Based on this database, we revealed six domain families encoded within vgrG loci, which are either fused at the C-terminus of VgrG/N-terminus of T6SS toxin or encoded by an independent gene. Among them, DUF2345 was further validated and shown to be indispensable for the T6SS effector delivery and LysM was confirmed to assist the interaction between VgrG and the corresponding effector. Together, our results implied that these widely distributed domain families with similar genetic configurations may be required for the T6SS effector recruitment process.Type VI secretion systems (T6SSs) deliver effectors into target cells. Besides structural and effector proteins, many other proteins, such as adaptors, co-effectors and accessory proteins, are involved in this process. MIX domains can assist in the delivery of T6SS effectors when encoded as a stand-alone gene or fused at the N-terminal of the effector. However, whether there are other conserved domains exhibiting similar encoding forms to MIX in T6SS remains obscure. Here, we scanned publicly available bacterial genomes and established a database which include 130,825 T6SS  vgrG database was established, which revealed six widely distributed domain families with similar genetic configurations. These families may be required for the T6SS effector recruitment process.A  T6SS effectors can be classified into “specialized” and “cargo” effectors depending on the modality of the delivery system8. In the case of a specialized effector, the toxin domain is fused to the structural components of T6SS complex, such as VgrG, Hcp or PAAR proteins10. In contrast, cargo effectors are associated with the structural components through non-covalent interactions.The type VI secretion system (T6SS) is a molecular device that delivers effectors to kill neighboring prokaryotic and eukaryotic cells using a contractile injection mechanism13. T6SSs deliver effectors into target cells with the help of some proteins, including adaptors, co-effectors, and etc. The adaptor proteins (sometimes referred to as chaperones) may have variable functions relating to the effectors, such as the proper folding/unfolding, stability and recruitment to the secretion apparatus15. Similar to the well characterized chaperone/adaptors in the type III secretion system (T3SS) and type IV secretion system (T4SS), three T6SS adaptor families have been characterized, including DUF4123 (Tec/Tap), DUF1795 (DcrB/Eag) and DUF2169. These domains are sometimes encoded adjacent to vgrG and facilitate the interactions between the VgrG and the cognate effector specifically. These adaptor proteins are not secreted but are indispensable for the delivery of their cognate effectors18.Effector delivery of specialized secretion systems is important for many virulent phenotypes caused by pathogenic bacteriavgrG neighborhood and can be used as markers for T6SS effectors, such as MIX (Marker for type sIX effectors) and FIX (Found in type sIX effector) domains19. The MIX domain, encoded by a single gene, can be secreted along with the cognate effector via the T6SS and has been termed as a T6SS co-effector. Fused at the N-terminus of the effector, MIX domain is also necessary for the secretion of the toxin21. Considering the diversity of well characterized T6SS effectors, it is possible that more conserved domains may be encoded that assist the effector delivery of T6SS in this complex secretion system.Different from classic adaptors that are encoded as single proteins, several conserved domains were identified primarily located at the N-terminus of toxins in the T6SS 22. Previous studies revealed that not only T6SS effector and immunity proteins but also many T6SS-related proteins, including the aforementioned adaptors and co-effectors, are frequently encoded in the vgrG neighborhood23.T6SS propels its “tube-spike complex” through the membranes of target cells in order to deliver effector proteins. As central components of the spike complex, VgrG proteins fall into two classes, “structural” and “evolved” VgrGs. Evolved VgrG proteins are defined as polymorphic toxins with conserved N-terminal VgrG domains and variable C-terminal toxin domains http://www.mgc.ac.cn/dbVgrG/), which contains 130,825 VgrG protein sequences from 45,041 Gram-negative bacterial genomes. In-depth analysis of this dataset revealed that, besides the MIX domain, six domain families within the vgrG loci are not only encoded as independent genes, but also fused at the C-terminus of VgrG and/or the N-terminus of toxin, including DUF2345 (cl01733), FIX-like (cl41761), LysM (cl21525), 5 (cl33691), PG_binding_1 (cl38043) and PHA00368 (cl30808) domains. These families are widely distributed among diverse bacterial genomes and associated with dozens of different toxins. Further experiments confirmed that DUF2345 and LysM containing proteins assist the interaction between VgrG and the corresponding effector, which are potentially important in the discrimination process of T6SS effectors.Here, we used a comprehensive bioinformatic approach to scan currently available bacterial genomes and created a VgrG database . A bioinformatic scan for conserved domains confirmed that the VgrG domain (accession: COG3501) was present in all 872 verified VgrG proteins in addition to 472 Afp8 proteins available from the dbeCIS database26. Importantly, the identified domains found in 861 (99%) verified VgrGs range between 451 and 750 amino acids, whereas there are only 10 (2%) Afp8 proteins that fall within this size range  are homologous to VgrG proteins, thus representing a potential confounding influence on the integrity of the datasetnge Fig. . We thernge Fig. . Using t27, supporting the validity of our approach.To further characterize the VgrG proteins identified above, we constructed a maximum-likelihood (ML) phylogenetic tree based specifically on the sequences of the conserved VgrG domains Fig. . Using tvgrG loci were identified, which are widely distributed among various bacteria . A domain family was reported if it was present in both of two encoded forms: “stand-alone” gene  and fused to either the C-terminus of VgrG or the N-terminus of a toxin . Finally, to further explore the presence of these domain families within vgrG loci in finer detail, we extended our search without the limitation of linkage to known toxins to identify more candidate domain-containing proteins within vgrG loci , FIX-like (cl41761), LysM (cl21525), 5 (cl33691), PG_binding_1 (cl38043) and PHA00368 (cl30808) were successfully identified  and MIX domain (3.14%), 31.33% of vgrG loci encode at least one of the six conserved domain families identified here. In contrast, only 28.09% of vgrG loci do not include any of the adaptor/MIX/conserved domains mentioned above  had been reported to assist the interaction between VgrG and its cognate effectors. We further screened these adaptor families encoded within 29. Nevertheless, among the aforementioned six conserved domains, the DUF2345 domain is the most frequently identified in vgrG loci , AKO63_2954 (DUF2345PAR) and AKO63_2955 (M35PAR), corresponding to the VgrG domain, the DUF2345 domain and the M35  toxin domain of PA0262 (VgrG2bPA), respectively. Similarly, Q094_05019 (VgrGPS) of P. aeruginosa PS42 encodes VgrG domain, whereas Q094_05020 encodes N-terminal DUF2345 domain and C-terminal M35 domain. AlphaFold v2.0 predicted that VgrGPAR, VgrGPS and VgrG domain of VgrG2bPA have the same conformation , PS42 locus (VgrGPS and Q094_05020) and VgrG2bPA form similar trimmer structure, which implies that these three complexes might endow similar biological functions  relieved this growth defect. Crucially, truncation of the M35 domain of VgrG2bPA restored growth, which indicated that DUF2345 in itself is not toxic to E. coli. Intraspecies P. aeruginosa competition assays were also performed to determine whether the DUF2345 domain could affect the function of VgrG2bPA  was not affected, complemented in the ΔVgrG2bPA strain, VgrG2bPAΔDUF2345 could only be detected in the cells, but not in the supernatant   Fig. . ImportaPAR Fig. . These rTaken together, DUF2345 domain is indispensable for the delivery of its cognate toxin via fusion at the C-terminus of VgrG or encoded as a single gene.vgrG, we then investigated whether the sequences of VgrG domains showed a correlation with those of DUF2345. An iterative procedure was devised to hierarchically cluster the 52,277 VgrG domains and their cognate DUF2345 domains, respectively. At the 30% amino-acid sequence similarity cutoff, VgrG domains form three major clusters and ten outliers, whereas DUF2345 domains were classified into 37 distinct groups  and Kalk_10460 (LysMG15) exhibited no toxicity when they were expressed in E. coli   Fig. .PA, which implied that LysM may mediate the interation between VgrG and toxin  and BRPE_05220 (LysM and NLPC_P60) form similar trimmer structure with VgrG2bIn sum, encoded at downstream of LysM containing gene or fused at the C-terminal of LysM domain, toxin interacts with VgrG in a LysM dependent manner implying LysM may assist the loading of its cognate effector onto the secretion apparatus.13. Most of the Rhs superfamily are linked to FIX-like (cl41761) and 5 (cl33691) domains. LysM domains are mainly correlated with Lyz_like, NlpD and NLPC_P60 superfamilies. As these domain families identified in this study, including FIX-like (cl41761), LysM (cl21525), 5 (cl33691), PG_binding (cl38043) and PHA00386 (cl30808), share a similar genetic organization and correlation with downstream toxins as the DUF2345 domain, it is reasonable to speculate that they would also function in the T6SS effector discrimination.The DUF2345 containing proteins exhibit specific correlation with their downstream diverse toxins Fig. . NotablyProteobacteria genomes, they are rarely encoded in the genomes of Vibrionaceae and Rhodospirillaceae bacterial families. In contrast, the PG_binding_1 domain is limited to the genomes of γ-proteobacteria, including the families of Chromatiaceae, Sinobacteraceae and Vibrionaceae. In general, although these conserved domains are widely encoded among various bacteria, their distributions exhibit obvious taxonomic specificity, which is coincident with their corresponding cognate effectors as shown in Fig. The overall distribution of the six conserved domain families was then analyzed Fig. . It is ivgrG19. Encoded as a single protein or fused at the N-terminus of effector, a MIX containing protein is secreted with its cognate effector for recipient bacterial killing, which is apparently different from classic adaptor proteins21. In this study, we created an open access VgrG database and identified six conserved domains, which exhibit similar genetic-context as MIX. Further bioinformatic and experimental approaches revealed the common characteristics of these six conserved domains.Widespread among the Gram-negative bacteria, T6SS employs diverse effectors to manipulate neighboring bacterial and/or eukaryotic cells. As an essential component of T6SS injection apparatus, VgrG can deliver