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Microalgae are unicellular photosynthetic organisms that can be grown in artificial systems to capture CO2, release oxygen, use nitrogen- and phosphorus-rich wastes, and produce biomass and bioproducts of interest including edible biomass for space exploration. In the present study, we report a metabolic engineering strategy for the green alga Chlamydomonas reinhardtii to produce high-value proteins for nutritional purposes. Chlamydomonas reinhardtii is a species approved by the U.S. Food and Drug Administration (FDA) for human consumption, and its consumption has been reported to improve gastrointestinal health in both murine models and humans. By utilizing the biotechnological tools available for this green alga, we introduced a synthetic gene encoding a chimeric protein, zeolin, obtained by merging the γ-zein and phaseolin proteins, in the algal genome. Zein and phaseolin are major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris) that accumulate in the endoplasmic reticulum (ER) and storage vacuoles, respectively. Seed storage proteins have unbalanced amino acid content, and for this reason, need to be complemented with each other in the diet. The chimeric recombinant zeolin protein represents an amino acid storage strategy with a balanced amino acid profile. Zeolin protein was thus efficiently expressed in Chlamydomonas reinhardtii; thus, we obtained strains that accumulate this recombinant protein in the endoplasmic reticulum, reaching a concentration up to 5.5 fg cell-1, or secrete it in the growth medium, with a titer value up to 82 µg/L, enabling the production of microalga-based super-food.

In eukaryotes, many proteins contain an N-terminal signal peptide that allows their translocation into the endoplasmic reticulum followed by secretion outside the cell according to the classical secretory system. However, an increasing number of secreted proteins lacking the signal peptide sequence are emerging. These proteins, secreted in several alternative ways collectively known as unconventional protein secretion (UPS) pathways, exert extracellular functions including cell signaling, immune modulation, as well as moonlighting activities different from their well-described intracellular functions. Pathways for UPS include direct transfer across the plasma membrane, secretion from endosomal/multivesicular body-related components, release within plasma membrane-derived microvesicles, or use of elements of autophagy. In this review we describe the mammals and plants UPS pathways identified so far highlighting commonalities and differences.

The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum cha- perone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

The interaction between organisms and their environment generates evolutionary forces able to modify a living species. The process of gene expression, from the transcription of information encoded in a gene to the synthesis of a functional polypeptide, largely relies on evolution, which plays a significant role in determining the factors that control and regulate gene expression. Nowadays, chloroplasts are the result of a complex evolutionary history, elicited by the intracellular cohabitation of an ancient photosynthetic cyanobacterium inside a mitochondriate eukaryotic cell. In this paper, we try to describe the recent investigations on mechanisms that regulate chloroplast translation, both in Chlamydomonas reinhardtii and in land plants. After a general description of plastid translational machinery, ribosome structure with related proteins and nuclear-encoded proteins regulating plastid translation in these organisms, we focus on specific examples of chloroplast translation regulation in the green lineage. In the end, we provide a comparison between plastid translation regulation in green algae and land plants, showing that in both cases chloroplast gene expression is prevalently regulated at post-transcriptional and translational level, although with different strategies.

The present study aimed to investigate the enzymatic potential of Silybum marianum leaves to bioconvert phenolic acids produced in S. marianum callus into silymarin derivatives as chemopreventive agent. Here we demonstrate that despite the fact that leaves of S. marianum did not accumulate silymarin themselves, expanding leaves had the full capacity to convert di-caffeoylquinic acid to silymarin complex. This was proven by HPLC separations coupled with electrospray ionization mass spectrometry (ESI-MS) analysis. Soaking the leaf discs with S. marianum callus extract for different times revealed that silymarin derivatives had been formed at high yield after 16 h. Bioconverted products displayed the same retention time and the same mass spectra (MS or MS/MS) as standard silymarin. Bioconversion was achieved only when using leaves of a specific age, as both very young and old leaves failed to produce silymarin from callus extract. Only medium leaves had the metabolic capacity to convert callus components into silymarin. The results revealed higher activities of enzymes of the phenylpropanoid pathway in medium leaves than in young and old leaves. It is concluded that cotyledon-derived callus efficiently produces compounds that can be bio-converted to flavonolignans in leaves tissue of S. marianum.

