Fucus vesiculosus, Fucus distichus subsp. evanescens, Fucus serratus, Laminaria digitata, Saccharina latissima) were harvested and fucoidans isolated by hot-water extraction. Fucoidans were tested in several concentrations . Effects were measured on a uveal melanoma cell line (OMM-1) (oxidative stress), retinal pigment epithelium (RPE) cell line ARPE19 (oxidative stress and VEGF), and primary RPE cells (VEGF). Oxidative stress was induced by H2O2 or tert-Butyl hydroperoxide (TBHP). Cell viability was investigated with methyl thiazolyl tetrazolium (MTT or MTS) assay, and VEGF secretion with ELISA. Affinity to VEGF was determined by a competitive binding assay. Results: All fucoidans protected OMM-1 from oxidative stress. However, in ARPE19, only fucoidan from Saccharina latissima was protective. The affinity to VEGF of all fucoidans was stronger than that of heparin, and all reduced VEGF secretion in ARPE19. In primary RPE, only the fucoidan from Saccharina latissima was effective. Conclusion: Among the fucoidans from five different species, Saccharina latissima displayed the most promising results concerning oxidative stress protection and reduction of VEGF secretion.Background: Fucoidans are interesting for potential usage in ophthalmology, and especially age-related macular degeneration. However, fucoidans from different species may vary in their effects. Here, we compare fucoidans from five algal species in terms of oxidative stress protection and vascular endothelial growth factor (VEGF) interference in ocular cells. Methods: Brown algae ( Fucoidans are sulfated polysaccharides found in the cell walls of brown algae. Common to all fucoidans is a high amount of L-fucose, yet their structures are complex and variable among different species . FucoidaFucoidan could be of interest for the development of new AMD therapeutics, since it has been described to be anti-inflammatory, blood lipid-reducing and, most importantly, protective against oxidative stress and VEGF-inhibiting . Fucus vesiculosus) exhibits interesting effects on RPE cells including reduction of VEGF secretion and reduction of angiogenesis [Our group has previously shown that commercially available fucoidan , Laminaria digitata (LD), Fucus serratus (FS), Fucus vesiculosus (FV), and Fucus distichus subsp. evanescens (FE) in terms of two important factors for AMD development, i.e., oxidative stress and VEGF secretion in ocular cells, as well as their binding affinity to VEGF. For this comparison, the algal material of all five species were harvested in summer, identically prepared, and then extracted according to the same standardized protocol, leading to the fucoidans SL, LD, FS, FV, and FE.In the current study, we compared the fucoidans of five species of brown algae , 200 µM (85.67 ± 17.02%) and 400 µM (81.00 ± 15.51%) showed no effect on cell survival, 1000 µM displayed a significant reduction of cell viability compared to the control   a. A concSaccharina latissima, incubation of OMM-1 treated with 1 mM H2O2 and SL fucoidan induced significant changes according to ANOVA testing. Incubation with 1 mM H2O2 resulted in a reduction of cell viability to 68.75% (±5.07). Incubation with 1 µg/mL induced no significant protection (72.00 ± 3.04%), while 10 µg/mL, 50 µg/mL, as well as 100 µg/mL all significantly increased cell viability  . The differences between the groups treated with LD fucoidan were significant in ANOVA testing. Incubations with any concentration of LD fucoidan tested resulted in a highly significant protection of cell viability   . In ANOVA testing, significant differences between the groups could be detected. FS fucoidan increased cell viability significantly, but viability remained considerably low  . In ANOVA testing, significant differences between the groups could be detected. All concentrations of FV fucoidan resulted in a significantly increased viability of cells  . In ANOVA testing, significant differences between the groups could be detected. FE fucoidan significantly increased viability when used at 1–50 µg/mL  but not at 100 µg/mL (55.00 ± 22.63%) (In the experiments concerning the fucoidan from < 0.001) a. In the± 7.03%) b. Testin< 0.001) c. When t< 0.001) d. Finall 22.63%) e. p < 0.001; 50 µg/mL p < 0.001), significantly higher than that of FE , and significantly higher than FS . FV was significantly more effective than FE  and significantly more effective than FS . Finally, SL was significantly more protective than FE  and more protective than FS . FE and FS, however, displayed no statistically significant differences , 400 and 1000 µM H2O2 significantly reduced cell survival . However, none of these concentrations reduced cell survival to approximately 50% , while 10 µg/mL, 50 µg/mL, and 100 µg/mL significantly increased cell viability   . ANOVA testing showed statistically significant differences between the groups. Again, none of the tested concentrations of FS fucoidan conferred any protection. On the contrary, 10–100 µg/mL FS fucoidan significantly reduced the viability of the cells  . ANOVA testing showed statistically significant differences between groups. However, none of the tested concentrations of FV fucoidan induced a significant change in cell viability  . ANOVA testing showed statistically significant differences between groups. While FE fucoidan in concentrations of 1 µg/mL (61.00 ± 3.08%) and 10 µg/mL (61.50 ± 4.06%) displayed no influence on cell viability, both 50 µg/mL and 100 µg/mL FE fucoidan significantly reduced cell viability  (In the experiments concerning the fucoidan of  < 0.01) a. In the 16.55%) b. Testin< 0.001) c. When t 17.69%) d. Finall< 0.001) e. Saccharina latissima showing protection, Laminaria digitata and Fucus vesiculosus displaying no influence, and Fucus serratus and Fucus distichus subsp. evanescens showing an additional toxic effect on the cells. Taken together, the results differ profoundly from those found with OMM-1 cells, with only p < 0.01), from FS fucoidan (10 µg/mL (p < 0.01)–100 µg/mL (p < 0.001), and from FE fucoidan . The differences between SD fucoidan and FV fucoidan are not significant. Similar results are obtained with LD fucoidan, which does not significantly differ from FV fucoidan, but does differ from FE fucoidan and FS fucoidan in all concentrations tested . FV fucoidan differs from FE fucoidan at 10–100 µg/mL  and from FS fucoidan at all concentrations . FE fucoidan differs from FS fucoidan in the lower concentrations   .p < 0.001) in a collection time of 4 h, as tested in ANOVA and subsequent t-test. The effect of the five different fucoidans on the secretion of VEGF was compared in two different RPE cell types, the human cell line ARPE19 and primary RPE cells derived from porcine eyes. Untreated ARPE19 cells secreted considerably and significantly less VEGF, with a mean of 416.33 pg/mL (±415.27), in a collection time of 24 h compared to primary porcine RPE cells, which secreted a mean of 2386.88 pg/mL (±824.81) (Fucus vesiculosus (Sigma) with both ARPE19 cells and primary RPE cells (see below). Sigma fucoidan was applied to the cells and supernatant was harvested after 1 day, 3 days, and 7 days. After 7 days, the experiment was terminated and cell viability was measured. Using Sigma fucoidan on ARPE19 cells for one day, no statistical difference could be found in ANOVA . After 3 days of incubation, a strong VEGF reduction could be seen, which was significant in ANOVA and was significant at concentrations of 50 µg/mL and 100 µg/mL in further testing  (p < 0.001). For day 3 and day 7, both 50 µg/mL and 100 µg/mL displayed a significant reduction of VEGF    a. In ad< 0.001) b. No sig< 0.001) c.Based on these results, we investigated VEGF content after 3 days of incubation with fucoidan for all following experiments. VEGF content was normalized to cell viability and displayed in relation to an untreated control.Saccharina latissima showed a reduction of VEGF when applied in concentrations of 10–100 µg/mL     and 50–100 µg/mL , but not 10 µg/mL (0.77 ± 0.60)   a. Fucoid< 0.001) b. Simila< 0.001) c. Fucoid ± 0.60) d. Fucoid< 0.001) e. p < 0.05) or from FE fucoidan (10 µg/mL and 100 µg/mL both p < 0.05), while there were significant differences compared to SL fucoidan in all concentrations tested . SL fucoidan significantly differed from FV fucoidan in all concentrations but 10 µg/mL , while it differed from FE and FS fucoidan at concentrations of 1 µg/mL and 100 µg/mL (both p < 0.001). FV fucoidan did not display any significant differences from FS fucoidan, and only at 100 µg/mL when compared to FE fucoidan (p < 0.05). Finally, FE and FS fucoidan did not display any significant differences  generally displaying the strongest effect. The differences between the different fucoidans are not as profound as those found in protective effects and are generally limited to effects seen at certain concentrations. LD fucoidan differs only slightly from FV or FS fucoidan . This effect was no longer seen after 7 days ; 100 µg/mL 73.75 ± 27.21%)  . After 3 days, however, a significant reduction could be detected in ANOVA and was found in all concentrations assessed  d. In add < 0.01) e. No sig < 0.01) f. Based Saccharina latissima reduced the VEGF content of RPE cells at a concentration of 10 µg/mL , while the other concentrations showed no significant effect       a. None o< 0.001) b. Fucoid ± 0.64) c. Fucoid < 0.01) d. Fucoid < 0.01) e. Saccharina latissima showed a reductive effect on VEGF content in the supernatant. When comparing the different fucoidans, significant differences were only found between SL and LD fucoidan (at a concentration of 10 µg/mL) and between FV and FS fucoidan (at a concentration of 50 µg/mL)  . In the pathogenesis of the exudative form of AMD, VEGF is