both specialized effectors and cargo effectors. T6SS adaptor  and co-effector (MIX) genes have been found encoded adjacent to 33. In addition to the conserved VgrG domain, the enteroaggregative E. coli (EAEC) VgrG (PDB 6sk0) contains a DUF2345 domain which adopts a β-helical fold similar to the gp5-like domain6. Previous studies reported that, fused at the C-terminus of VgrG, both DUF2345 and its downstream TTR domain are involved in the specific interaction with their effectors and DUF2345 proteins and so was termed as an internal adaptor of T6SS cargo effectors30. Our results revealed that DUF2345 domains correlated well with their downstream toxins, which further confirmed these previous studies. In the case of fusion at the C-terminus of VgrG, DUF2345 is inevitably to be secreted by T6SS. Whether the upstream and downstream regions of DUF2345, i.e. TTR, are always associated with DUF2345 would be interesting for further inverstigations.To date, the structures of several VgrG proteins have been reported, which exhibits a canonical trimeric spike-forming region containing gp27 and gp5-like domains36. Interestingly, although LysM containing protein is an essential component of the eCIS particle, its protein homolog is not present in the structural components of T6SS, a subtype of contractile injection system32. Further phylogenetic analysis reveals the diversity of T6SS-related LysM domains that is distinct from the eCIS-related LysM domains. Considering LysM domains are not exclusively linked to T6SS-associated loci in bacterial genomes, it implies that some  of these domain-containing proteins might be multi-functional. Our study implied that, either when fused at the N-terminus of the effector or encoded as a distinct gene, LysM can assist loading the effector into the secretion apparatus through interaction with VgrG. Further structural and evolutionary studies are required to elucidate this new role of LysM in the bacterial secretion system.LysM domains are found in a variety of enzymes involved in bacterial cell wall degradation and may have a general peptidoglycan binding function (CDD accession: cl21525). However, this domain is also encoded in fusion forms with phage baseplate components37. T6SS co-effectors can be secreted in a T6SS dependent manner whereas adaptors are not translocated. RIX-containing proteins function as a secreted adaptor which enables the T6SS-mediated delivery of other cargo effectors by a previously undescribed mechanism38. Intriguingly, we found some vgrG loci encode both adaptor and conserved domains described in this study, such as MIX, FIX and LysM 21 and MIX, LysM and PG_binding domains are in one protein of Vibrio sp. CL6 (WP_061036948.1)39. Although our results indicated that LysM domain is involved in the interaction between VgrG and its cognate toxin, the phylogenetic tree showed that there are potential differences among VgrG, eCIS and phage-related LysM domains  DUF2345-encoding genes are in fact not linked to known toxin genes  was used for cloning procedures, and BL21(DE3) (TransGen Biotech) was used for protein expression and purification. P. aeruginosa strains were used for bacterial competition assays. All cultures were supplemented with antibiotics where necessary. The antibiotic concentrations were as followed, ampicillin (50 μg/mL), kanamycin (50 μg/mL), chloramphenicol (34 μg/mL), gentamicin (50 μg/mL) or tetracycline (50 μg/mL). Gene expression was induced with 1 mM IPTG.Bacterial strains . Therefore, based on a statistical histogram of their length distribution only 872 (92%) VgrGs with length of 601~1100 aa were retained and included as a positive control dataset of “typical” VgrG proteins . Several VgrG-associated protein domains were successfully identified within one or more VgrGs, but the conserved domain recognized by CDD’s COG3501 PSSM was the only conserved domain detectable in all of the 872 VgrG proteins. With this in mind, we used the presence of the VgrG domain (accession: COG3501) as a key marker for VgrG proteins in the follow-up study. However, previous studies revealed that the Afp8 proteins of eCISs elements are homologous to VgrG proteins. Indeed, we also identified 472 VgrG domain-encoding Afp8 proteins available from dbeCIS database26, which were collected to serve as the negative control dataset for further VgrG screening.A total of 946 verified VgrG proteins  were downloaded from the SecReT6 database ins Fig. . This da43. Of note, annotated Afp8 proteins available from the dbeCIS database were further excluded  of the positive dataset encode a VgrG domain span of 451~750 aa, whereas only 10 (2%) of the negative dataset had VgrG domains that fit into this range Fig. . Therefoded Fig. .44. FastTree program v2.1 was then employed to construct the maximum-likelihood (ML) phylogenetic tree under the Whelan Goldman (WAG) models with gamma optimization45. The resulting phylogenetic tree was annotated and represented via the iTOL online server46.Based on the aforementioned VgrG screening criteria each of the identified VgrG protein carries a 451~750 aa VgrG domain, which provided an ideal molecular marker for phylogenetic analysis. An in-house Perl script was utilized to extract the corresponding domain sequence from each VgrG. For brevity only one representative domain sequence was kept for VgrGs from the same bacterial genus with identical sequence. The remaining non-redundant domain sequences were fed to the Clustal-Omega program to generate multiple sequence alignment27. Therefore, we retrieved the existing classification information of the associated T6SS from the SecReT6 database for the 872 verified VgrGs. Then, we mapped the established type/subtype information onto the VgrG-based ML tree to propagate the current T6SS classification to all of the newly identified VgrGs.The current SecReT6 database has classified T6SS systems into eight types/subtypes based on the phylogeny of TssB http://www.mgc.ac.cn/dbVgrG/)  of each vgrG locus. Moreover, the hmmsearch program from the HMMER3 package47 was applied to identify the other 12 known components of T6SS based on the conserved domains summarized by the SecReT6 database27.In order to facilitate future studies on the VgrG proteins as well as VgrG-related adaptors or effectors we constructed a publicly accessible online database, named dbVgrG . pETduet and RSFduet were used to express genes (Supplementary Table 48. Two DNA regions upstream (~700 bp) and downstream (~700 bp) of the target region were amplified by PCR using the specific primer pairs, which were cloned into pK18mobSacB. The plasmids were transformed into E. coli S17-1 λpir. After mobilization into P. aeruginosa by conjugation with E. coli S17-1 λpir, conjugants were selected using Luria Bertani broth supplemented with 200 μg/mL gentamicin and 34 μg/mL chloramphenicol. Plating onto LB agar containing 20% (w/v) sucrose selected for strains that had undergone double-recombination, excising the plasmid. Only colonies showing both kanamycin resistance and gentamicin sensitivity were selected. All mutants were confirmed by PCR and sequencing.In-frame deletion mutants were created as described previouslyFor western blots, samples resolved by SDS-PAGE were transferred onto PVDF membranes (Millipore), blocked with 5% skimmed milk for 30 min at room temperature. After that, the blots were incubated with the indicated primary antibodies overnight at 4 °C. The membrane was washed three times with Tris Buffered Saline with Tween 20 (TBST), then were incubated with horseradish peroxidase-conjugated (HRP-conjugated) secondary antibodies for 1 hr at room temperature. After a further three washes in TBST, the antibody-bound proteins were detected using SuperSignal West Femto Chemiluminescent Substrate Kit  following the manufacturer’s protocol.P. aeruginosa secretion assays were conducted as previously described with corresponding modifications49. Cultures were inoculated into 5 mL Luria Bertani broth at OD600 = 3.0. Supernatants were discarded from cells by centrifugation at 5000 × g at 4 °C. Bacterial pellets were resuspended in sterile PBS and sub-cultured with 50-fold dilution in M9 medium to an OD600 of 1.0. The supernatant and cells were separated by centrifugation. The supernatant was filtered through a 0.22 μm filter . One-fifth of the volume of 100% trichloroacetic acid was added into the supernatants. The fractions were incubated 2 h at −20 °C before centrifugation at 15,000 × g and 4 °C for 30 min. The resulting protein pellets were washed three times with ice-cold acetone and resuspended in SDS protein sample buffer and then incubated at 100 °C for 10 min. For the cell pellet samples, 1 mL of culture was centrifuged and the total cells were harvested. Both the supernatant and pellet were analyzed by western blot analysis.30. Briefly, donor and recipient strains were grown overnight and washed with sterile PBS. The mixture was spotted onto a nitrocellulose membrane overlaid onto a 3% LB-LS agar plate . If needed, 0.5 mM IPTG was included in the medium for induction of cloned genes. The plates were incubated at 37 °C. Bacterial cultures were scraped off and suspended in 1 mL LB medium. After serial dilution, donor and recipient colonies were counted on selective LB agar plates and changes of the donor-recipient ratios were determined.Bacterial competition assays were performed as described previouslyE. coli strain BL21 (DE3) containing pET22 b(+) expressing targeted proteins to the periplasm were serially diluted in LB medium at 10-fold. A total of 2.5 μL of this bacterial dilution was spotted on LB agar containing 0.25 mM IPTG. Images were taken after 24 h growth. E. coli strains harboring pET22 b(+) derivatives encoding both effector and immunity proteins were performed in a similar way.Overnight cultures of 600, 1 mM IPTG was added for a 16 h induction at 20 °C. Bacterial pellets were collected and resuspended in IP-lysis buffer  containing a protease inhibitor PMSF. Cells were sonicated and centrifuged at 15,000 rpm for 20 min at 4 °C to remove cell debris. The supernatants were incubated with Anti-FLAG beads (Invitrogen). Bound proteins were eluted with elution buffer. Protein samples were separated by SDS-PAGE and analyzed by western blot. Anti-FLAG , anti-MYC  and anti-Stag  monoclonal antibody at a radio 1:1000 was used for Western blot analyses.PETduet and RSFduet plasmids were used for expression of VgrG, conserved domain-encoding proteins and effector proteins with FLAG/MYC/Strep tags in BL21 (DE3). After initial growth to 0.6 ODvgrG gene in the adjacent genomic location. Therefore, we introduced the following operational criteria for the enrollment of genes downstream of vgrG: (i) for each vgrG locus a maximum of three continuous downstream genes were included for efficiency; (ii) valid downstream genes needed to be encoded on the same strand as vgrG; (iii) the intergenic region of all adjacent genes should <1 kb; and (iv) known components of the T6SS operon and any annotated pseudogenes were excluded. The CD-search