogenetic ablation of renin-expressing cells, located at the anterior mesenteric artery of 3-day-old larvae, caused a loss of tone, due to diminished contractility. The ren −/− mesonephric kidney exhibited vacuolated cells in the proximal tubule, which were also observed in Ren1 c−/− mouse kidney. Fluorescent reporters for renin and smooth muscle actin ( tg(ren:LifeAct-RFP; acta2:EGFP )), revealed a dramatic recruitment of renin lineage cells along the renal vasculature of adult ren −/− fish, suggesting a continued requirement for renin, in the absence of detectable angiotensin metabolites, as seen in the Ren1 YFP Ren1 c−/− mouse. Both phenotypes were rescued by alleles lacking the potential for glycosylation at exon 2, suggesting that glycosylation is not essential for normal physiological function. Conclusions: Phenotypic similarities and transcriptional variations between mouse and zebrafish renin knockouts suggests evolution of renin cell function with terrestrial survival.

Effects of Acute Ammonium Nitrate Levels Caused by Agricultural Activities on Four Amphibian Species in The Eastern Black Sea Region

The tadpoles of four amphibian species, namely the Marsh Frog (Pelophylax ridibundus), the Iranian Long-Legged Frog (Rana macrocnemis), the Caucasian Parsley Frog (Pelodytes caucasicus) and the Variable Green Toad (Bufotes variabilis), were exposed to acute concentrations (0 to 500 mg/L) of ammonium nitrate to assess the lethal effects (larval growth, abnormalities, mortality, and LC50 values). Eggs of each species were obtained from clean and polluted habitats in the same region and the tadpoles for experiments were provided from those eggs in the laboratory conditions. Although there was some variability between different populations of the same species or between different species in the observed effects, acute levels of ammonium nitrate caused decreased growth rate and increased abnormalities and mortality in general. Among the 4 amphibian species, the Variable Green Toad was the most damaged one in terms of growth reduction (on average 77-83%), and abnormality rates, and the most damaged one in terms of mortality rates was the Marsh Frog (on average 61-72%). Additionally, the species with the lowest concentration of ammonium nitrate, which killed half of its population, was the Marsh Frog. LC50 values for two populations of Marsh Frog were 37 and 59 mg/L. As a result of our research, it was determined that the acute fertilizer levels caused by agricultural activities in the region had very important harmful effects for all the species we examined. In this context, it can be said that very important environmental and biodiversity problems may occur if certain precautions are not taken regarding the use of the fertilizers and if the awareness of the farmers using these fertilizers cannot be raised.

Southern pine beetle-specific RNA interference exhibits no effect on model nontarget insects

The efficacy and high specificity of the RNA interference pathway has prompted its exploration as a potential molecular management tool for many insect pests, including the destructive southern pine beetle, Dendroctonus frontalis Zimmermann, in which gene knockdown and mortality via double-stranded RNAs (dsRNAs) have already been demonstrated in the laboratory. The nucleotide sequence of dsRNAs requires an exact match of at least 16 nucleotides with the targeted messenger RNA to trigger knockdown of that gene. This allows vital genes in a target pest to be silenced and mortality induced while reducing the probability of adverse effects in nontarget organisms. However, prior to utilization in forest ecosystems, demonstration of the specificity of dsRNAs through laboratory bioassays evaluating potential nontarget effects on model insects is required for proper risk assessment analyses. Consequently, we evaluated three SPB-specific dsRNAs for lethal effects, sublethal effects (larval growth rate, adult emergence or adult fecundity), and relative gene expression in three model nontarget insects representing key functional guilds, including a predator, herbivore, and pollinator. The SPB-specific dsRNAs had no effect on survival of our nontarget insects. Additionally, no sublethal effects were found and the gene expression analyses corroborated bioinformatic analyses in finding no gene knockdown. Our findings support the high specificity of RNAi technology and provide support for its development and deployment for protection of conifer forests against SPB with minimal nontarget concerns.

Diverging life cycle patterns of two Diamesa species (Diptera, Chironomidae) in High Arctic streams, Svalbard

The fauna of streams in the High Arctic, dominated by chironomids, is shaped by extreme environmental conditions that represent the physiological limits for benthic invertebrates. Despite their ecological importance, little is known of chironomid life histories, development strategies and the key abiotic drivers limiting larval growth in High Arctic streams. We investigated the larval development and growth in three High Arctic rivers with contrasting water sources, thermal regimes and nutrient characteristics. Populations of the larvae of Diamesa bohemani (Goetghebuer 1932) and Diamesa aberrata (Lundbeck 1898) from two sampling occasions in July and August 2016 were morphometrically analysed to determine life history patterns and instream productivity. Water temperature differences lead to diverging development patterns on local spatial scales. The lowest larval growth was in a groundwater/snowmelt fed stream with low food concentration and quality, suggesting that stream productivity is not primarily water source dependant, but is dependent on the nutrient supply. Glacially influenced streams are clearly more productive than previously assumed, resulting in comparable secondary production to groundwater/snowmelt-fed streams.