an important factor ,6. VEGF Fucus fucoidans bound significantly more strongly to VEGF than heparin (p < 0.01) . By adding increasing concentrations of unlabeled heparin, the detection of biotinylated heparin decreased (data not shown). As expected, incubation with a mixture of 0.5 µg/mL heparin and 0.5 µg/mL biotinylated heparin reduced the signal to 49.96% (±7.28) and thus the binding of biotinylated heparin to VEGF. All five fucoidans showed a significant affinity to VEGF compared to the control, tested in ANOVA and subsequent testing  . Moreove < 0.01) . Althoug < 0.01) .Fucoidans are of great interest to biomedical research, especially considering their possible application in age-related macular degeneration . HoweverIn this study, we have compared fucoidans from five different brown algae species harvested in summer and obtained from the same supplier . The preparation of the algal material as well as the extraction and purification of the fucoidans were performed in parallel according to a standardized procedure. Therefore, this study is well equipped to reliably examine the effects of fucoidans from different algal species. The aim was to compare the beneficial properties of fucoidans with regard to age-related macular degeneration.In our study, we focused on two major factors important for the development of age-related macular degeneration, i.e., oxidative stress and VEGF. While interference with these is without doubt of great interest, we are aware that other activities, e.g., anticomplementary, anti-inflammatory effects, or influence on lipid metabolism, are also potentially beneficial for impairing AMD pathogenesis and are of high interest for further testing.Fucus vesiculosus [We have previously shown that OMM-1 cells are protected by commercially available fucoidan . In conticulosus , as welliculosus  and, as Fucus fucoidans showed an ROS scavenging effect (manuscript in preparation) but did not protect ARPE19 cells from oxidative stress. Little is known about oxidative stress pathways in uveal melanoma cells, but an increased susceptibility due to the reduction of superoxide dismutase activity in uveal melanoma cells has been described . There iSaccharina latissima fucoidan) could be explained via these pathways. As mentioned above, uveal melanoma cells have been described to have a reduced SOD activity [However, independent of all of the results from simple cell-free assays, fucoidans may exhibit antioxidative activity by cellular effects. Accordingly, fucoidan has been shown to increase the expression of superoxide dismutase (SOD) in several experimental models and activate the transcription factor nuclear factor erythroid 2-like 2 (Nrf2), the “master regulator” of the antioxidative stress response ,30,31,32activity . Hence, activity . Indeed,activity  and Nrf2activity . It is factivity . TherefoFucus vesiculosus additively reduced VEGF expression when co-applied with the VEGF inhibitor bevacizumab [A major contributor to the pathology of wet AMD is the growth factor VEGF, and its inhibition is the only current treatment option for AMD patients. We have previously shown that commercially obtained fucoidan from acizumab . On the acizumab , with diacizumab . The intacizumab ,39. Furtacizumab ,40,41.In our current study, we were able to demonstrate antagonization as well as a reduction of VEGF secretion in ARPE19 cells, in which all five fucoidans were effective. In primary porcine RPE cells, however, only SL displayed a significant effect. Fucoidan was found to influence Stat3-regulated promoters, which includes the promoter of VEGF . But it The amount of VEGF secreted by primary RPE cells in this study was much higher than that of ARPE19 cells . In the presence of such high VEGF concentrations, the VEGF antagonizing mode of action obviously became ineffective, explaining the discrepant results in ARPE19 cells and RPE cells. Therefore, the effect of SL on VEGF secretion in primary RPE cells is even more remarkable.Saccharina latissima. Among the tested fucoidans, SL had the highest degree of sulfation, the highest molecular weight, and the highest degree of purity (under submission). But these parameters cannot explain its superiority or the ranking of the other fucoidans. Fucoidans from Saccharina latissima have been previously shown to be highly biologically active compared to those from other brown algae species [Saccharina latissima have been shown to inhibit angiogenesis in tumor models [Our data clearly show a positive effect of the tested fucoidans in terms of oxidative stress protection and VEGF inhibition, with the most promising fucoidan extracted from  species ,16. Furtr models . HoweverIn addition, VEGF secretion and oxidative stress are also involved in the pathomechanisms of diabetic retinopathy ,49. TherSaccharina latissima turned out to be most suitable for further investigations. In conclusion, we compared fucoidan from five brown algae species in terms of three activities that are considered promising for the treatment of AMD, i.e., their capacity for oxidative stress protection, inhibition of VEGF secretion, and binding affinity to VEGF. Based on these three basic parameters, the fucoidan from The uveal melanoma cell line OMM-1  was a kiPrimary RPE cells were prepared as previously described ,52. In bSaccharina latissima , Fucus vesiculosus , Laminaria digitata , Fucus distichus subsp. evanescens , and Fucus serratus , all harvested in summer and provided by Coastal Research & Management, Kiel, Germany. The fucoidans were extracted as previously described [v/v) ethanol, and was then extracted with aqueous 2% calcium chloride for 2 h at 85 °C under reflux conditions. The supernatants of the raw extracts were concentrated and precipitated with ice-cold ethanol in a final concentration of 60% (w/w). After centrifugation, the sediments were dissolved in demineralized water, dialyzed, and lyophilized. In addition, commercially available fucoidan from Sigma  was used. Fucoidan was solved in Ampuwa bidest , and then further diluted with appropriate cell medium for the cell experiments and phosphate buffered saline (PBS) for the VEGF binding assay, filtered through a 0.2 µm filter , and applied to the cells in final concentrations of 1, 10, 50, and 100 µg/mL. Fucoidans were extracted from dried stocks of the species escribed . Briefly2O2) to induce oxidative stress-related cell death, as previously shown [2O2 resulting in approximately 50% cell viability. In order to assess this, OMM-1 cells were treated with different concentrations  of H2O2 for 24 h and cell viability was investigated by MTS assay (see below). To investigate the potential protective effects of the different fucoidans, a concentration of 1 mM H2O2 was chosen. Cells were treated with fucoidan  30 min prior to the application of H2O2. OMM-1 cells were treated with hydrogen peroxide  induced a cell death of about 50% after 24 h, as detected by MTS assay. In addition, the cell death rate was highly variable after H2O2 incubation (see results). Therefore, we tested tert-Butyl hydroperoxide (TBHP), a more stable inducer of oxidative stress in RPE cells [Corresponding experiments to find the appropriate HPE cells , at concMTT assay is a common method in cell research  and was ® AQueous One Solution Cell Proliferation Assay ). The cell viability assay was performed in 96 well plates in phenol red-free medium with the same supplements described above. In each well, 20 µL of MTS solution was added for 1 h.The MTS assay is a commercially available viability assay and was used according to the manufacturers’ instructions  for 1 day, 3 days, and 7 days. A cell viability assay (MTT) was conducted after 7 days. According to these results, an incubation time of 3 days was chosen for experiments with the five different fucoidans. The medium was changed 24 h prior in ARPE19 cells and 4 h prior in primary RPE cells and the supernatant collected. Measured VEGF content was normalized for cell survival and is depicted in relation to that of untreated control cells.VEGF was detected in the supernatants of ARPE19 and primary RPE cells using commercially available ELISA kits  according to the manufacturer’s instructions. To establish the parameters of VEGF ELISA, we investigated time-dependent VEGF secretion in ARPE19 and primary RPE cells in the presence and absence of commercially available fucoidan from The affinity to VEGF of the test compounds was investigated with a competitive VEGF-binding assay using biotinylated heparin. In addition to the fucoidans, heparin  was tested.The wells of a 96-well Nunc-Immuno MaxiSorp microplate  were coated with 0.1 µg recombinant human VEGF 165 (R&D Systems Cat. 293-VE/CF) dissolved in 100 µL PBS overnight at 4 °C. After washing with PBS, the coated wells were blocked for 90 min at 37 °C with 100 µL of 5 mg/mL bovine serum albumin  and subsequently washed three times with PBS. During the blocking, 65 µL of 1 µg/mL heparin, biotin conjugate  in PBS, and 65 µL of 1 µg/mL test compounds in PBS were preincubated at 4 °C. For the blank and the 100% binding value, 100 µL PBS and 100 µL of 0.5 µg/mL biotinylated heparin in PBS, respectively, were preincubated at 4 °C. Aliquots of 100 µL of these solutions were pipetted into the coated microplate wells and incubated for 2 h at 37 °C with gentle agitation. After three washing steps, 100 µL of streptavidin alkaline phosphate conjugate  was incubated for 1 h at 37 °C with gentle agitation. The next steps involved three washings with PBS and incubation with 100 µL p-nitrophenyl phosphate substrate system  for 30 min in the dark. The reaction was stopped by addition of 25 µL 3 N NaOH, and the absorbance was measured at 405 nm. Blank values in the absence of biotinylated heparin were subtracted from the measured values. The reduction of the binding of biotinylated heparin by the test compounds is indicated as a percentage in relation to the binding of the biotinylated heparin alone. t-test was conducted. A p value of <0.05 was considered significant. All bars represent mean and standard deviation.All experiments testing fucoidans were independently repeated at least six times, experiments for establishing oxidative stress response were repeated at least three times, and the VEGF binding experiments were performed in duplicates on three different days. Statistics were calculated using Statistica 7  and Microsoft Excel . A Friedman’s ANOVA was performed, and, if a significant difference between groups was detected, a subsequent Student’s