of the CDD database42 was then employed to screen for conserved domains among the 280,581 downstream genes in addition to the C-termini of VgrG proteins  with the E value cutoff of 10−5. Additional domains were identified by the HHpred server manually50. The hierarchical sequence clustering of the VgrG domains, DUF2345 domains and other toxin domains were individually conducted by the CD-HIT v4.6.551 following the protocol described previously43. The relationships between domains/clusters were visualized by the SankeyMATIC web server (https://sankeymatic.com/).Previous studies showed that VgrG-associated effectors, adaptors and co-effectors were usually encoded downstream of the vgrG and downstream toxin/effector genes. A collection of 928 experimentally verified exotoxins and effectors available from the well-established bacterial virulence factor database VFDB52 were used to identify possible toxin/effector-related conserved domain families by CD-search ; and Case III: domains encoded by a toxin gene downstream of the vgrG .Many VgrG-related adaptors/co-effectors were expected to be flanked by the upstream vgrG or N-terminus of toxin. In addition, to exclude possible false positives due to unexpected domain recognition we considered only domains with more than five instances present in at least two different bacteria genera. The taxonomic distribution of co-effector or conserved domains was visualized by the GraPhlAn tool53.After careful manual curation of the output domain lists for all three cases based on literature review and current annotations of reported domains, we finally accepted potential conserved domains that were present in not only a single gene but also either C-terminus of n, and the mean ± SD are reported and described in the figures and figure legends. GraphPad Prism 6 was used for all statistical analysis. All experiments reporting statistical significance were analyzed by one-way ANOVA with Dunnett’s test. Statistical significance is denoted in figures by asterisks .Values of Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


The ‘Signuredda’ bean is a local genotype of pulse with particular technological characteristics, cultivated in Sicily, Italy. This paper presents the results of a study to evaluate the effects of partial substitutions of durum wheat semolina with 5%, 7.5%, and 10% of bean flour to prepare durum wheat functional breads. The physico-chemical properties and the technological quality of flours, doughs, and breads were investigated, as well as their storage process up to six days after baking. With the addition of bean flour, the proteins increased, as did the brown index, while the yellow index decreased. The water absorption and dough stability according to the farinograph increased from 1.45 in FBS 7.5%, to 1.65 in FBS 10%, for both 2020 and 2021, and from 5% to 10% supplementation for water absorption. Dough stability increased from 4.30 in FBS 5%-2021 to 4.75 in FBS 10%-2021. According to the mixograph, the mixing time also increased. The absorption of water and oil, as well as the leavening capacity, were also examined, and results highlighted an increase in the amount of water absorbed and a greater fermentation capacity. The greatest oil uptake was shown with bean flour at 10% supplementation (3.40%), while all bean flour mixes showed a water absorption of approximately 1.70%. The fermentation test showed the addition of 10% bean flour significantly increased the fermentative capacity of the dough. The color of the crumb was darker, while the crust became lighter. During the staling process, compared with the control sample, loaves with greater moisture and volume, and better internal porosity were obtained. Moreover, the loaves were extremely soft at T0 (8.0 versus 12.0 N of the control). In conclusion, the results showed an interesting potential of ‘Signuredda’ bean flour as a bread-making ingredient to obtain softer breads, which are better able to resist becoming stale.  Legumes belong to the Fabaceae or Leguminosae family , which rPhaseolus vulgaris L.) represents 50% of the grain legumes consumed worldwide as a source of human nutrition, and is also widely used for animal feed [The increased interest in the health aspects and beneficial properties of food has directed research over time towards the formulation of food products enriched with functional components that are able to improve foods from nutritional and health points of view . In partmal feed ,14. Aboumal feed . This stWheat re-milled semolina (SC-100%) was supplemented with different percentages of ‘Signuredda’ bean flour at 5% (FBS 5%), 7.5% (FBS 7.5%), and 10% (FBS 10%), according to Różyło et al. and Spina et al. ,17 for tFor the samples, both pure  and the The analysis of the nutritional and mineral content of the pure flour of the ‘Signuredda’ bean was carried out during the two years of harvest. Although in the literature there are still no data concerning this genotype, from a nutritional point of view, the recorded results are in line with the study of Marletta et al.  on the fThe colorimetric parameters were evaluated on the control sample, on the different supplements, and the corresponding bean flour. The results are expressed according to the following parameters: brown index (100-L), red index (a*), and yellow index (b*). The results are shown in Regarding the brown index, significantly different values were observed between the control sample (10.26) and samples obtained with the addition of the bean flour.In particular, the latter recorded significantly higher values, compared to the control, which means that the integration of semolina with ‘Signuredda’ bean flour resulted in a browner color of the flour compared to semolina.The bean flour for the year 2021 displayed a higher brown index (14.49) than the flour in 2020 (13.63). Samples obtained with a supplement of 7.5% bean flour recorded the highest values of the brown index. In particular, FBS sample 7.5%-20 recorded the highest value (12.98), while the lowest value was recorded for FBS sample 5%-21 (11.49).The red index (a*) showed significant differences, both between the control sample and the samples obtained with the addition of bean flour, and between the latter and the 100% bean flour. In particular, the latter, compared with the integrated samples with different percentages, showed much higher values of a*, between 1.08 and 1.29, which are close to those found by other authors for different varieties of beans ,25.The data reported in Regarding the yellow index (b*), the highest results were recorded in the sample SC-100% (17.17), in line with what was reported by other authors , and difAs can be seen from the analysis of the data presented in For almost all variables, except the brown index, the other color parameters were not significant. The brown index remained almost unchanged over the two years.In the present study, the technological properties of the samples obtained from a mix of bean flour and semolina at different percentages and the control sample (SC-100%) were evaluated. As shown in The bean flour spiked samples showed significantly higher water absorption than the SC-100% sample. As reported in This positive trend of increased water absorption following an increase in the percentage of bean flour supplementation has been found by other authors. Hoxha et al.  recordedIt is conceivable that a longer stability time could result in a dough with higher tolerance to mixing and flexibility during mixing .With regard to the dough development time, defined as the time between the first addition of water and the moment at which the dough reaches optimal consistency , our stuIn particular, from The water absorption capacity is an extremely important parameter since it can significantly affect some characteristics of the bread, such as the product humidity, the retrogradation of the starch, and the subsequent bread ageing . By compThe water and oil absorption capacities were evaluated to verify the processing aptitude of both the SC-100% control sample and the samples to which bean flour were added.2O/g d. m., with the FBS 7.5-2020 sample, which had an integration of 7.5%. This is a rather common trend that is observed in semolina samples enriched with an ingredient with a high protein content [2O/g d. m. It is important to emphasize, however, that this ability could also be influenced by other parameters, as observed by other authors [Regarding this property, as shown in  content . Legume  authors ,37. A hi authors .The 7.5% integration of bean flour achieved a plateau in terms of water absorption, presumably due to the interaction of semolina with the proteins and fiber of bean flour. The oil absorption capacity represents an indication of the rate at which the protein binds to fat in food formulations . Many auThe samples obtained from the integration of semolina with bean flour, relating to the year 2020, recorded an increasing trend of absorption as the percentage added increased. The highest value (3.40 g/100 g d. m.) was found in the FBS 10%-2020 sample, supplemented with 10% bean flour. The same integration obtained different data in the years 2020 and 2021, with a reduction in absorption in the FBS 10%-2021 sample, which recorded a value of 2.37 g/100 g d. m. This absorption process is related to the action of the non-polar side chains of proteins, which trap the oil .With regard to the water and oil absorption of pure bean flour, it is possible to observe, for both the years 2020 and 2021, a higher predisposition to water absorption. This trend is in line with what was found for other legume flours .The leavening test was carried out both on the SC-100% sample and on the mix of semolina and bean flour, obtaining the trend shown in After cooking, the physical properties of both the SC-100% control samples and the mixes were evaluated. The specific volume, specific gravity, porosity, and internal structure of the bread were evaluated, and the results are reported in Regarding specific weight, the samples supplemented with different percentages of bean flour recorded slightly lower values than the SC-100% sample, except for FBS 5%-2021, while among the different samples the values were constant. The FBS 5%-2021 sample reported the highest value and FBS 7.5%-2021 the lowest value.2, which becomes trapped between the meshes of the gluten [The bread porosity is influenced by mixing and leavening processes . Specifie gluten , releasie gluten .Starting from the SC-100% control sample, with an index equal to 7, which results in a less developed porosity of the crumb, an improvement was observed following integration with bean flour, with breads showing a higher alveolature. The best value of 5 was recorded in FBS 5%-2020 and FBS 7.5%-2020 samples. No irregularities in the internal structure were reported in any of the samples obtained in this study.The data obtained suggest a positive correlation between the use of Signuredda bean flour and the physical characteristics of the bread, reporting a clear difference