Northeast Arctic Cod and Prey Match-Mismatch in a High-Latitude Spring-Bloom System

By combining an ocean model, a nutrient-phytoplankton-zooplankton-detritus-model and an individual-based model for early life stages of Northeast Arctic cod we systematically investigate food limitations and growth performance for individual cod larvae drifting along the Norwegian coast from spawning grounds toward nursery areas in the Barents Sea. We hypothesize that there is food shortage for larvae spawned early and late in the 2-monthlong spawning period, and to a larger degree to the north and south of the main spawning grounds in the Lofoten. Model results for three contrasting years (1995, 2001, and 2002) show that spawning early in the season at spawning grounds in the Lofoten and farther north is favorable for larval growth close to their size- and temperature-dependent potential. Still, both early and late spawned larvae experience slower growth than individuals originating closer to the time of peak spawning late March/early April. The reasons are low temperatures and shortage in suitable prey, respectively, and this occurs more frequent in areas of strong currents about 1–2 months post hatching. In particular, late spawned larvae grow relatively slow despite higher temperatures later in the season because they are outgrown by their preferred prey.

Leaf Induction Impacts Behavior and Performance of a Pollinating Herbivore

Flowering plants use volatiles to attract pollinators while deterring herbivores. Vegetative and floral traits may interact to affect insect behavior. Pollinator behavior is most likely influenced by leaf traits when larval stages interact with plants in different ways than adult stages, such as when larvae are leaf herbivores but adult moths visit flowers as pollinators. Here, we determine how leaf induction and corresponding volatile differences in induced plants influence behavior in adult moths and whether these preferences align with larval performance. We manipulated vegetative induction in four Nicotiana species. Using paired induced and control plants of the same species with standardized artificial flowers, we measured foraging and oviposition choices by their ecologically and economically important herbivore/pollinator, Manduca sexta. In parallel, we measured growth rates of M. sexta larvae fed leaves from control or induced plants to determine if this was consistent with female oviposition preference. Lastly, we used plant headspace collections and gas chromatography to quantify volatile compounds from both induced and control leaves to link changes in plant chemistry with moth behavior. In the absence of floral chemical cues, vegetative defensive status influenced adult moth foraging preference from artificial flowers in one species (N. excelsior), where females nectared from induced plants more often than control plants. Plant vegetative resistance consistently influenced oviposition choice such that moths deposited more eggs on control plants than on induced plants of all four species. This oviposition preference for control plants aligned with higher larval growth rates on control leaves compared with induced leaves. Control and induced plants of each species had similar leaf volatile profiles, but induced plants had higher emission levels. Leaves of N. excelsior produced the most volatile compounds, including some inducible compounds typically associated with floral scent. We demonstrate that vegetative plant defensive volatiles play a role in host plant selection and that insects assess information from leaves differently when choosing between nectaring and oviposition locations. These results underscore the complex interactions between plants, their pollinators, and herbivores.

Using simulated temperature regimes to test growth and development of an invasive forest insect under climate change

Temperature and its impact on fitness are fundamental for understanding range shifts and population dynamics under climate change. Geographic climate heterogeneity, behavioural and physiological plasticity, and thermal adaptation to local climates makes predicting the responses of species to climate change complex. Using larvae from seven geographically distinct wild populations in the eastern United States of the non-native forest pest Lymantria dispar dispar (L.), we conducted a simulated reciprocal transplant experiment in environmental chambers using six custom temperature regimes representing contemporary conditions near the southern and northern extremes of the US invasion front and projections under two climate change scenarios for the year 2050. Larval growth rates increased with climate warming compared to current thermal regimes and responses differed by population. A significant population-by-treatment interaction indicated that growth rates increased more when a source population experienced the warming scenarios for their region, especially for southern populations. Our study demonstrates the utility of simulating thermal regimes under climate change in environmental chambers and emphasizes how the impacts from future increases in temperature can be heterogeneous due to geographic differences in climate-related performance among populations.