Summary Photosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR‐Cas9 target‐specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse‐chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function., Significance Statement LPA2 is a nuclear‐encoded protein that is suggested to be involved in PSII assembly in vascular plants. Here, using CRISPSRS/CAS9 mutagenesis, we identified and investigated an LPA2 homolog in Chlamydomonas reinhardtii. lpa2 mutants were characterized by a strong reduction in the accumulation of photosystem II core subunits, reduced photosystem II activity, increased photosensitivity and impairment in photoautotrophic growth, revealing the critical role of LPA2 in the assembly of photosystem II supercomplexes in green algae.

There is a need to enhance the production of bioactive secondary metabolites and to establish new production systems, e.g., for liver-protective compounds of Silybum marianum seeds. Quantifying and identifying the produced phytochemicals, and examining their protective effects against genotoxic agents, is of great interest. This study established a protocol for the qualitative and quantitative production of hepatoprotective compounds in cotyledon-derived Silybum marianum callus through optimized supplementation of the MS medium with the growth regulators 2,4-D, benzylaminopurine, myoinositol, and asparagine. High-performance liquid chromatography (HPLC) coupled with electrospray ionisation mass spectrometry (ESI-MS) allowed for identification and quantification of the produced compounds. None of the growth medium combinations supported a detectable production of silymarin. Instead, the generated calli accumulated phenolic acids, in particular chlorogenic acid and dicaffeoylquinic acid, as revealed by HPLC and mass spectrometric analysis. 4-Nitro-o-phenylenediamine (NPD) was employed in the AMES-test with Salmonella typhimurium strain TA98 because it is a potent mutagen for this strain. Results revealed that callus extract had a high anti-genotoxic activity with respect to standard silymarin but more evident with respect seed extract. The callus produced chlorogenic acid and dicaffeoylquinic acid, which revealed higher bioactivity than silymarin. Both compounds were not formed or could not be detected in the seeds of Silybum marianum Egyptian ecotype.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Photosystems must balance between light harvesting to fuel the photosynthetic process for CO2 fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment‐binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR‐Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching., The functional role of the Photosystem II monomeric antenna complexes CP29 and CP26 was investigated in Chlamydomonas reinhardtii generating single and double mutants by CRISPR‐Cas9 genome editing demonstrating their key role in non‐photochemical quenching of both Photosystem I and II.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