from what is reported in the literature by Bresciani and Marti .The color represents an important quality parameter for bread, since it greatly affects the degree of acceptability of the product by consumers. It is important to underline that color does not depend only on factors related to the raw material, such as the presence of pigments, but can also be influenced by other factors .Bakery products, in fact, can be subjected to chemical reactions during cooking, such as the Maillard reaction, which then affects the color of the finished product.The color was measured in terms of brown index, a*, and b*. The breads showed differences in color that are expressed in the values reported in Samples were stored and analyzed to evaluate the evolution of the physical properties during storage. The measurements were carried out immediately after cooking (T0), on the first day after baking (T1), and on the fourth day (T4) and the sixth day of storage (T6). In particular, the moisture content, volume, weight, height, and hardness of the bread were determined .A progressive moisture reduction was observed in all the analyzed samples during the storage period. It is possible to observe a statistically significant difference in the moisture values ranging from T0 to T6 during the storage period.Regarding the volume, it is important to underline that at T0 almost all bread samples obtained from the mix of bean flour and semolina recorded higher values than the SC-100% sample. This means that the use of bean flour contributed to the formation of a more voluminous bread than that obtained using only semolina. In particular, in our study, the most significant increase in volume was recorded using an integration rate ranging from 5% to 7.5%. During the storage period, from T1 to T6, as well as for moisture content, a decreasing trend was also observed in bread volume measurement, which was already significant at T1, after one day of storage. The FBS5%-2020 sample recorded the greatest volume reduction at T6, while a smaller decrease was observed with SC-100% compared to all the other samples. During the 6 days of storage, a significant difference in bread volume measurement from T0 to T6 was highlighted for all samples, in particular for the FBS5%-2020 sample, with the exception of FBS10%-2020, which did not show significant results. The weight and height of the loaf were analyzed as physical parameters. The evaluation of the staling process carried out on each sample during storage showed statistically significant differences from T0 to T6 for weight, while regarding height, only the FBS10%-2021 sample recorded significant differences.Regarding the texture, from As observed for the other parameters, the statistical evaluation of the texture results also highlighted significant differences for all samples during the storage period.Sicilian durum wheat semolina produced by the agricultural cooperative society ‘Valle del Dittaino’ a.r.l. in Assoro   was used for the tests, and also used in the production of bread ‘Pagnotta del Dittaino DOP’. This semolina, called ‘re-milled semolina’, was subjected to a greater grinding than that used for the production of pasta, which makes it smaller in grain size, due to a higher hydration rate . Re-millThe soil was prepared with winter work (20–25 cm deep) and 2 subsequent low works. Sowing was undertaken by hand on 25 March 2020 and 20 March 2021, spreading the seed at a depth of about 5 cm in the soil, in rows 100 cm apart. No fertilization was undertaken during sowing. When the second true leaf appeared, fertilization was carried out with a liquid organic nitrogen fertilizer called ‘Fantastic soil’ from organic farming . An irrigation and fertigation system with drippers and 8-day irrigation shifts was installed in April. Only in the case of excessive rains, favorable to fungal diseases, were treatments with wettable sulfur carried out.Phaseolus vulgaris L. with a determined growth and a creamy-white pod. The seeds are reniform with a firm, nut-colored, white ileum around which there is a reddish ring. The cotyledons are light yellow. It is a bean that cooks easily, and it does not need to be soaked. It is very mellow and tasty, and has various uses: soups, in salads after boiling, ground to obtain flour, etc. Although it does not have a brown integument, it leaves the cooking water dark in color. It is a genotype with a strong bond with the territory of origin , where it is grown in a still-restricted area.The ‘Signuredda’ bean is a local variety of The authors decided to use this local variety of bean because it is easily grindable, thanks to its slightly waxy, light-yellow cotyledons, which give rise to a flour very similar in color to that of semolina, and its firm tegument, which shatters easily. Furthermore, based on the results of preliminary rheological and technological tests on the doughs of the mixes with durum wheat semolina, it was seen that it had good potential for making bread.‘Signuredda’ bean seeds were milled with a Cyclotec type 120 mill  equipped with a 500 µm sieve .The moisture content of bean seeds, flours and breads was determined by oven drying  at 103 °C until constant weight, according to the AOAC method 935.25 . The anaThe results are expressed as percentage relative humidity, as described by He et al. .The water binding capacity and the oil binding capacity were determined following Kahraman et al.  and Du eA CR 200 colorimeter  was used for color evaluation. The CIELab colorimetric model was adopted by expressing the results according to the coordinates L*, a* (indicating the change from green to red), and b* (indicating the change from blue to yellow) ,60. The -Phase of absorption of added water and formation of the gluten mesh;-Stability, in which disulfide bonds are broken and reformed continuously;-Gluten mesh rupture and curve slope .The evaluation of the quality of dough in terms of mechanical resistance was carried out using farinographic analysis, which consists of three specific moments:® software, according to the method AACC 54–21.02 [The curves were obtained using 300 g of semolina in the dough chamber, at different percentages of integration of the various flour mixes, using the Farinograph of Brabender , equipped by Farinograph54–21.02 .The analyses were carried out in triplicate.The strength of the dough, also linked to the quantity and quality of gluten, was evaluated using a mixograph which, together with the farinographic analysis, can provide a sufficiently descriptive measurement of the characteristics of the dough under study ,64,65,66The method described by Canale et al.  was foll(a)Control dough: 50 g semolina; 1.5 g yeast; distilled water;(b)5% dough: 47.5 g semolina; 2.5 g bean flour; 1.5 g yeast; distilled water;(c)7.5% dough: 46.25 g semolina; 3.75 g bean flour; 1.5 g yeast; distilled water;(d)10% dough: 45 g semolina; 5 g bean flour; 1.5 g yeast; distilled water.The following quantities were used for the preparation:The analyses were carried out in triplicate.Breads were obtained according to the official AACC 10-10.03 methodology, described by Canale et al. . The forThe following physical characteristics were evaluated on the breads obtained: volume, height, weight, moisture, texture, porosity of the crumb, color of the crumb, and crust. The bread volume was determined by the rape seed displacement method according to the AACC 10–05 method . The heiThe Zwick Z 0.5 Röell texturimeter, Ulm, Germany, was used to measure the bread hardness. This was equipped with an 8 mm diameter cylindrical aluminum probe and configured with 50 g pre-load, 100 mm/min pre-load speed, 20%forced shutdown threshold. The resulting peak force was measured in Newtons (N). The central bread slices of each loaf were visually compared with the eight Dallmann reference images representing the cross-section of breads with different crumb structures. Crumb porosity was evaluated based on the 8-degree Mohs scale modified by Dallmann  where, fBread was stored for 7 days at 25 °C and packed in cardboard. On the day of baking, 1 day, 4 days, and 6 days later, the loaf moisture, volume, weight, height, and hardness were determined to evaluate the stale rate, according to the AACC method 10–10.03 . The moi® Centurion XVI software package . One-factor and two-factor analyses of variance (ANOVA), followed by Tukey’s HSD test , was carried out on all physicochemical, technological, and breadmaking attributes and the bread staling process. The following two factors were considered: 1. the year of production of the bean seeds; 2. the amount of bean flour. A one-factor analysis determined the interaction of the factors studied, while a two-factor analysis analyzed each factor’s influence (or lack of influence) individually. The statistical analysis was performed using the StatgraphicsThe results of the present study showed that the addition of ‘Signuredda’ bean flour to durum wheat semolina can change the technological characteristics of the doughs, as well as the qualitative characteristics of the bread produced. In particular, the mixes obtained with various integrations of bean flour recorded higher values on the farinograph than those recorded by semolina alone, regarding the water absorption, attributing to the capacity of the legume proteins to absorb a greater amount of water. The development time of the dough was shorter than that required for the semolina, while the stability increased. Regarding the leavening tests, these data demonstrated a greater leavening capacity of the doughs obtained from semolina integrated with bean flour compared to those with semolina alone. Therefore, it is possible to confirm that the use of bean flour favors the leavening process and the increase in the volume of the dough.The data collected on the finished product, after cooking, indicated the possibility of using ‘Signuredda’ bean flour, partially replacing the durum wheat semolina, for the production of biofortified bread. In particular, it was possible to obtain with the bean flour a bread with good chemical-physical characteristics. This was particularly the case regarding the volume and porosity parameters, which tend to increase as the percentage of substitution increases, making them superior to those achieved with durum wheat semolina bread alone. The crust color was darker in the bread obtained with the highest percentage of integration (10%), while the other percentages were similar to those from bread made with semolina alone.Our promising results allow us to state that the replacement percentage of 7.5% was the best result, as it recorded the best values for almost all parameters.Concerning the staling process, even if the breads integrated with bean flour showed a greater decrease in volume and moisture content, our results indicate staling was at the levels of the control bread. The texture values were lower than those of SC-100%, indicating that the breads with added bean flour are softer and more resistant to staling, and therefore keep better and for longer.