In 2011, Benin’s National Malaria Control Programme (NMCP) organized a nationwide mass distribution campaign of LLINs throughout the country. Following this intervention, it was important to assess whether the level of susceptibility of malaria vectors to insecticides had remained the same as compared to the pre-intervention period. The current study investigated this.Anopheles gambiae sensu lato (s.l.) larvae from the 4 study districts were reared to adulthood and WHO susceptibility tests were conducted. The insecticides tested were deltamethrin (0.05%), permethrin (0.75%), bendiocarb (0.1%) and DDT (4%). Molecular species identification as well as, the characterization of the kdr L1014F mutation were also performed in the An. gambiae s.l. complex using PCR method.Larval collections were conducted in Ifangni, Sakété, Pobè and Kétou districts located in Plateau department, Southeastern Benin before (2009) and after (2012–2013) LLIN distribution. An. gambiae s.l. to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) was observed post-LLIN distribution, respectively: ,  and, . By contrast, susceptibility of vectors to bendiocarb (0.1%) remained the same  pre- and post-intervention. An increase in the kdr L1014F frequency was observed post-LLIN distribution [F(kdr) = 0.91)] compared to the pre-intervention period [F(kdr) = 0.56], p < 0.0001. Anopheles coluzzii and An. gambiae were the two molecular species identified in the study area.Overall, a significant decrease in mortality rates of kdr L1014F mutation after the intervention stressed at the time, the need for the development and implementation of effective insecticide resistance management strategies. At present, an update of the vectors resistance status in the area is also necessary for decision-making.The decrease susceptibility to pyrethroids and DDT as well as, the increase in the frequency of the  Plasmodium falciparum, the parasite responsible for the most severe form of human malaria, is Anopheles gambiae sensu lato (s.l.)  Fig. . In all on) Fig. .Fig. 2VaWith deltamethrin, the mortality rate pre-intervention was 100% in all districts. However, this rate decreased to 78.9% (95% CI 73.3–84.5%) in Ifangni (p < 0.0001), 86.8% (95% CI 80.6–93%) in Kétou (p = 0.0001), 82.1% (95% CI 76.7–87.6%) in Pobè (p < 0.0001) and 78.1% (95% CI 72.2–84.1%) in Sakété (p < 0.0001) post-intervention  vs 70.1% (95% CI 63.5–76.7%), p < 0.0001]. However, in the other three districts, although a slight decrease in susceptibility to permethrin was observed post-intervention, this effect was not significant ,  and,   vs 4.4% (95% CI 2.6–6.2%), p < 0.001].An increase in phenotypic resistance to DDT post-intervention was also observed in Ifangni , Sakété (0%), Pobè  and, Ketou  compared to the pre-intervention period where mortality rates were 62% , 36% , 48%  and, 45% , respectively  results revealed a proportion of 29.65% of An. gambiae and 70.35% of An. coluzzii after cumulating data of all 4 districts. The frequency of An. gambiae was of 7.14% in Ifangni, 20.83% in Sakété, 28% in Pobè and 65.52% in Ketou (Table Table ed Table . Pre-intention 202–2013 rekdr L1014F frequencies were relatively low in Ifangni [f(kdr L1014F) = 0.03] and Pobè [f (kdr L1014F) = 0.58] whereas they were very high in Sakété [f (kdr L1014F) = 0.90] and Ketou [f (kdr L1014F) = 0.92]  = 0.95, p < 0.0001] and Pobè  (Table kdr L1014F frequency increased from 0.56 pre-intervention to 0.96 post-intervention (p < 0.0001)  Table . By cont3] Table . The evo1) Table .An. gambiae s.l. from Plateau department, the kdr L1014F frequencies were compared between dead and live mosquitoes from susceptibility tests carried out with deltamethrin in 2013. In all districts, the results show that the kdr L1014F frequencies of dead mosquitoes were similar to those of live ones (p > 0.05)  and molecular species (p = 0.072) on the mosquito mortality is observed.Table An. coluzzii, the kdr L1014F frequency was positively associated to the mortality rate (RR > 1) but, the association is not significant (p > 0.05) (Table An. gambiae, it was not possible to test the association as no RS or SS genotype was observed (Table In t p > 0.0 (Table 5An. gambiae s.l. was the main malaria vector in the Plateau department as previously showed by Padonou et al. [An. gambiae and An. coluzzii post-intervention, whereas pre-intervention, only An. coluzzii was found. The detection of An. coluzzii only, over the pre-intervention period could presumably be due to the fact that, the data collection covered a shorter period as compared to the post-intervention period which spanned 2 years. This fully justified the low number of vector specimens sampled for PCR analysis pre-intervention. Thus, it is possible that An. gambiae were present at a very low frequency during the pre-intervention period and that a greater sample size of vectors may have demonstrated their presence. Moreover, the post-intervention data might have been generated with adult mosquitoes having emerged from larvae collected in highly diverse breeding sites as compared to the pre-intervention period. Hence, data collected during the post-intervention period may have provided a more representative capture of vector diversity and the species present in the study sites.The current study evaluated the evolution of insecticide resistance of malaria vectors in the Plateau department following mass distribution of Olyset nets in 2011 by the NMCP. Overall, u et al.  in Ouémékdr L1014F frequency could be due to increased use of pyrethroid LLINs following the mass distribution by the NMCP in 2011. Similar observations have been made in Kenya and Niger, respectively by Stump et al. [Post-intervention, the decrease in susceptibility to permethrin, deltamethrin and DDT combined with the significant increase in p et al.  and Czehp et al. . Indeed p et al.  as well p et al.  observedAn. gambiae s.l. suggest that IRS with carbamate insecticides could effectively control An. gambiae s.l. in the Plateau Department. A combined intervention of pyrethroid LLINs and IRS with bendiocarb could be particularly effective in improving the impact of control whilst delaying the onset of resistance. However, the emergence of carbamate resistance in Atacora, a department in Northern Benin [The decrease susceptibility to pyrethroid insecticides as well as the continued susceptibility to bendiocarb observed post-intervention in rn Benin , emphasikdr L1014F mutation as well as the molecular species were non-significantly correlated with the mortality rate to deltamethrin, which suggests that if they had an impact, it was in a very low way. The results are similar to those from Reimer et al. [kdr L1014F mutation, a combination of other resistance mechanisms might explained the pyrethroid resistance observed in An. gambiae s.l. This is confirmed by the similarity of the kdr L1014F frequency in dead and live mosquitoes of the 4 surveyed districts. A non significant association between the kdr L1014F mutation and the mortality rate to deltamethrin was also observed in An. coluzzii while, the opposite result was obtained by Ibrahim et al. [The logistic regression performed reveals that the r et al.  in Camerm et al.  with lamAn. gambiae s.l. collected in Missérété, a neighbouring site of the Plateau Department [An. gambiae s.l. in Covè, another neighbouring district of the Plateau department [The presence of mono-oxygenase mediated pyrethroid resistance has been demonstrated in partment . In addipartment . It is, An. gambiae s.l. to pyrethroids and DDT, as well as the increase of the frequency of the kdr L1014F mutation constituted an alert to the NMCP which should at the time, consider development and implementation of an effective resistance management strategy. At the molecular level, it would have been of interest to perform insecticide resistance intensity tests, and Taqman PCR assays to evaluate the contribution of metabolic enzymes and N1575Y mutation to vector resistance to pyrethroids. At present, the effectiveness of a strategy for combatting malaria vectors in the area requires an update of their resistance status.The data of the current study provide important information on vector resistance to insecticides in the Plateau Department, following mass deployment of LLINs. The decrease of the susceptibility of


