A Dangerous Couple: Sequential Effect of Phosphorus Flame-Retardant and Polyurethane Decrease Locomotor Activity in Planarian Girardia tigrina.

Understanding the interplay among organophosphorus flame retardants (OPFRs), microplastics, and freshwater organisms is crucial for unravelling the dynamics within freshwater environments and foreseeing the potential impacts of organic pollutants and plastic contamination. For that purpose, the present research assessed the exposure impact of 10 mg L-1 flame-retardant aluminium diethylphosphinate (ALPI), 10 µg mg-1liver microplastics polyurethane (PU), and the combination of ALPI and PU on the freshwater planarian Girardia tigrina. The exposure to both ALPI and PU revealed a sequential effect, i.e., a decrease in locomotor activity, while oxidative stress biomarkers (total glutathione, catalase, glutathione S-transferase, lipid peroxidation) and metabolic responses (cholinesterase activity, electron transport system, and lactate dehydrogenase) remained unaffected. Despite this fact, it was possible to observe that the range of physiological responses in exposed organisms varied, in particular in the cases of the electron transport system, cholinesterase activity, glutathione S-transferase, catalase, and levels of total glutathione and proteins, showing that the energetic costs for detoxification and antioxidant capacity might be causing a lesser amount of energy allocated for the planarian activity. By examining the physiological, behavioural, and ecological responses of planarians to these pollutants, insights can be gained into broader ecosystem-level effects and inform strategies for mitigating environmental risks associated with OPFRs and microplastic pollution in freshwater environments.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa.

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Endophytic colonization of five Trichoderma species and their effects on growth of a Eucalyptus hybrid.

The study aimed to evaluate the effectiveness of endophytic colonization via leaf and root inoculation of five Trichoderma species in a Eucalyptus hybrid, as well as the effects of inoculation on plant growth. The experimental design was completely randomized in a 6 × 2 factorial scheme. Plant growth was evaluated during the experimental period at three different times: 20 days after inoculation (d.a.i), 40 d.a.i., and 60 d.a.i. A statistical difference was observed between the inoculation methods during each period and between the Trichoderma species. Plants inoculated with T. asperellum showed the greatest growth among the treatments. Root-inoculated plants produced the greatest growth response. This showed that the presence of Trichoderma in the roots assisted in nutrient assimilation, promoted greater plant growth, when compared with leaf-inoculated plants. Evaluation of the effectiveness of endophytic colonization was performed at each sampling period by collecting leaf samples, and at 60 d.a.i., by collecting leaf, stem, and root samples. T. longibrachiatum and T. harzianum were isolated from leaves at 20 d.a.i., with an increase in the number of colonized plants throughout the evaluation of leaf-inoculated plants. In root-inoculated plants, treatment with T. longibrachiatum, T. harzianum, and T. asperellum presented the highest endophytic colonization in the stem and root samples (at 60 d.a.i.).

Cell death and changes in primary metabolism: the onset of defence in Eucalyptus in the war against Leptocybe invasa.

BACKGROUND: Here, we investigated changes in primary metabolism and cell death around oviposition sites in two hybrid clones of Eucalyptus with different degrees of resistance to Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae), as well as tolerance to water deficiency. RESULTS: We showed that apices of the resistant clone with oviposition had a higher content of amino acids, organic acids and the compound putrescine compared with those of the susceptible clone with oviposition. By contrast, apices of the resistant clone with oviposition had lower sugar and pyruvate organic acid content than those of the susceptible clone with oviposition. Small areas of necrosis were induced around the oviposition sites in the stem apices of Eucalyptus 24 h after infestation. The resistant clone developed larger necrotic areas that showed progressive increases 24-72 h after infestation compared with the susceptible clone, in which cell death was significantly lower and no changes were observed in necrotic area over time. Thus, the programmed death of cells around the egg, modulated by several amino acids, is likely the first defence response of Eucalyptus against L. invasa. CONCLUSION: Our results serve as the basis for the early identification of key metabolites produced in plants in defence against galling insects. © 2022 Society of Chemical Industry.

Differential development times of galls induced by Leptocybe invasa (Hymenoptera: Eulophidae) reveal differences in susceptibility between two Eucalyptus clones.

BACKGROUND: The significance of morphological responses of hosts on susceptibility against gall-inducing insects is relatively unknown, especially in planted forests. Here, we investigate the temporal morphological responses (gall development) induced by the invasive gall wasp Leptocybe invasa and the subsequent insect development in two Eucalyptus clones. RESULTS: Our results identified a novel stage of gall development, not previously reported, termed here 'brownish ring'. In both hosts similar gall development stages were observed. Although L. invasa oviposited in both clones, comparison of external morphological traits of galls over time revealed a differential response in the number of galls between clones. Comparison of the developmental time of each gall and insect stage between clones suggests that plant defense mechanisms against L. invasa are activated shortly after oviposition by the wasp, yet before gall formation. CONCLUSION: Gall number is an important parameter that should be used to measure host susceptibility among Eucalyptus clones. To the best of our knowledge, this is the first study showing differential morphological responses induced by a galling insect, even before gall formation, revealing differences in susceptibility between different plant hosts. These findings provide insight into the use of early stages of gall formation by L. invasa to prevent invasion and establishment of this pest.