Atta vollenweideri leaf-cutter ants. Mandible movements from live restrained ants were reconstructed in three dimensions using a multi-camera rig. Rigid body kinematic analyses revealed strong evidence that mandible movement occupies a kinematic space that requires more than one rotational degree of freedom: at large opening angles, mandible motion is dominated by yaw. But at small opening angles, mandibles both yaw and pitch. The combination of yaw and pitch allows mandibles to ‘criss-cross’: either mandible can be on top when mandibles are closed. We observed criss-crossing in freely cutting ants, suggesting that it is functionally important. Combined with recent reports on the diversity of joint articulations in other insects, our results show that insect mandible kinematics are more diverse than traditionally assumed, and thus worthy of further detailed investigation.Insects use their mandibles for a variety of tasks, including food processing, material transport, nest building, brood care, and fighting. Despite this functional diversity, mandible motion is typically thought to be constrained to rotation about a single fixed axis. Here, we conduct a direct quantitative test of this ‘hinge joint hypothesis’ in a species that uses its mandibles for a wide range of tasks: This article is part of the theme issue ‘Food processing and nutritional assimilation in animals’. As illin vivo observations, or detailed analyses of the evolutionary history of mandible morphology )rphology . In the rphology , and arerphology –25. Howerphology ,26, mandrphology ,25,27–31rphology , cockroarphology  and beetrphology ,35, haverphology ,36,37), rphology ,25,37.Another consequence of the emergence of dicondylic mandible joints is a putative simplification of possible mandible motion. A single condyle that articulates like a ball-and-socket joint allows three rotational degrees of freedom (DoF). Two condyles, in turn, can form two ball-and-socket joints, which are then thought to constrain mandible motion to rotation about a virtual axis that connects their centres . Using  ) DLTdv8a ,65, alon (d)A. vollenweideri worker was selected from the foraging box. We selected six ants within a narrow body mass range to minimize potential size effects (20.4–23.6 mg). Each ant was positioned on a custom 3D printed mount which featured a clamp, designed to physically restrain the head-capsule during the recordings. The mounted specimen was chilled on an ice block for 1–2 min to reduce activity during subsequent marker placement. Markers were added to enable consistent and accurate tracking, using a small insect pin dipped in correction fluid . Two constraints informed marker placement: (1) to increase the accuracy of downstream kinematic analyses, the marker positions needed to be far away from each other and yield non-collinear line elements, at least one of which is approximately perpendicular to a perceived dominant axis of rotation (see below); (2) markers needed to remain visible on at least three cameras throughout the movement. Markers were added to the second and last tooth , as well as at a coordinate near the mid-point of the mandible, opposite to the masticatory margin of the left mandible . The mounted and marked ant was kept on the ice block until the set-up was ready for acquisition; this helped to reduce active mandible movements during the wait period, which often led to loss of the markers via mandible scraping. Preliminary trials indicated that the brief cooling period had no discernible impact on subsequent behaviour. Some ants readily displayed biting movements, so the cameras were triggered, and around 2000–3000 frames were captured (corresponding to 30–50 s recording time); otherwise, individuals were encouraged to move their mandibles by a gentle puff of air. Ants used for the trials were released back to the foraging box after filming was completed.For each trial, one mandible a,c,d. ThAll recordings contained multiple mandible movement sequences, and were reviewed post-trial in ImageJ v. 1.53j . Frame s (e)R). Three non-collinear points, A, B and C, on the rigid body then suffice to determine vB/A, vC/A and rC/A are the instantaneous relative velocity and relative position vectors for points A, B and C, respectively. The location of the IHA can then be found relative to any reference point X on the body via an expression that again involves the angular velocity and the instantaneous linear velocity of point X )the IHA .In principle, rotational joint DoFs can then be inferred by inspecting the temporal variation of the orientation and location of the IHA throughout a motion sequence. For a hinge joint, for example, the IHA degenerates to a single rotational axis with constant orientation and location. However, the instantaneous relative and absolute velocity vectors, required to estimate the orientation and location of the IHA, are generally unknown, and thus need to be approximated via the average relative velocities over a finite time, equal to the instantaneous velocities only in the limit of an infinitesimal time step . ,80. 75,8Atta mandibles, we instead developed a method that leverages the advantages of cost functions, but can estimate the orientation of the rotational axis independent of joint translation. The general idea is to estimate the axis with fixed orientation and location that describes as much of the observed kinematics as possible—we will call this axis the best continous axis of rotation, or bCAR. The hypothesis of a dicondylic hinge joint will then be assessed quantitatively by statistical comparison of the explained versus observed mandible kinematics.Because leaf-cutter ant mandibles are small (length of about 1.8 mm), we anticipated that even best-case signal-to-noise ratios may be such that direct calculation of the angular velocity vector would yield noise-ridden estimates of the IHA, preventing robust conclusions on rotational joint DoFs through inspection of its temporal variation. In order to conduct a direct test of the hinge joint hypothesis in mc, across N frames :2.2CL=∑Dp are the average distances between each mp and mc across all frames. If the mandible rotates about a fixed point, then all markers populate the surfaces of concentric spheres with radii Dp, and minimization of equation , around the putative joint centre (see above).Here, equation  yields t 1c, see  for a moequation , becauser and r be an arbitrary vector that connects two points on a rigid body, and let this body rotate by a movement angle θ about an axis with fixed orientation α, and thus rp that connects two points on a rigid body forms a constant angle αp to d). Crucially, this angle is invariant to translation of either the body or the rotational axis. αp for P − 1 independent vectors spanned by P markers across N frames:kth time instant, and In order to determine the orientation of the rotational axis,  formula . Let r babout R^ d. CruciaP = 3 markers and N = 2 frames close in time, minimization of equation mp and mc across all recorded frames per individual. The magnitude of this variation was then assessed by comparing it with (i) the accuracy of the distance measurements on the calibration target ; (ii) the size of one image pixel (7 μm); (iii) the lengths of the mandible (about 1.8 mm) and of the effective mandible in-lever .In order to test how well mandible kinematics can be described as rotation about a fixed point, we assessed the variation of the distance between  μm, see ); and ; and the ratio between the observed and predicted distance moved by the mandible, from here on referred to as distance metric. The observed distance is the total distance covered by the individual markers; the predicted distance is the sum of distances between the marker locations projected onto the plane of rotation.To test whether mandible kinematics can be accurately described as rotation about an axis with fixed orientation, we extracted the residuals resulting from the minimization of equation —the diffequation , with th & Slate , implemeb). All kinematic calculations were conducted in Python v. 3.9.13, and all statistical analyses were conducted in R v. 4.1.2. All data reported in the text correspond to mean ± standard deviation unless otherwise indicated.In order to compare results across ants, we converted individual-specific movement angles into global opening angles independent of the orientation of the rotational axis; we defined the opening angle as the angle spanned by the vector that connects the joint centre with the most distal tracking point and the normal of the sagittal plane figureb. All ki (g)a). The model, consisting of a ‘head-capsule’ and a ‘mandible’, was designed in Autodesk Fusion 360  to have similar dimensions to a 20 mg worker ant head-capsule and mandible, and was printed using a stereolithography resin printer . The mandible was held in place within the head-capsule by an insect pin that served as rotational axis; rotation about this axis was induced with a gentle push from a fine-tip paintbrush (n = 3 instances from one model). The resulting movement was recorded using the set-up described above, and the 3D coordinates of the following landmarks were extracted for each frame: three points on the mandible ; two points on the longitudinal axis of the insect pin to serve as the ground truth rotational axis; and two points on the head-capsule to define a horizontal vector orthogonal to the rotational axis. The data were then subjected to the same analysis as described above to estimate the error associated with the 3D reconstruction and tracking, and the accuracy of the rigid body kinematic analysis.In order to validate the experimental and analytical methods, we tested them on experimental data obtained from a well-defined physical model with a hinge joint inset in a. The mo (h)A. vollenweideri worker with a body mass of 23.7 mg, obtained for a previous study [3D printed models and computer animations were used to intuitively explore and visualize mandible kinematics . To thius study , and simus study , were exus study . After f. 3 (a)a). The vma and ala resemble ball-and-socket joints; the dma, in contrast, is elongated and akin to a hinge . The cuticular surface of the ala is partially covered in a dense field of short spine-like protuberances, which terminate in either a single peak or multiple peaks (figure 2a(iii)). A region of the ala surface that is microscopically smooth and completely devoid of protuberances is exposed when the mandible is fully closed ). Long slender hairs are present on the lower  half of the ala, but not on its dorsal half (figure 2a(iii)). Both the vma and the dma cuticular surfaces lack the spine-like protuberances. The slender hairs are present, but with lower density (figure 2a(iii)).The mandible interfaces with the head-capsule via the mandible stem. The stem bears the ventral mandibular articulation (vma), the atala  and the dorsal mandibular articulation (dma), which together define the joint a. The vm a hinge a,b. The  (b)A. vollenweideri worker ants draw their mandibles from approximately fully open to almost fully closed. This motion visually resembles transverse adduction . Mandibles were sometimes closed sufficiently to overlap, in particular when cuts were initiated at the free leaf edge, or when workers attempted to cut thick leaf veins . Notably, there was no apparent pattern of preference for either the left or right mandible ending up as superior. Indeed, we observed swaps in superiority during in vivo cutting of thick leaf veins, leaf lamina and thin PDMS films . That both the left and right mandible can be superior suggests that the kinematic space accessible to the mandibles may not be limited to rotation about a fixed axis: mandible ‘criss-crossing’ likely requires mandible pitch , in addition to yaw . We also observed this pitch movement in experiments with restrained live ants : when a worker ant was mounted such that her head was held steady and the left mandible was physically prevented from opening, the mandible moved downward upon interaction with the right mandible (figure 3c). When the right mandible re-opened, the left mandible sprang back to its initial position (figure 3c (iv)). This joint elasticity was retained in a freshly dissected ant head: when a gentle torque was applied to a closed mandible using tweezers, it pitched approximately about the horizontal axis of the vma, before returning to the original position upon removal of the torque (figure 