Xiphopenaeus has been marked by many changes in the last three decades. The taxonomic status of the Atlantic species has a low resolution, and many species are still undefined and grouped as cryptic species. Here we employed an integrative approach to define the species of Xiphopenaeus and the morphological characters needed to differentiate them. We combined the analyses of two molecular markers (COI and 16 S rDNA), scanning electron microscopy and light microscopy. Based on specimens from 17 localities from the Atlantic and Pacific oceans, we detected five divergent genetic groups, three in the Atlantic  and two in the Pacific . Male secondary sexual characters were able to differentiate four out of the five genetic groups. Group A1 corresponds to X. kroyeri, and A2 and A3 correspond to new species. We redescribed the genus and two new species are described and illustrated: Xiphopenaeus dincao nov. sp. (A2) and Xiphopenaeus baueri nov. sp. (A3). Since the holotype of X. riveti was missing and the specimen analysed from group P2 was a female, the status of the species of Xiphopenaeus from the Pacific remains unresolved.After being stable for nearly a century, the taxonomic history of the genus  The discovery of cryptic species has been increasing in the last decades due to the development of molecular tools, which indicates that they might be more common in the animal kingdom than previously thought3. An example of an unresolved taxonomic issue involving cryptic species occurs in penaeid shrimps of genus Xiphopenaeus Smith, 1869. This genus was described in 1869 based on the type-species Xiphopeneus hartii Smith, 1869, whose individuals came from the south of Bahia, Brazil. Later, X. hartii became a junior synonym of Penaeus kroyeri Heller, 1862 from Rio de Janeiro, and named Xiphopeneus kroyeri . For nearly 40 years X. kroyeri was the only known species of the genus. However, in 1907, a new species was described from the coast of Peru, Xiphopeneus riveti Bouvier, 19074. In 1969, the International Commission on Zoological Nomenclature (Opinion 864) changed the genus spelling to Xiphopenaeus5.Cryptic species are two or more species considered as one due to their highly similar morphologyXiphopenaeus kroyeri has an Atlantic distribution from North Carolina (USA) down to Rio Grande do Sul (Brazil). Xiphopenaeus riveti occurs in the Pacific from Sinaloa (Mexico) to Paita (Peru)4. Due to the high morphological similarity between X. kroyeri and X. riveti their taxonomy remained controversial: X. riveti was considered as a variety of X. kroyeri, or its sister species7. Perez-Farfante & Kensley4 revised the suborder Dendrobranchiata and considered X. riveti as a junior synonym of X. kroyeri. Thus, the genus was again considered monotypic and X. kroyeri its only valid species8. Gusmão et al.9 addressed the issue again, using molecular data from PCR/RFLP, isoenzyme polymorphisms and Cytochrome C Oxidase Subunit I sequences. They proposed the revalidation of X. riveti from the Pacific plus the existence of cryptic speciation in Xiphopenaeus kroyeri, which contained two entities they call: Xiphopenaeus sp. 1 and Xiphopenaeus sp. 2. These entities were not formally described, and neither were the morphological characters that could differentiate the species (synapomorphies). Eight years later, Piergiorge et al.10 came to the same conclusions, also using molecular techniques, but once again, no definitive morphological characters were given to differentiate these entities. Recently, Kerkhove et al.11, have detected Xiphopenaeus sp. 2 in north part of South America (Guiana and Caribbean Ecoregions), and pointed out differences in the colour of individuals between the two species.Xiphopenaeus kroyeri sensu lato represents ~40% of the total shrimp fisheries in the Brazilian coast12 and often accounts for 90% of the shrimp biomass captured in shallow waters, up to 20 m of depth15. Moreover, the species is the second most important fishery resource of the southeast of Brazil, and the most targeted shrimp in the State of São Paulo, were the catch reach the maximum in early 1980s  and then start to decrease until 2000 (629 tons), since then the biggest catch occurred in 2012 18. However, in 2018 X. kroyeri becomes the most exploited fishery resource in this state 19.Xiphopenaeus kroyeri sensu lato has been constantly studied, despite the lack of a definitive taxonomic solution to define and/or distinguish the entities that are known to exist . In the last 13 years, more than 50 articles have been published on diverse subjects — population biology, ecology, fishery biology, physiology, toxicology, sperm ultrastructure, bycatch — considering X. kroyeri as the only species of Xiphopenaeus in the Atlantic. Certainly, this was caused by the lack of knowledge on the morphological characters that could be used to differentiate the two species.Naturally, given the commercial importance of this resource, 22. Recently, the combination of molecular tools and the detailed morphological analyses of the reproductive structures has proved to be very effective to solve taxonomic problems and to differentiate and describe cryptic species of decapod crustaceans24. Therefore, considering the controversial taxonomic history of Xiphopenaeus explained above and its high economic importance, here we employed an integrative analysis and combined molecular and morphological tools to characterize the species that constitute the genus Xiphopenaeus, based on specimens from 17 localities, 15 in the Atlantic and two in the Pacific cost of America. We provide a detailed redescription of the genus and the description of two new species.The correct taxonomic identification of important fishery resources is essential to enhance biodiversity knowledge and to develop adequate management and conservation strategiesst codon base, 2 in the 2nd, and 96 in the 3rd; and 93 variable sites were phylogenetically informative , from our double-genes analyses, were used to construct the phylograms. A total of 91 sequences of the COI gene (barcoding region) were obtained. The final alignment had 596 base pairs. There were 111 variable sites: 11 in the 1ormative . The aveormative . An averMaximum likelihood phylogram based on the COI (barcoding region) gene indicated the existence of five groups, three from the Atlantic Ocean  and two from the Pacific (P1 and P2).The phylogram of the concatenated COI and 16S sequences supported the same division into five groups Figs. . Both tret al.9, based on the Palumbi region of the COI gene25, indicate that our clade A1 corresponds to Xiphopenaeus sp1, and clade A2 corresponds to Xiphopenaeus sp 2. Clade P2 corresponds to Xiphopenaeus riveti, and clades A3 and P1 are two genetic groups previously undetected  and a higher distance between groups. The lowest between-group distances were between A2 and A3, 2.7−3.3%, and the highest were 12.9−13.5%, between A1 and P2 , and the highest, between A3 and P2 (3.2–3.6%) , legs and mouthparts did not point to substantial variation capable to differentiate the genetic groups. The number of rostral teeth varied from four to six, but most individuals had five, irrespectively of the group. Individuals bearing four were seen only in groups P1 and A3, while six teeth were seen only in groups A1 and A2. There was individual variation regarding the teeth shape, rostrum length (in relation to the carapace), groove and carina depth, but they did not lead to a separation of the groups. The presence and size of the teeth of the dorsomedial carina in the fourth, fifth, and sixth abdominal segments also varied. Although in A1 they were smaller and less frequent, this character was also not enough to separate the groups.appendix masculina, however, were much more informative and separated the individuals according to the genetic groups. Thus, we decided to update the general description of Xiphopenaeus given by Perez-Farfante & Kensley4 adding a detailed redescription of these two male structures. The description of the appendix masculina is given for the first time on the literature on Xiphopenaeus.The petasma and Suborder Dendrobranchiata Spence Bate, 1888Superfamily Penaeoidea Rafinesque-Schmaltz, 1815Family Penaeidae Rafinesque-Schmaltz, 1815Xiphopenaeus Smith, 1869Penaeus – Heller, 1862, Sber. Akad. Wiss. Wien, 45(1): 425.Xiphopeneus Smith, 1869, Trans. Conn. Acad. Arts Sci., 2(1): 27. For details see Perez-Farfante & Kensley (1997), p. 149–152.Integument glabrous. Rostrum long, considerably overreaching antennular peduncle, usually longer than carapace in adults, sinuous, styliform anteriorly; armed with dorsal teeth only, situated basally; epigastric tooth distinctly separated from first rostral. Carapace with orbital angle well marked, antennal, and hepatic spines present; pterygostomian angle produced but lacking spine; postocular sulcus well marked; clearly distinct orbito-antennal sulcus; short, almost indistinct cervical sulcus, clearly distinct orbito-antennal sulcus; hepatic sulcus and sharp hepatic carina reaching only base of pterygostomian region and posteriorly merging with long branchiocardiac sulcus and carina, respectively; longitudinal suture extending to about mid length of carapace, transverse suture lacking (in adults). Abdomen with sixth somite bearing interrupted cicatrix. Telson unarmed. Antennule lacking parapenaeid spine; antennular flagella long, dorsal (twice or more as long as carapace) longer than ventral. Palp of first maxilla entire, gradually tapering distally, produced into small, triangular, setose proximolateral lobule, broad, setose proximomesial and quite small, acute setose distomesial lobules, latter armed with slender spine; distolateral row of small spines present on ventral surface. Fourth and fifth pereopods long, much longer than third, subflagelliform, each with multiarticulate dactyl. Basial and ischial spines on first pereopod only.Thelycum closed, with single plate of sternite XIV smooth, broad, not produced anteriorly into pair of flaps, its anterolateral hoods much reduced; anterior sternal invagination nearly as broad as sternite, forming spacious pocket extending as far as posterior thoracic ridge; median protuberance of sternite XIII also broad but quite short. Paired seminal receptacles (spermatheca) is bilobed, each consisting of large posterior lobe and small anterolateral one; sinuous slit-like opening