Plastids are sites of biochemical and biological processes that are fundamental for plant life. The genome of these endosymbiotic organelles encodes for almost one hundred of the three thousand proteins that make up the chloroplast proteome. Genes coding for plastid multi-subunit protein complexes derive from both nuclear and plastid genomes, so it is clear that there is the need of a highly integrated coordination between this two subcellular compartments. The coordination between the nucleus and the plastome takes place at many different levels, including the modulation of nuclear and plastid transcription, RNA processing and translation, post-translational modifications and protein targeting. In addition, the plastid retains a whole series of mechanisms for the preservation of its protein balance (proteostasis), including proteases and molecular chaperones (Figure 1). Plastids have largely abandoned transcriptional control switching predominantly to translational and post-translational control of their gene expression, but some transcriptional regulation is known to occur. Transcription of plastid genes is performed by two different types of RNA polymerases: plastid-encoded RNA polymerase (PEP) and a nuclear-encoded RNA polymerases (NEP). Liebers et al. (2017) propose that targeted changes in plastid transcription, mostly by controlling the relative activities of NEP and PEP enzymes, impact the establishment of the plastid proteome and these represent key determinants for the transitions between the different plastid types. The transcriptional regulation mechanisms are still far from being completely elucidated. An unusual light- and stress-responsive promoter (psbD LRP), regulated by a AAG-box immediately upstream of the -35 element, has been recently mapped. Shinmura et al. (2017) analysed psbD LRP promoter regions in eleven embryophytes, at different evolutionary stages, from liverworts to angiosperms. This analysis identified conserved features of the promoter and facilitated study of its emergence and evolution in plant species. Among the proteins that regulate plastid gene expression, the nucleus-encoded proteins of the mitochondrial transcription termination factor (mTERF) family have been recently identified. Information on their function is only beginning to emerge. Xu et al. (2017) investigate the function of the chloroplast-associated mTERF. They report that these proteins are localized to chloroplast nucleoids and identify two of them involved in the salt stress response. The import of plastid precursor proteins into plastids is another checkpoint affecting plastid proteostasis that is regulated in response to the fluctuating environmental conditions. This fine regulation ensures the optimal functioning of important biological processes taking place in this cellular compartment. The import of plastid precursor proteins is mediated by two distinct translocation complexes called TOC and TIC, located respectively at the outer and at the inner envelope membrane of chloroplasts. The individual steps involved in protein translocation and the corresponding regulation mechanisms used by plants to modulate protein import are reviewed by Sjuts et al. (2017). Upon transition from an endosymbiont to a plant cell organelle, the plastid retains a set of mechanisms that involves enzymes and proteins of prokaryotic origin which are responsible for protein maturation, post-translational modification, correct folding, protein abundance control and removal of misfolded or damaged components. These processes ensure that plastid proteins are ready to exert their biological mission and require an intricate system of signals from nucleus to plastid and backwards. Plastid-derived signals can regulate availability of nuclear-encoded plastid precursors controlling their de novo synthesis, and targeting. Recently, an important player in the chloroplast-to-nucleus retrograde communication has been identified: the protein GENOMES UNCOUPLED1 (GUN1). Recent studies indicate that GUN1 might play a role in the coordination of translation, import and degradation of plastid proteins. The molecular function of different GUN1 partners has been reviewed by Colombo et al. (2016), highlighting its potential role in plastid proteostasis. Another important mechanism during nuclear-plastid interaction is the degradation of multiple components through the ubiquitin-proteasome system. It has become increasingly clear that, together with feedback regulation of nuclear gene expression by plastid-derived signals, this mechanism avoids the accumulation of non-imported proteins in the cytosol. In addition to the anterograde signaling pathway, recent studies in A. thaliana demonstrated that also the retrograde signaling pathway can be subjected to ubiquitin-proteasome regulation. Hirosawa et al. (2017) review recent advances in understanding how the ubiquitin-proteasome system regulates the nuclear-plastid interaction and plastid biogenesis. The maturation of plastid proteins is another highly regulated process that in some cases needs complex apparatuses to occur. This is the case of c-type cytochromes that require a multicomponent assembly pathway for their maturation, as reviewed by Gabilly et al. (2017). The regulation of this pathway has not yet been clarified. Cline et al. (2017) describe the functional characterization of the CCS2 gene of Chlamydomonas reinhardtii required for cytochrome c assembly. The authors discuss the possible functions of CCS2 in the heme attachment reaction. A wide range of post-translational modifications (PTMs) contribute to finely regulate the biological processes that take place in plastids. PTMs alter the physicochemical properties of the plastidial proteins thus affecting their function. Grabsztunowicz et al. (2017) review the current knowledge on the PTMs regulating important metabolic processes in chloroplasts such as DNA replication and gene expression, photosynthetic carbon assimilation and starch metabolism and report the known physiological effects of these modifications. Considering that there is limited knowledge of the combined action among the molecular mechanisms that regulate plastid proteostasis, the goal of this Research Topic is to bring together a set of articles that contribute to our understanding of how transcription, translation, and post-translational modifications (including protein targeting) maintain plastid proteostasis. As a consequence, this Research Topic will help us understand more deeply how plastids function.

The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence.

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

Endoplasmin is a molecular chaperone of the heat-shock protein 90 class located in the endoplasmic reticulum and its activity is poorly characterized in plants. We assessed the ability of endoplasmin to alleviate stress via its transient overexpression in tobacco protoplasts treated with tunicamycin, an inhibitor of glycosylation and inducer of the unfolded protein response (UPR). Endoplasmin supported the secretion of a model secretory protein but was less effective than BiP, the endoplasmic reticulum member of the heat-shock protein 70 family. Consistently, immunoprecipitation experiments with in vivo radioactively labelled proteins using an antiserum prepared against Arabidopsis endoplasmin showed that a much smaller number of newly synthesized polypeptides associated with endoplasmin than with BiP. Synthesis of endoplasmin was enhanced by UPR inducers in tobacco seedlings but not protoplasts. As BiP synthesis was induced in both systems, we conclude that the UPR acts differently, at least in part, on the expression of the two chaperones. Endoplasmin was not detectable in extracts of leaves and stems of the Arabidopsis endoplasmin T-DNA insertion mutant shepherd. However, the chaperone is present, albeit at low levels, in shepherd mutant callus, mature roots and tunicamycin-treated seedlings, demonstrating that the mutation is leaky. Reduced endoplasmin in the shepherd mutant has no effect on BiP protein levels in callus or mature roots, leaves and stems, but is compensated by increased BiP in seedlings. This increase occurs in proliferating rather than expanding leaf cells, indicating an important role for endoplasmin in proliferating plant tissues.

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.