3d(i–iv) and electronic supplementary material, video SV3)—a motion visually resembling the pitch observed in live ants (figure 2d(iv); electronic supplementary material, video SV4). Thus, the mandible joint articulation appears to permit pitch in regions of mandible overlap.When cutting bramble leaf lamina,  (c)a,b and electronic supplementary material, videos SV5 and SV6). In order to formally assess whether the overall motion patterns can be explained by rotation about a fixed axis, we subjected kinematic data from six A. vollenweideri specimens to quantitative kinematic analysis.The 3D kinematic space occupied by mandible movements mirrored the qualitative observation of mandible movements during unrestrained cutting and biting: mandible motion is dominated by transverse adduction, but mandibles appeared to both pitch and yaw in regions of mandible overlap . The standard deviation of these distance indicates negligible deviation from rotation about a fixed point: it corresponds to about three times the accuracy of the calibration target dimensions, about one image pixel, less than 1% of the mandible length, less than 5% of the effective in-lever, and is in good agreement with the standard deviation obtained from a physical hinge joint model, which serves as a proxy for experimental noise . Furthermore, the derived joint centre was in close proximity to the initial guess informed by morphology (putative joint centre): it was shifted toward the sagittal plane by 0.067 ± 0.037 mm, or 5–10 pixels . To test the hypothesis that ant mandibles rotate about a single axis with fixed orientation, we estimated the orientation of the axis that minimizes the variation of the angle spanned with two mandible-fixed line elements across the kinematic space—the bCAR (see equation (n = 6). Qualitative inspection of the residuals suggested that this result emerged from a systematic widening of the residuals in the region of mandible overlap, hinting at heteroscedasticity (figure 4d). In contrast, for widely open mandibles, the residuals were small and consistently close to the expected zero line . In order to account for this systematic pattern in our analysis, we next determined the largest movement range for which the residuals were uncorrelated, continuous, independent and normally distributed. To this end, we iteratively truncated the kinematic space to smaller and smaller movement ranges, starting from the region of mandible overlap where residuals appeared inflated. We then re-calculated the bCAR, residual angles, and the movement angle range after each truncation step, and re-tested the residual angles until all assumptions were met for both line elements . This procedure suggested that mandible kinematics were consistent with rotation about a single fixed axis as long as opening angles were larger than b and electronic supplementary material, video SV2). The average cut-off angle was subsequently used to split the kinematics space of each individual into two characteristic regions. For each truncated region, the bCAR, the standard deviation of residuals and the distance metric were extracted separately, and compared with the global values across the entire kinematic space.To test the hypothesis that ant mandibles rotate about a fixed point, we estimated the location of a joint centre as the coordinate that minimizes the variation in the distances between the joint centre estimate and mandible markers across the kinematic space (see equation ). A suite). First, both the standard deviation of the angle residuals and the distance metrics for all three markers decreased significantly . To put both values into perspective, the corresponding results for the physical hinge joint model were e). Second, the orientation of the bCAR was highly consistent across individuals, with a mean deviation about the global average of n = 6, f). In all individuals, the rotational axis was approximately aligned with the dorso-ventral head axis (deviation of f). Given that the global orientation estimate is influenced by tracking error, fitting error, inaccuracy in head landmark placement, and biological variation, the small deviation across individuals strongly suggests a robust estimate of a consistently oriented bCAR in this region of the kinematic space.For opening angles greater than 68°, which encompass most of the mandible movement range, the inference that a single axis of rotation sufficiently explains the mandible kinematics was supported further by two independent pieces of evidence e. First,ectively e. Second∘ n = 6, f. In alln = 6). Thus, rotation in this region is multi-axial, consistent with the initial speculation based on qualitative observations of mandible kinematics. In further support of this conclusion, both the standard deviation and the distance metrics in this region increased upon truncation c); second, fitting a single rotational axis to data from opening angles larger than 68° resulted in small and randomly distributed residuals of the cost function defined by equation (e); and third, the standard deviation of the residuals and the distance metrics in this region of the kinematic space were small, and comparable to those obtained from a physical hinge joint model (e).The majority of the kinematics space occupied by mandible movements reflected mandible yaw, and can be accurately described as rotation about a single dominant axis of rotation. This conclusion is supported by three independent lines of evidence. First, the distance between mandible markers and a derived joint centre is highly consistent across the kinematic space c; secondequation   and electronic supplementary material, video SV3). However, alternative orientations may bring other functional benefits. In general, if the rotational axis has components in more than one of the three body planes, the mandible opening angle will have components in all three planes—mandibles will yaw, roll and pitch with respect to head-fixed reference planes. Thus, bi-axial mandible movement with respect to head or body planes, as observed in a trap-jaw and a ponerine ant [per se. Instead, it may merely suggest an inclined rotational axis, advantageous because it may allow ants to handle objects of different sizes with different parts of their mandibles [The functional implications of the rotational axis for bite mechanics are well appreciated, but the behavioural and functional significance of its orientation with respect to a head- or mandible-fixed coordinate system has received less attention ,50–52. Ir areas’ . Mandiblr areas’ . In silv insects , or the le gapes , which arine ant ,50, is n (b)e). Thus, our results provide strong evidence against the null hypothesis of a simple hinge joint in A. vollenweideri ants. How is mandible pitch actuated, and what is its biological function?Qualitative observations of mandible movements during cutting and biting suggested that mandibles can pitch: both the left and right mandibles can be superior when mandibles are closed, and swaps in position occurred across repeated closing cycles figures e. Thus, A. vollenweideri is actuated by a single pair of antagonistic muscles. Each muscle is attached to the mandible via its own apodeme, and thus has one main line of action [Atta, the net muscle vector is almost perfectly aligned with the anterior–posterior axis [Odontomachus brunneus trap-jaw ants: during rapid mandible closure, mandibles first followed a circular trajectory, but out-of-plane movements occurred as mandibles overlapped [Each mandible in f action . Activatf action ,55), it ior axis ,86, and erlapped . Re-direA. vollenweideri, a similar effect may help to keep the blades tightly connected during scissor-cutting. Indeed, in both living and dead specimens of A. vollenweideri, the gap between the two mandibles was narrow (figures 2a(i) and 4b), and mandibles of dead ants sprang back to their original position after pitch was induced by application of an external torque, indicating joint elasticity. Second, during the cutting of tough materials such as primary or secondary leaf veins [figure 3b and supplementary material in [Based on our observations of both unrestrained and restrained cutting, we suggest three functional benefits of mandible pitch. First, leaf-cutter ants frequently use their mandibles like scissors, for example when they initiate cuts at the free edge of a leaf lamina ,93. As aaf veins , mandiblerial in ), remini (c)A. vollenweideri mandible joints have more than one DoF. Mandible joints with multiple DoFs were likely the original condition in early hexapods, which possessed monocondylic mandibles actuated by multiple muscle groups [A. vollenweideri, joint articulations also depart from classic dicondyly: the ventral articulation (vma) resembles a ball-and-socket joint, but the dorsal articulation (dma) is elongated, cylindrical, and thus conspicuously distinct from the vma in shape (a). Without the coupling of dorsal and ventral ball-and-socket joints to constrain the mandible—as observed in obligate dicondylic taxa—additional DoFs become accessible. On the basis of these observations, it is evident that neither facultative dicondyly, obligate dicondyly, nor secondary monocondyly adequately describes the mandibles of A. vollenweideri. Instead, we propose that they represent ‘secondary facultative dicondyly,’ characterized by the following conditions: (i) two condyles exist, but have distinct morphology; (ii) mandible kinematics involve more than one DoF, but the dominant movement is rotation about a single fixed axis, actuated by a single antagonistic pair of muscles; and (iii) additional DoFs emerge through changes in sustainable joint reaction throughout the kinematic space, or via physical interactions between overlapping mandibles. Recently, an elongated dma has been identified as a characteristic trait of the Formicidae [We have demonstrated that e groups . Silverfe groups ,24,25, we groups ,22. These groups . These me groups . In lighe groups . In A. vin shape a. Withourmicidae ,96, raisJaw kinematics in vertebrates have received significant attention, but the equivalent problem in insects remains quantitatively unexplored. There is an increasing body of evidence that suggests that insect mandible kinematics are considerably more diverse than traditionally appreciated—representatives of at least two insect families that were thought to have simple hinge joints are capable of more complex mandible movements. Why did some neopteran taxa shift away from obligate dicondyly? Perhaps the more diverse mandible movement repertoire of wingless hexapods confers functional advantages that remain under-appreciated . Quantit