lying on anterior end of posterior lobe, connecting with median pocket and extending laterally dorsal to posterolateral extremity of median protuberance.Petasma formed by the union of the endopods of the first pleopod pair, joining the two dorsolateral lobes by the cincinuli Fig. , and beaappendixmasculina is subcircular   Figs. –9.FigurePeneus Kroyeri Heller, 1862b: 425; Plate 2, fig. 51. [Rio Janeiro]Xiphopeneus hartii Smith, 1869: 27, 40; Plate 1, Fig. Holotype: Rio de Janeiro, 1♂, col. Kröyer, NHMW 342 Fig. .et al., 3♂ (CCDB 5339) – São Paulo, Ubatuba, Praia do Cedro, 23°32′38,4″S − 45°09′54″W, 22/VII/2013, R.C. Costa et al., 4♂ (CCDB 5019) – Santos, 24°04′55,″S − 46°16′56,8″W, 24/X/2011, R.C. Costa et al., 2♂, 2♀ (CCDB 3663) – Santa Catarina, Penha, V/2014, R.C. Costa et al., 3♂, 1♀ (CCDB 5292).Brazil: Rio Grande do Norte, Baía Formosa, 06°21′23,3″S − 35°00′24,7″W, 25/IV/2014, col. M. Lopes & A. Carvalho-Batista, 6♂, 3♀ (CCDB 5337) – Alagoas, Maragogi, Praia de Maragogi, 09°00′48,59″S − 35°13′14,46″W, 05/X/2013, col. F.L. Mantelatto & F.B. Mantelatto, 1♂, 3♀ (CCDB 5338) - Sergipe, Aracajú, Praia do Atalaia, 26/VII/2013, col. G.L. Hirose, 1♀ (CCDB 5246) – Espírito Santo, Marataízes, 20°59′S − 40°47′W, 20/VI/2012, Col. F.L. Carvalho, D. Peiró & R. Robles, 3♂, 1♀ (CCDB 3985) – Rio de Janeiro, Macaé, 22°23,44′S − 41°44,57″W, 21/VII/2014, R.C. Costa Petasma: In dorsal view, the posterior margin of the proximal region of the distolateral projection is straight Figs. , 8A. TheAppendix masculina subcircular with a rounded projection towards the body median part and bearing two spines in the dorsal surface .Xiphopenaeuskroyeri, deposited in the Museum of Natural History of Vienna (Austria), indicated that this species corresponds to our clade A1 and also to Xiphopenaeus sp.1 detected by Gusmão et al.9 Based on the material examined here, this species seems to be the very abundant. It was the most abundant species in almost all localities of the northern, southern, and southeastern coasts of Brazil.The comparison with the holotype of  Figs. and 11.FXiphopenaeus sp. 2 — Gusmão et al. 2006: 491, 496–500, Figs et al. 2019: 853–858, Figs Xiphopenaeus sp. II — Piergiorge et al. 2014: 349–353, Figs Brazil: Alagoas, Maragogi, Praia de Maragogi, 09°00′48.59″S − 35°13′14.46″W, 05/X/2013, col. F.L. Mantelatto & F.B. Mantelatto, 1♂ (MZUSP 39350).Brazil: Alagoas, Maragogi, Praia de Maragogi, 09°0′48.59″S − 35°13′14.46″W, 05/X/2013, col. F.L. Mantelatto & F.B. Mantelatto, 5♂, 2♀ (CCDB 6499) - São Paulo, Cananéia, X/2014, R.C. Costa, 2♂, 5♀ (CCLC 418).Brazil: Amapá, Oiapoque, Estuário do Rio Oiapoque, Parna Cabo Orange, 04°22′17.6″N–51°24′26.4″W, 22/VIII/2013, col. I.M. Vieira, A.G. Santiago & E.G. Oliveira, 1♂ (IEPA 1618) – Pará, Vigia, Ponta Seca, 0°51′45.00″S – 48°7′50.00″W, 19/XI/1994, col. M.P. Barros, 2♂ (MCP 2024).appendix masculina is subcircular but less rounded than in X. kroyeri and its protruding tip has many spine rows .ColombiaXiphopenaeus sp. 211. Besides the description, this is the first record of this taxon to Amapá, Pará and Alagoas. Despite the vast list of works in the literature that studied biology of X. kroyeri, it was not possible to identify in which of these studies were used specimens of the described new species and thus the synonymic list was short.This species refers to the specimens of clade A2, previously known as Figures −13.FigurMexico: Tabasco, 18°31′40.25″N – 93°19′43.95″W, 05/XI/2014, col. Ku, M.A.M., 1♂ (MZUSP 39351).Mexico: Tabasco, 18°31′40.25″N – 93°19′43.95″W, 05/XI/2014, col. Ku, M.A.M., 5♂, 3♀ (CCDB 5461).USA: Isle Dernier, Louisiana, 16/XI/1992, col. Bauer R.T., 3♂ (CCDB 5394) – Puerto Rico: Mayagüez Bay, XII/1985, col. R.T. Bauer (CCLC 0417). Brazil: Amapá, Oiapoque, Estuário do Rio Oiapoque, Parna Cabo Orange, 04°22′17.6″N – 51°24′26.4″W, 22/VIII/2013, col. I.M. Vieira, A.G. Santiago & E.G. Oliveira, 1♀ (IEPA 1617) – Pará, Vigia, Ponta Seca, 0°51′45.00″S – 48°7′50.00″W, 19/XI/1994, col. M.P. Barros, 1♀ (MCP 2024).appendix masculina is subcircular but less rounded than in X. kroyeri , USA ; Puerto Rico, Brazil .Xiphopenaeus dincao nov. sp. The main character that differentiates these two species is the ventral surface of the appendix masculina covered with spines in Xiphopenaeus baueri nov. sp. The two species seem to occur in sympatry, at least in the north of Brazil, which may hamper their differentiation. In the Gulf of Mexico, however, Xiphopenaeus baueri nov. sp. is the only known species of Xiphopenaeus. Despite the vast list of works in the literature that studied biology of X. kroyeri, it was not possible to identify in which of these studies were used specimens of the described new species and thus the synonymic list was not provided.The individuals of our clade A3 correspond to a new species, undetected in previous studies. The shape of the petasma is very similar to that of Costa Rica: Puntarenas, Sierpe, Terraba, GPS coordinates unknown, VI/2013, J.S. Vargas, 4♂, 1♀ (CCDB 5247).Appendix masculina subcircular , our study also indicates the existence of a third species from the Atlantic (Xiphopenaeus baueri nov. sp.) and at least two entities  from the Pacific. This last species (group P2) could not be described since only a female was available, and diagnostic morphological characters from the petasma are needed.Our results revealed that the genus 30. The between-group distances resulting from the phylogenetic analyses of COI and 16S were in the range of interspecific distances expected in decapods. Moreover, the genetic groups were supported by morphological differences in the male secondary sexual characters. Within the order Decapoda, the highest intraspecific distances known for the COI gene (barcoding region) are ~2%, while the interspecific distances between congeneric species are usually higher than 5%, reaching 10% in many cases, and even more than 30%32. For instance, in shrimps of the family Penaeidae from the coast of Egypt and in species of the genus Farfantepenaeus, the interspecific distances varied from 3 to 20%34. Thus, most interspecific distances detected here are above the threshold used to separate congeneric species based on the barcoding region.The use of molecular tools, in addition to the morphological ones, was extremely useful to strengthen and deepen our study, which confirms their usefulness in the taxonomic identification and to solve taxonomic and phylogenetic problemsXiphopenaeus dincao nov. sp. and Xiphopenaeus baueri nov. sp. for the COI gene are in between the intraspecific and interspecific thresholds of Decapoda. Even though intraspecific distances of up to 2.7% were reported for the penaeid shrimp Artemesia longinaris Spence Bate, 1888, it was less than 1% in other Atlantic Dendrobranchiata shrimps, such as Pleoticus muelleri , Farfantepenaeus paulensis , Farfantepenaeus brasiliensis , and Farfantepenaeus subtilis 38.The genetic distance between Xiphopenaeus dincao nov. sp.; 0.0–0.3% in Xiphopenaeus baueri nov. sp.) were much lower than the interspecific (2.7–3.3%), thus, there is a well-defined intraspecific gap. The DNA Barcoding technique uses this gap to identify the species43, and prove to be effective in the case of Xiphopenaeus.The intraspecific distances  is an interesting and novel result since it has been undetected in previous studies. Unfortunately, we obtained DNA sequences only from a female from Tehuantepec, Mexico, warranting further descriptions. However, the genetic distances between P2 and the other Pacific specimens (P1), from Sierpe, Costa Rica, reinforce the idea that these two groups are indeed two distinct species. Further studies, with a more comprehensive sampling including more localities are needed to understand their geographic range in the Pacific. There is likely an overlap in the distribution range of these two species because the specimens from Panama, analysed by Gusmão et al.9, belong to our group P2, while the specimens from P1 came from Costa Rica, which is close to Panama. Since the holotype of X. riveti could not be located (it is probably lost), its comparison with specimens of these two groups was not possible.The existence of a second species besides X. kroyeri and Xiphopenaeus dincao nov. sp. (as Xiphopenaeus sp. 1 and Xiphopenaeus sp. 2) along the Brazilian coast has been reported in Natal, Rio Grande do Norte and Ubatuba, São Paulo9, and later in Cananéia, São Paulo and in Caravelas, Bahia10. Here we show that they also overlap in Maragogi and Baía Formosa. Xiphopenaeus dincao nov. sp. and Xiphopenaeus baueri nov. sp. occurs in sympatry in Vigia and Oiapoque 9 and in São Luís, Maranhão (Brazil)46, the geographic distribution of these three species seem to overlap in the northern Atlantic coast of South America. In the present study, Xiphopenaeus dincao nov. sp. and Xiphopenaeus baueri nov. sp. were collected in the North of Brazil between Caracas and São Luís, Maranhão. Kerkhove et al.11 found X. kroyeri and Xiphopenaeus dincao nov. sp. (as Xiphopenaeus sp. 2) in Colombia and Guianan Marine Ecoregion. Therefore, based on our data, and on Gusmão et al.46, Piergiorge et al.10 and Kerkhove et al.11 the known geographical distribution of the species of the genus Xiphopenaeus is shown in Fig. An overlap between the geographical distribution of que Fig. . ConsideXiphopenaeus until its definitive closure circa 2.8 million years ago47. The low genetic distances seen between Xiphopenaeus dincao nov. sp. and Xiphopenaeus baueri nov. sp. suggest that these species diverged more recently. Xiphopenaeus baueri nov. sp. was the only species found in the Gulf of Mexico and probably originated there. The Quaternary sea level changes created or strengthened the isolation between this region and the Caribbean, warranting