Pruning is a common practice in citrus for various reasons. These include controlling and shaping the canopy; improving phytosanitary health, productivity, and fruit quality; and facilitating operations such as harvesting and phytosanitary treatments. Because pruning is an expensive operation, its need is sometimes questioned. However, it has been proven to be particularly important in Mediterranean citriculture, which is oriented towards producing fruits for a high-quality demanding fresh market. Herein, we summarize and explain the pruning techniques used in Mediterranean citriculture and refer to the main purposes of each pruning type, considering citrus morphology and physiology. Pruning is a common cultural practice in citrus and one of the most expensive orchard maintenance operations. Even so, technical information on citrus pruning is relatively scarce, especially in peer-reviewed scientific journals.In the Scopus database, there are only 70 articles on citrus pruning [Search by: TITLE-ABS-KEY “citrus” OR “mandarin” OR “poncirus” AND TITLE “pruning”]. In this database, a yearly average of only four papers were published in the last ten years; none of these was a review. Therefore, a review of the cutting methods and the needs of the main citrus species and cultivars and a critical analysis of pruning are needed to put together and summarize the current knowledge on and technical aspects of citrus pruning.Citrus fruits are cultivated worldwide, but the technologies that can be used depend on the edaphoclimatic conditions and production objectives. The Mediterranean basin is a subtropical area where citrus have adapted and have significant economic and cultural importance. Moreover, citrus are now an essential part of the Mediterranean landscape and diet ,2.This review will address general aspects of citrus pruning. It will focus, in more detail, on the practices used in Mediterranean countries, according to the authors’ experiences after several years of work on pruning trials.Although pruning is an ancestral practice, it originated many years after agriculture was consolidated as a major activity of humankind. Reports from the time of Ancient Greece indicate that the first “pruning” was performed by livestock  and by meteorological events (wind and hailstorms). Farmers began to observe that these “pruned” trees behaved differently from unpruned trees. As a result, farmers tried to reproduce this livestock-based and meteorological “pruning” artificially. From then on, knowledge continued to evolve, and pruning is now considered an essential practice for many crops ,4.Pruning consists of the removal/suppression of plant parts such as branches, roots, and buds. Some authors limit the definition of pruning to the removal of vegetative or fruit-bearing organs, provided that such removal affects the physiological behavior of the plant in question . PruningPruning allows tree growth management and development using cuts and affects the plant’s physiological behavior . TherefoThe plant’s phenological cycle must always be considered when planning to prune it; thus, knowing the stages of the citrus plant’s phenological cycle, such as shoot growth, flowering, and fruit development, is fundamental .In citrus, flowering and vegetative growth depend on the formation of shoots, which arise from apical and axillary buds. Shoots can be of five different types, as illustrated in In subtropical climates, there are usually three periods of shoot growth per year: spring, summer, and autumn . FloweriIn subtropical climates, flowering and other related aspects can be used to group cultivars according to their bearing habits: (i) single-annual-bearing cultivars (SB), with regular production only once a year, every year; (ii) multiple-annual-bearing cultivars (MB) that produce more than once a year, every year; and (iii) alternate-bearing cultivars (AB) with abundant yields one year and scarce or no yields the next.The shoots formed in spring develop from the axillary buds of the previous year’s branches that were formed in summer and autumn  7,13]. ,13. 7,13Fruit development occurs after flowering and fruit set. Fruit development has three main stages  15]:  shoSMS cultivars form short and usually multiple shoots (more than one shoot per node) a. MediteLSS shooting cultivars form long, usually solitary shoots (only one shoot per node) c. Lemon IS cultivars form shoots that are not as long as in LSSs but are longer than those of SMS cultivars; sometimes, they form more than one shoot per node b. Most oThere are also cultivars exhibiting characteristics that lie somewhere between these main types of shooting habits, and not all the shoots on a tree are characteristic of that shooting type of cultivar.2 is photosynthetically reduced to carbohydrates. Those carbohydrates not readily used by the plant’s cell metabolism are stored, mainly in leaves and branches, with a smaller amount being stored in the roots. The maximum amount of carbohydrates the tree stores is reached just before spring flush :A t:A t95]:AIn alternate-bearing cultivars, a high fruit load in one year leads to the severe suppression of flowering the following year . TherefoRecovery pruning is a strong-to-very-strong-intensity pruning carried out after several years without regular maintenance pruning ,97. It aThe fruit development period lasts for over one year in late cultivars such as the orange trees ‘Valencia Late’, ‘Ovale’, and ‘Dom João’. No matter when such a cultivar is pruned, pruning results in fruit removal, which directly reduces the subsequent yield. Therefore, many growers avoid pruning, postpone it, or prune too little to prevent production losses while also reducing the operation’s cost. When maintenance pruning is not performed or is improperly performed, trees tend to form large crowns with dense foliage on the peripheries a 41,78],7841,78]There are several limitations related to oversized canopies: dense foliage outside limits the aeration and light entry through the canopy ,89,98; tUsing recovery pruning, some scaffold and large branches should be removed with thinning cuts b to openThe checking of the branches to be removed must be done from the inside of the tree canopy, where it is possible to better evaluate branch structure and distribution. Recovery pruning is also an excellent opportunity to remove large-diameter or poorly positioned branches left by inadequate or poorly executed formative pruning ,73.Recovery pruning reduces the next year’s yield  in late-Rejuvenation pruning aims to rejuvenate trees and can be performed on aged and depleted yet healthy trees . It remoAlthough old, the trunk and scaffold branches contain adventitious buds from which new shoots can emerge. New branches can form from these new shoots, allowing canopy renewal b. After Rejuvenation pruning causes a strong imbalance between the above- and below-ground parts of the plant . TherefoThere are other brief pruning operations that some growers may consider necessary, such as cleaning pruning, phytosanitary pruning, and water sprout removal.Cleaning pruning removes dead branches affected by biotic or abiotic stress . It is a straightforward operation that does not require as much expertise as other pruning types. Deadwood removal can be performed at any time of the year ,11383,11Topping should be performed before the trees in question get too tall and should be included in the orchard maintenance pruning program. When the canopy is too high, harvesting and phytopharmaceutical treatments are more difficult ,113. LowLonger intervals between topping operations are more expensive because the cut is harder, and wear and tear on cutting equipment is higher. Yield losses due to low-intensity topping are insignificant unless the trees are very large, especially since fruit density tends to be higher in smaller trees .Topping should be performed in a flat cut  whenever trees are small, narrow, or were hedged in an open angle. If the hedging angle is sufficient in narrow rows, topping can be performed in one pass down the row ,114,117.The optimal tree height depends on the distance between the trees, the hedging cutting angle, and the trees’ width. The cutting height can range from 3 m to 6 m. Commonly, low-cutting heights are done to increase fruit size or to renew declining trees.Topping stimulates the formation of new shoots .Phytophthora, which causes a high rate of fruit rot. Moreover, when the skirts are too close to the ground, their branches and fruits are often damaged by passing machinery, herbicide application, and other cultural practices.Tree skirts consist of the lower branches, which are closer to the ground . In someSkirting is a pruning practice that eliminates the skirt’s lower part.C. unshiu) [Skirting prevents the foliage and fruits from being too close to the ground ,113, thu unshiu) .If mechanical pruning operations are frequent, each vegetation removal is light; additionally, frequent interventions avoid large diameter cuts, minimizing wear on pruning blades . The amoWhen pruning is performed mechanically in an orchard, it is essential to establish a pruning program . This plThe seven mechanical pruning strategies examples listed in Strategy 1 (THH/THH) consists of completely pruning all sides of the tree each year. This strategy can be a good option for early, vigorous cultivars. Early cultivars can be pruned before spring bloom, preventing flower or fruit removal and stimulating spring bloom. Vigorous cultivars may benefit from annual pruning because less vegetation is removed, cuts are smaller, and the wear on pruning equipment is less than with a lower pruning frequency. In one study, this pruning strategy did not reduce ‘Navel Foyos’ orange tree production, and the costs were 82% lower than those of annual pruning performed exclusively by hand . For theStrategy 2 (THH/--) consists of completely pruning all sides of the tree in alternate years. Strategy 2 can be a solution for less vigorous cultivars whose growth does not need to be controlled as much. In a study, this pruning strategy did not change yield, fruit size, and fruit quality in ‘Washington Navel’ orange compared to only manual or mixed  pruning. On the other hand, ‘Salustiana’ orange yield decreased by 17% compared to manual pruning, but there were no differences in fruit size and quality (average of 4 years of results) .Strategy 3 (TH/TH) consists of pruning each side of the tree in alternate years while pruning the top annually. This strategy may be appropriate for cultivars that grow vertically or produce vigorous vertical branches and where control over vertical growth is more urgent than control over side growth. This strategy was the fastest, the cheapest, and the most productive for the ‘Fino 95′ lemon in a study . This stStrategy 4 (HH/T) consists of cutting both sides of the canopy or topping in alternate years. This strategy may be appropriate for cultivars of medium vigor, where the annual pruning of the entire canopy is not warranted.Strategy 5 (H/H/T) consists of alternately pruning each side of the canopy or the top every three years. This strategy can be used for low-growing cultivars where more frequent pruning is unnecessary.Strategy 6 (HH/HH) consists of annually pruning both sides of the canopy. It may be appropriate for vigorous, open-growing (strongly spreading or drooping) cultivars where topping is unnecessary. This strategy was 56% cheaper than strategy 1 and 88% cheaper than manual pruning (4-year average) for the ‘Clemenules’ clementine. No differences in fruit size or yield (4-year average) were observed compared to strategy 1 or manual pruning .Strategy 7 (T/T) consists of topping every year. It may be appropriate for vigorous, upright-growing cultivars. In a study, this strategy led to the formation of larger fruits in the grapefruit ‘Star Ruby’ .Mechanical pruning can be used as a complement to manual pruning. There are many different possible strategies, and it is up to the farmer to define a program that considers manual and mechanical pruning operations and their frequency .One of the most frequent strategies is to mechanically prune the skirts (skirting) while the rest of the canopy is pruned manually. Another possible strategy is controlling the trees’ height using a regular topping operation while the middle part of the canopy is pruned manually . Other gDepending on its intensity, pruning may produce significant waste  ,127. TheThis practice consists of leaving the pruning waste in the orchard inter-rows to be mechanically shredded a. After The crushed waste decomposes, promoting soil enrichment with organic matter . This in2), methane (CH4), carbon monoxide (CO), and other gases, as well as tiny particles (smoke), into the atmosphere [Waste incineration in the orchard is an outdated practice that must be avoided and is justified only in exceptional cases. Burning waste releases large amounts of carbon dioxide (COmosphere ,137. Themosphere , banned mosphere .There are other uses for pruning waste that are less common but still possible ,141. LeaThe objectives of citrus pruning are to control tree development and shape, increase fruit quality and yield, facilitate disease and pest control, reduce production costs, and mitigate alternate bearing. Understanding the general aspects of citrus morphology and physiology is crucial for designing appropriate pruning actions. Training systems and pruning procedures, techniques, and intensity must be carefully selected and executed considering the tree, edaphoclimatic conditions, and pruning objectives in question. Mechanical pruning is an alternative to strictly manual pruning as it can significantly reduce the pruning costs. Still, it has some disadvantages compared to manual pruning such as tree and fruit damage that negatively impacts the fruit production for the fresh market as a part of Mediterranean citriculture. Pruning generates waste that must be disposed of properly. Conversely, pruning waste may be a resource for soil enrichment and is valued as a raw material for essential oils, animal feed production, and other uses. Pruning affects a plant’s physiological behavior and is crucial for managing citrus trees’ growth, development, and production. Well-planned and well-executed pruning is essential for the prosperity of a citrus orchard.