the exchange of migrants51. This isolation pattern has also been observed in other crustaceans such as the hermit crabs Clibanarius vittatus  and C. simetricus , the first restricted to southeastern coast of the United States and Gulf of Mexico, and the second distributed along the Caribbean and South America52. Furthermore, in Atya scabra , individuals from the Gulf of Mexico are isolated from those from the Caribbean and Brazil, which share haplotypes among them although genetic divergences remain within the intraspecific level53.It is likely that the successive openings and closures of the Panama Isthmus allowed the diversification of the genus 54. Here, morphological differences supporting the separation of the groups identified in the molecular analyses were found in the male secondary sexual characters — petasma and appendix masculina. Previously, the petasma of Xiphopenaeus has been described without many details on the distolateral projections56. Fransozo et al.57, who studied the petasma development of X. kroyeri in Ubatuba, did not report morphological differences among the individuals. The photo provided by the authors corresponds to the shape described here to Xiphopenaeus dincao nov. sp. Burkenroad6 described a difference between individuals from Bahia (Brazil) and Louisiana (USA), concerning the “small tooth” of the distolateral projection, probably referring to the distal region of the distolateral projection. Considering the known geographical distribution of Xiphopenaeus . Thus, this information could facilitate and pave the way for further studies on the population biology and ecology of these cryptic species. According to Dall et al.7 the morphology of these structures are unique in each species. Using the stereomicroscope, it is possible to see the interspecific differences in the spines of the ventral face of the appendix masculina, which can also be enhanced using dyes like methyl blue.Microanatomic details may reveal crucial information to differentiate cryptic speciesXiphopenaeus is composed of at least five species, by combining morphological and molecular tools. Four out of five species can be differentiated based on the morphology, and two of them are described herein (Xiphopenaeus dincao nov. sp. and Xiphopenaeus baueri nov. sp.). Further studies addressing the status of the species from the Pacific and investigating the existence of more cryptic species are strongly encouraged.To conclude, we demonstrate that the genus Xiphopenaeus from 17 localities: 15 from the Atlantic Ocean and two from the Pacific 65. Two molecular markers from mitochondrial genes, cytochrome c oxidase subunit I (COI) barcoding region68 (N = 91) and 16 S rDNA67 (N = 15) were used. Additionally, the Palumbi region of COI25 (N = 9) was used to allow the comparison of our specimens to those used by Gusmão et al.9 The amplification reactions contained bovine albumin 1% (Sigma), 10X Taq Buffer (Thermo Scientific), MgCl2 (25 mM), betaine (5 M), dNTPs (1.25 mM each), primers (10 or 20 µM) (Supplementary table Thermus aquaticus polymerase (5 U µl−1) (Thermo Scientific), 1.0–5.5 µl of extracted DNA (50−100 ng ml−1), and distilled and deionized water to complete 25 µl.To extract DNA, muscle tissues were dissected from the abdomen and two common techniques were employed: the salting-out methodThe thermal cycler settings used for the amplification for COI were: 4 min at 94 °C for initial denaturation; 35 cycles of 30 s at 94 °C, 30 s at 40–46 °C, 60 s at 72 °C; and final extension for 10 min at 72 °C. For the 16 S they were: 4 min at 94 °C for initial denaturation; 40 cycles of 30 S at 48 °C; and final extension for 10 min at 72 °C. The results were checked in a 1.5% agarose gel stained with GelRed. Amplicons were purified with the SureClean Plus kit (Bioline USA Inc.) and sequenced using the BigDye Terminator Mix in an ABI 3730 XL DNA Analyzer (Applied Biosystems), following the manufacturer’s instructions.69. Consensus sequences were aligned separately for each gene with the software MUSCLE (Multiple Sequence Comparison by Log-Expectation)70 on the online platform EMBL-EBI (European Molecular Biology Laboratory — The European Bioinformatics Institute)71 with the default parameters. The presence of stop codons in the COI sequences, which could indicate the occurrence of pseudogenes, was ruled out using the online translation tool EMBOSS Sixpack available in the EMBL-EBI Portal73.Forward and reverse sequences were aligned to obtain the consensus sequence using BioEdit v.7.0.5et al.74 using the software DAMBE. Highly variable regions were removed from the aligned sequences using Gblocks76 available online through the Castresana Lab, Animal Biodiversity and Evolution Program (http://molevol.cmima.csic.es/castresana/index.html).The substitution saturation of all sequences was previously tested with the saturation test of Xia 77 available in the Cyber Infrastructure for Phylogenetic Research (CIPRES) website78. The default parameters of RAxML were used to perform the analysis for the GTR model. To measure the consistency of the topology we selected the option to automatically determine the number of bootstraps to be used in the RAxML79. Consequently, 1000 bootstrap pseudo-replicates were run, and only the values > 50% were reported.The phylograms based on the Maximum Likelihood criterion were constructed in the program RAxML-HPC2 on X-SEDE80 based on COI (barcoding region) and 16S rDNA sequences separately, using MEGA 6.0681. We also included five other sequences of Dendrobranchiata species in our alignments to serve as external groups to rout the distance and phylogenetics analysis  sputtering, properly placed on stubs with carbon adhesive tape and sputter-coated with gold (50 nm) in a Denton Vacuum Desk II sputtering. Micrographs were obtained in a Jeol JSM 5410 scanning electron microscope. Voucher specimens were deposited in CCDB  and in collection of Museu de Zoologia da Universidade de São Paulo . Ten samples containing two cryptic species were mixed and then checked using the morphological differences obtained from both stereomicroscopy and SEM. All samples were submitted again to the molecular protocol to validate the morphological characters.For the scanning electron microscopy (SEM), the petasma and supplemantary tablesuplementary figures


Cervus nippon) overabundance on dung beetle larvae feeding habits in Japan. δ15N values were notably higher in raccoon dog and badger dung than in that of other mammals. A dung beetle breeding experiment revealed that the δ15N values of dung beetle exoskeletons that had fed on deer feces during their larval stage were significantly lower than those of beetles that had fed on raccoon dog feces. The δ15N values of the adult exoskeleton were significantly lower in a deer high-density area than in a low-density area in large dung beetles only. It is possible that the high-quality feces, such as those of omnivores, preferred by the large beetles decrease in availability with an increase in deer dung; large beetles may therefore be unable to obtain sufficient high-quality feces and resort to using large amounts of low-quality deer feces. Small dung beetles may use the easily obtained feces that is in high abundance and they may also use deer feces more frequently with increases in deer density. These findings suggest that a larval resource shift associated with deer overabundance may affect ecosystem functions such as soil nutrient cycling and seed dispersal.Because animal feces contain organic matter and plant seeds, dung beetles (Scarabaeinae) are important for the circulation of materials and secondary seed dispersal through burying feces. Dung beetles are usually generalists and use the feces of various mammals. Additionally, the larval stages have access to feces from only one mammal species leaving them susceptible to changes in animal fauna and variations in animal populations. Here, we explain the effects of resource availability changes associated with sika deer ( In terrestrial ecosystems, decomposers maintain the material cycle system  by decomposing and converting organic materials such as plants and animal carcasses into inorganic materials , 2, 3, 4Typically, dung beetles have generalist diets, therefore resource shifts are easy. They depend on changes in the population density of individual animal species, and with these changes dung beetles may select higher quality feces  or more abundant feces , 12. HowCervus nippon) in Japan, which has directly or indirectly impacted ecosystems because of overgrazing of several plant types [In recent years, there has been an overabundance of sika deer  and badger (Meles anakuma) population densities by increasing the earthworm abundance, their main food resource. Conversely, rodents have declined in the understory, thereby negatively affecting predators such as red foxes (Vulpes vulpes) and Japanese martens (Martes melampus) [Ursus thibetanus) have also changed, as they eat more deer meat [The overabundance of sika deer affects other, sympatric, mammals. Understory reduction by the deer, the deposition of their dung, or both, increases the raccoon dog (elampus) , 28. Theeer meat . OverabuOur objective here was to study the relationship between the increase in sika deer population density and the diet of dung beetles and changes in the dung beetle community. We hypothesized that dung beetle larvae would use more sika deer feces in areas with a high density of sika deer, because deer feces would be easier to obtain in these areas. We also hypothesized that this trend would be more notable in the larger dung beetle species, because large dung beetles need large amounts of feces. Because deer live in herds, we expected that increased size or abundance of these herds would make their feces easier for large dung beetles to find. If the larger dung beetles change their resource use to sika deer feces, then there would possibly be a change in ecosystem functioning. Specifically, larger dung beetles play a role in bioturbation through moving large quantities of earth to the soil surface during nesting, have a positive effect on permeability, and perform secondary seed burial for several plant seed sizes . Becauseδ15N increasing [δ15N in herbivore and carnivore feces or used δ15N to estimate dung beetle larval diets [To evaluate the dietary changes of larval dung beetles, we focused on the adult exoskeleton. Generally, insects that completely metamorphose form an exoskeleton, the molecular composition of which varies depending on food ingested by larvae . Furthercreasing , 31. Thecreasing . Only a al diets , 19. We Quercus crispula, Acer mono, and Carpinus japonica. The sites were located at 850 to 1450 m above sea level, with an average altitude of 1120 m. Several medium to large mammalian species are found in the region, including sika deer, Asian black bear, Japanese macaque (Macaca fuscata), wild boar (Sus scrofa), badger, and raccoon dog.The study was conducted in the Ashio–Nikko mountain area of central Japan . We established 20 study sites, all in forested areas comprising deciduous trees, dominated by 2) (HD) and low (<5 deer/km2) (LD); 10 study sites were set up in each of the different sites of deer density, which were at least 5 km apart among sites and they were interspersed  to collect fecal samples. We surveyed the routes once every 2 weeks, twice in each study area. All fresh feces of medium to large mammals found on or along the route were collected. The feces were identified by their smell and shape as from sika deer, Asian black bear, Japanese marten, badger, red fox, masked palm civet, wild boar, and raccoon dog. If we were unable to identify the animal species at the scene, we did not collect the fecal sample. Raccoon dogs and badgers use latrines  for defecation. In the surveys, we collected only new fecal samples.Feces obtained from the zoo-housed sika deer and raccoon dogs were used for the breeding experiment as were: exoskeletons (elytra) of the dung beetles that ate these feces during the larval stage in the breeding experiment; dung beetles collected from each study area ; and feces collected in each study area from each wild mammalian species were dried for 72 h at 60°C, powdered, and measured into 0.5-mg aliquots .Each sample was enclosed in a tin cup and combusted in a FlashEA1112 elemental analyzer  interfaced with a Delta V isotope-ratio mass spectrometer (Thermo Fisher Scientific) to determine the nitrogen isotope ratio.δ notation as parts per thousand (‰) relative to Rstandard, were calculated as follows:Rsample is the 15N/14N ratio of the sample and Rstandard is the nitrogen isotope ratio of the international standard (atmospheric N2). The nitrogen isotope ratios were calibrated against laboratory standards , which are traceable back to international standards. The analytical standard deviation (SD) of the stable isotope analysis was ± 0.15‰ for δ15N.The results, presented in We installed automatic cameras  at 10 locations at 500-m intervals along animal trails in each study area and took still images of mammals from June to September 2017. The cameras were installed two to five meters from the animal trails at heights of 30 to 50 cm. They were checked and the batteries replaced monthly. From these images, we calculated a relative abundance index . Because cameras along animal trails do not sample different species at random, the results are biased by such factors as activity patterns and changes in trail use. Thus, they can be used to assess species’ relative abundance between sites, but the data cannot be extrapolated to compare species population densities within defined areas. Thus, we estimated the relative abundance of same mammal species between areas. If the same species was photographed at the same location within 30 min, we discounted the second sighting.Paguma larvata; one individual, fed kiwifruit), and adult martens  from the Kanagawa Prefectural Natural Environment Conservation Center for the calculations. We also used adult Asian black bear  from the Institute of the Japanese Black Bear in Ani, Akita Prefecture. For wild boars, we assumed a body weight of 60 to 80 kg, but because of a lack of referenced data we used the fecal production value for adult domestic pigs [We multiplied the RAI of each animal species and the weight (in grams) of the feces produced by one individual of the species each day in each study area to estimate the relative amount of feces potentially provided by each species in each study area. The mean fecal production (fresh weight) per day per individual of each species during the summer was calculated from mammals in captivity or from data from a previous study (see below). We used adult sika deer , an adult Japanese macaque , adult raccoon dogs , adult badgers , adult masked palm civets  of the feces (deer and raccoon dog) used in the breeding experiment were compared by using Welch’s t-tests. To compare the δ15N values of the exoskeletons of dung beetles that, during their larval stage, ate the feces of deer or raccoon dogs during the breeding experiment, we used Student’s t-tests. The δ15N values of feces from each animal species in each study area were compared by using a linear mixed-effect model. We set the δ15N value of feces as the response variable and the deer density (high or low) as the fixed effect. The probability distribution was defined to follow a normal distribution pattern. We set each mammal species  as a random effect on the slope to evaluate whether the deer density effect varied among mammal species. This was done because it was possible that changes in deer density could have different effects on the diets of different types of mammal, such as herbivores (sika deer and wild boar) or omnivores . We also set mammal species as a random effect on the intercept to evaluate mammal species–related differences in δ15N values that were unrelated to deer density. We used the lme4 package [R2 to check the degree to which the fixed effect explained the response variable and the conditional R2 to check the degree to which the fixed effect plus the random effects explained the response variable. We also compared the δ15N values of the exoskeletons of dung beetles collected in each study area; for this we used Student’s t-test, Mann-Whitney’s U-test, or Welch’s t-test, depending on the variance of the population. For all analyses, we used the statistical analysis software R ver. 3.3.0 [To compare the mean number of each species of dung beetle per pitfall trap in each study area, we used Mann-Whitney’s  package  to creat package  to calcur. 3.3.0 .Onthophagus ater. The number of O. ater individuals per trap was significantly greater at HD than at LD (P = 0.041).We collected eight species of dung beetle, all tunneller species (no dweller), totaling 335 individuals : 7.6 ‰ ± 0.9 ‰; sika deer (n = 10): 3.4 ‰ ± 0.3 ‰, t = –12.70, df = 11.07, P = 6.06 x 10−8; δ15N values : t = –15.63, df = 12, P = 2.42 x 10−9; C. nikkoensis : t = –17.86, df = 14, P = 4.95 x 10−13; and O. lenzii : t = –26.65, df = 14, P = 2.13 x 10−13: Compared with sika deer feces, raccoon dog feces had significantly higher δ15N values in five species: P. auratus, Phelotrupes laevistriatus, Onthophagus fodiens, O. lenzii, and C. nikkoensis. We found that in P. auratus (HD (n = 6): 2.8 ‰ ± 0.7 ‰; LD (n = 6): 5.1 ‰ ± 1.3 ‰, Student’s t-test, t = 3.65, df = 10, P = 0.004) and P. laevistriatus (HD (n = 10): 2.9 ‰ ± 0.9 ‰; LD (n = 10): 4.1 ‰ ± 1.6 ‰, Student’s t-test, t = 2.12, df = 18, P = 0.049) the δ15N values were significantly lower in individuals caught at HD than in those from LD , 4.1 ‰ ± 0.7 ‰, LD (n = 10), 3.0 ‰ ± 2.1 ‰, Welch’s t-test, t = –1.44, df = 11.141, P = 0.18; O. fodiens: HD (n = 6), 3.0 ‰ ± 0.8 ‰, LD (n = 5), 2.5 ‰ ± 1.9 ‰, Mann-Whitney’s U test, Z = 1.28, P = 0.25; C. nikkoensis: HD (n = 10), 2.8 ‰ ± 0.7 ‰, LD (n = 10), 3.2 ± 0.7 ‰, Student’s t-test, t = 0.97, df = 18, P = 0.35, From among the dung beetles collected in the wild, we measured  from LD . In the δ15N values of wild mammal feces declined by 0.14‰ on average , and finally sika deer and wild boars (consumers of plant-based diets). Although badgers and raccoon dogs are omnivores, their summer food habits are heavily biased toward an animal-based diet [The  effects . However effects . The val effects , indicated value . Additio species . The val species . The 95% species ; however species . Therefosed diet . We therP = 0.001). There were no significant differences in the RAIs of raccoon dogs and badgers between the study areas. In both study areas the RAI of sika deer was the highest   than at HD  .The mean fecal production (fresh weight) per day per individual of each species during summer was 442.2 ± 34.5 g for sika deer; 15.2 ± 3.5 g for Japanese macaques; 41.2 ± 6.5 g for raccoon dogs; 49.4 ± 5.2 g for badgers; 29.4 ± 7.5 g for masked palm civets; and 9.9 ± 2.5 g for Japanese martens. For Asian black bears, the calculated mean fecal production per day per individual was 467.5 ± 46.8 g. For wild boars, the rate (as determined in domestic pigs) was 2.3 kg.To estimate the relative amounts of feces between LD and HD for each mammal species, we targeted those mammal species for which the RAI was greater than 0 at both LD and HD; we therefore excluded Japanese macaque , Asian black bear , and wild boar (HD: 0.0 LD: 0.0). Only the relative amount of sika deer feces was substantially greater at HD than at LD Click here for additional data file.