Intermittency represents a certain form of heterogeneous behavior that has interest in diverse fields of application, particularly regarding the characterization of system dynamics and for risk assessment. Given its intrinsic location-scale-dependent nature, wavelets constitute a useful functional tool for technical analysis of intermittency. Deformation of the support may induce complex structural changes in a signal. In this paper, we study the effect of deformation on intermittency. Specifically, we analyze the interscale transfer of energy and its implications on different wavelet-based intermittency indicators, depending on whether the signal corresponds to a ‘level’- or a ‘flow’-type physical magnitude. Further, we evaluate the effect of deformation on the interscale distribution of energy in terms of generalized entropy and complexity measures. For illustration, various contrasting scenarios are considered based on simulation, as well as two segments corresponding to different regimes in a real seismic series before and after a significant earthquake. Intermittency, generally understood as the pseudo-periodic occurrence of higher-level or variation episodes within a certain regular behavior, is considered a phenomenon of interest in very diverse fields of applications . Structural characteristics associated with such an effect, in relation to the underlying generating process, often constitute a primary objective in environmental studies, as they provide relevant information for the detection and prediction of critical events and for risk assessment.Different manifestations of intermittency have lead to various interpretations and formal definitions of this concept. These are essentially related to its genesis and, as a consequence, to the nature of its effects as a certain form of heterogeneous behavior. Depending on the domain where such heterogeneities occur, it is common to distinguish between ‘isolated’ types of intermittency, for heterogeneities in the spatial/temporal domain, and ‘non-isolated’ types of intermittency, associated with heterogeneous scaling, closer to the concept of multifractality . A commDeformation has been used, among other purposes, as an approach to define flexible classes of nonhomogeneous random fields from homogeneous ones :Fx(a)=Assilicos  explain It must be noted that the flatness factor is a special prominent case of general sparsity wavelet-moment-based measures of intermittency, defined byexample, ).As often mentioned in the literature, the CWT provides a complete description of a signal at all scales and locations, and it is then usually adopted when the user wants to depict the signal behavior at selected scale-space ranges. As is well-known, when an efficient non-redundant compression of information, yet allowing the signal reconstruction, is the objective, a particularly interesting alternative is given by the orthogonal discrete wavelet transform (DWT) .In our analysis on the effect of deformation in terms of the scalogram and related intermittency indicators, we use the CWT to give a complete representation for a ‘continuous’  scale-space range.A key aspect in the analysis through wavelets is the choice of the mother wavelet, since the main objective of this type of analysis is to extract, by convolution, certain characteristics of interest in the structure of a signal. Therefore, the wavelet function should be selected depending on the target features to be detected. For example, it is known that the Haar wavelet is appropriate to identify a sustained change in the signal level, while the Morlet wavelet is more appropriate to seek concentration in time–frequency of signal energy (see ). This iH are For a discrete probability distribution  entropy  is definTwo important generalizations of Shannon entropy, both based on a deformation parameter determining a power distortion on the argument distribution, are given by Rényi and Tsallis entropies.q of the discrete probability distribution Specifically, Rényi entropy  of orderq of the discrete probability distribution On the other hand, Tsallis entropy  of orderShannon entropy is the limiting case of Rényi and Tsallis entropies as Whilst Rényi entropy is extensive, meaning that is additive for independent systems, Tsallis entropy is non-extensive except for Based on Rényi entropy, a product-type generalized complexity measure can be defined asβ<∞  and (5) in (1), the b. On the other hand, if the deformation has a local effect of contraction  and (8), we can see that the Hence, when the signal is of ‘flow’-type, the energy is reduced where the deformation has local dilation properties, F, which averages squared values of the local intermittency measures (LIM) obtained within each single scale. In the case of a ‘level’-type signal x, and for a deformation As introduced in x, since the For a ‘flow’-type signal Similar results can be obtained, under analogous developments, for the general sparsity-wavelet-moment-based measures of intermittency (2).For a general non-linear deformation, with varying contraction or dilation effects, the local transfer of energy between scales leads, by the In this section, we study the multiscale effect of deformation on intermittency, first based on the analysis of several selected simulated scenarios. Secondly, two segments corresponding to different activity regimes in a real seismic signal are also analyzed and compared with this purpose.In this subsection, we first consider two versions of the ARMA modelF curves are plotted. In the case where the noise is Cauchy a well-defined interscale coherence of energy concentrations over time can be observed, and a strong global intermittency behavior is clear from the F curve. However, when the noise is Gaussian, the behavior is more regular, the F curve shows slightly significant values only for some higher scales.F curves are much less smooth. In what follows, for illustration purposes, all the analyses are only based on a Haar wavelet.As commented at the end of To study the effect of deformation of the time domain  on the distribution of the energy concentrations over time at different scales, we consider that the signals to be analyzed are observed at times 024]see .F curves corresponding to the original signal and its transformation after deformation for the series with Cauchy noise, it can be seen that intermittency levels are modified under deformation. A similar effect is observed for the series with Gaussian noise, though in this case, with the original signal showing slightly significant values only for some higher scales, its transformation displays intermittency effects for all scales.Firstly, the effect of deformation on a ‘level’-type signal is analyzed based on simulated realizations from ARMA model (9) with Cauchy or Gaussian white noise; see F curves have a global decay for the range of scales considered. Also, a clear increase of intermittency levels is observed after deformation, as can be seen in Secondly, we consider series generated from the ARIMA model obtained by double integration of ARMA model (9), again with Cauchy and Gaussian white noise, and study the effect of a ‘flow’-type deformation. In all cases, the Finally, a complementary analysis for intermittency is performed focusing on the assessment of temporal heterogeneities in the scale distribution of energy. For this purpose, as mentioned in However, there are clear differences between both scenarios corresponding to Cauchy or Gaussian white noise. For the original signals, the entropy values are locally more sensitive to the sliding steps, although showing a more stable average level, in the Cauchy case. Furthermore, deformation enhances the intermittency effects of extremal inputs from the Cauchy white noise through the ARMA model, noticeable in the heterogeneous structure of the entropy curves.An analogous analysis is performed under the ARIMA model, based on the realizations displayed in top plots of In this subsection, as an example with the aim of illustrating the interest of the aspects discussed above in a real situation, we consider the seismic signal  comprising the earthquake that happened on 6 April 2009, with epicenter near L’Aquila , with magnitude 6.3 on the Ritcher scale see .Since it is expected that the dynamics of the system differ before and after the earthquake, we select two segments for comparative analysis, respectively, beginning at 01:29:00  and 11:24:03 . Given the intrinsic nature of the signal, a ‘level’-type deformation according to Equation (10) is applied.F curves increase after deformation. While this effect is moderated for segment 1, yet with flatness values becoming larger than threshold 3 for most scales, there is a drastic increase for segment 2, particularly enhanced at lower scales.The structural differences between both periods are maintained when the deformation is applied, as can be observed in Regarding the wavelet entropy analysis of the interscale relative energy distribution, it is interesting to note, as can be seen in In this paper, we study the effect of time deformation on the structure of signals displaying an intermittent behavior. Regarding the nature of the signal, we distinguish the cases of ‘level’- and ‘flow’-type magnitudes. Specifically, under a wavelet-based approach, we analyze the interscale transfer of energy derived from local contraction or dilation properties of deformation, and its implications on well-known intermittency quantifiers such as the ‘local intermittency measure’ (LIM) and ‘flatness factor’ (F). Furthermore, variations in the heterogeneous interscale distribution of energy are assessed using Shannon, Rényi and Tsallis entropies, as well as the Rényi-entropy-based product-type generalized complexity measure.In relation to the underlying generating random process, for an illustration based on simulation, we consider different scenarios regarding the dynamics dependence structure , and the random inputs marginal distribution (Gaussian and Cauchy). In addition, as an example based on real data, we select two segments from a seismic series, respectively, before and after a big earthquake, hence corresponding to contrasting systemic activity regimes, and compare the effect of deformation on their intermittency behavior.We must emphasise that our methodological approach is essentially empirical, in the sense that the wavelet-based analysis and extraction of information by different indicators is performed on observed or simulated signal data, which can be seen as realizations of a random system. Although we do not address any inferential objective in this paper, it is implicit that a researcher can potentially use this analytical approach under such perspective; for instance, in the case of repeated observations, or under model-based conditional simulation.q in generalized entropy measures , and its potential usefulness for detecting intermittency levels from the inter/intrascale distribution of energy; extension and interpretation of results in terms of diversity and considering alternative complexity measures; intermittency analysis of spatial or spatiotemporal signals; risk analysis, e.g., using quantile-based risk measures ; sensitivity with respect to the deformation parameter example, ), on losexample, ,35,36).