Strobilanthes crispus which is increasingly marketed as an important herbal tea remains to be reported. This study evaluated the effects of conventional and new drying technologies, namely vacuum microwave drying methods, on the antioxidant activity and yield of essential oil volatiles and phytosterols. These drying methods included convective drying (CD) at 40 °C, 50 °C, and 60 °C; vacuum microwave drying (VMD) at 6, 9, and 12 W/g; convective pre-drying and vacuum microwave finish drying (CPD-VMFD) at 50 °C and 9 W/g; and freeze-drying (FD). GC–MS revealed 33 volatiles, and 2-hexen-1-ol, 2-hexenal, 1-octen-3-ol, linalool, and benzaldehyde were major constituents. The compounds β-sitosterol and α-linolenic acid were the most abundant phytosterol and fatty acid, respectively, in fresh S. crispus. The highest phenolic content was achieved with CD at 60 °C. The highest antioxidant activity was obtained with CD at 40 °C and VMD at 9 W/g. On the contrary, the highest total volatiles and phytosterols were detected with CD at 50 °C and VMD at 9 W/g, respectively. This study showed that CD and VMD were effective in producing highly bioactive S. crispus. A suitable drying parameter level, irrespective of the drying method used, was an important influencing factor.The preservation of active constituents in fresh herbs is affected by drying methods. An effective drying method for  Strobilanthes crispus, known as “pecah beling” or “Hei Mian Jiang Jun”, is native to various countries, such as Malaysia and Indonesia. Its leaves are used as traditional medicine in the form of infused fresh or dried leaves. The intake of its herbal preparation became popular as an alternative treatment to prevent diseases and increase overall well-being. Al-Henhena et al. (2015) showed that S. crispus extract is high in antioxidant activity because of various phenolic constituents, such as caffeic acid, ferulic acid, kaempferol, and luteolin [et al. 205 showed luteolin .Drying is an effective preservation step that involves reducing the moisture content of a freshly harvested material, thereby extending a product’s shelf life. Conventional drying methods, such as convective drying (CD) and freeze-drying (FD), are characterized by low drying rates, especially in the falling rate period . New dryIn VMD combined with CD, a material is pre-dried with CD and completely dried through VMD. This combined method, known as CPD-VMFD, maximizes the benefits of both drying methods. VMFD is usually applied at the end stage of drying to remove bound water from a product during the falling rate period, thereby effectively reducing drying time .w), and color of dried S. crispus. The fatty-acid profile was also determined to verify the active constituents in S. crispus. VMD methods are yet to be utilized to dry S. crispus. As such, this study also aimed to confirm the potential of this new innovative technology. Determining good drying applications is important for manufacturing new dried forms of S. crispus with high bioactivity. The drying kinetics were modeled with thin-layer models to integrate experimentally obtained data into industrial applications. Drying is also one of the most energy-intensive operations in the food industry. This study also considered the relationship of drying kinetics with the energy input of a drying operation and used specific energy consumptions to provide an indication of the cost effectiveness of S. crispus drying which is yet to be reported.This study aimed to evaluate the effects of different drying methods, namely conventional and new drying methods, on the drying kinetics, phenolic content, volatile and phytosterol content, antioxidant activity, water activity ) based on the highest 2R and the lowest root-mean-square error (RMSE).a is the model constant, k is the drying constant, and n is the dimensionless empirical constant.The drying kinetics of −1 from a low drying rate of 0.003 min−1 during CPD at 90 min. When the moisture content of the leaves was high during the initial drying period, the drying rate increased with time regardless of the drying methods. In CD, surface water evaporated easily to the surrounding air through external diffusion. The removal of surface water produced a moisture gradient in the material, and the removal of moisture at this stage depended on internal diffusion that occurred at a slow rate. In VMD and VMFD, the high drying rate observed at the beginning could be explained by the high amount of available water molecules, resulting in high microwave energy absorption. However, the amount of moisture that reduced as drying progressed corresponded to the low absorption of microwave energy, resulting in the decreasing drying rate.−1, respectively. In CD at 40 °C, 50 °C, and 60 °C, the maximum drying rates were 0.015, 0.022, and 0.031 min−1, respectively. In VMD, the drying duration of S. crispus leaves was reduced from 81% to 92%. When VMFD at 9 W/g was introduced to CPD at 50 °C, the drying rate increased from 0.003 min−1 to 0.044 min−1. This condition could be attributed to the volumetric heating of VMD in which high internal moisture could be effectively removed at a fast rate, thereby shortening the drying time from 150 min to 105 min.The maximum drying rates observed for VMD at 6, 9, and 12 W/g were 0.086, 0.115, and 0.173 min−1 to 0.047 min−1. On the contrary, when the microwave power increased from 9 W/g to 12 W/g, the average drying rate increased from 0.047 min−1 to 0.070 min−1. When hot air temperature increased by 10 °C, a marginal improvement was noted. The average drying rate also increased from 0.008 min−1 to 0.009 min−1 by increasing the hot air temperature from 40 °C to 50 °C. Similarly, the average drying rate increased from 0.009 min−1 to 0.010 min−1 when temperature increased from 50 °C to 60 °C.Increasing the parameter level, that is, microwave power and hot air temperature, led to an increase in the drying rates. As the microwave power increased from 6 W/g to 9 W/g, the average drying rate also increased from 0.035 minS. crispus despite the long dying duration of 150–180 min. Samples dried using CD at 40 °C  (ABTS): 5.08, and ferric-reducing antioxidant power (FRAP): 5.98 µM Trolox/100 g dry weight (dw)), and CD at 60 °C  achieved the highest antioxidant values that were significantly higher than those of FD . The long exposure to heat and oxygen did not reduce the antioxidant activity. In previous studies, antioxidant activities and polyphenol contents in heat-treated peppermint, oregano, Artemisia annua, and various Lamiaceae herbs, such as rosemary, marjoram, oregano, sage, thyme, and basil [The analysis of the antioxidant results revealed that CD showed potential for preserving antioxidant constituents in nd basil , increasS. cripus could achieve optimum activity at 50 °C. Therefore, phenolic and antioxidant compounds were likely degraded. Both drying methods which operated at the same temperature of 50 °C consistently yielded samples with low antioxidant activities and TPC values.CPD-VMFD  and CD at 50 °C produced the lowest ABTS, FRAP, and total phenolic content (TPC). A good agreement between these values is shown in It was observed that the FRAP values of fresh samples were lower than those of some dried samples. Thermal treatment in drying may have caused an easier release of cell constituents from plant cells . This isS. crispus. The TPC value obtained from the fresh sample was 1222 mg/100 g dw, comparable with the TPC value of 1262 mg/100 g dw described in a previous study [S. crispus, which contributed to the overall antioxidant activity. Antioxidant compounds derived from plants such as phenolic compounds and terpene derivatives are widely reported to exhibit antioxidative properties. In our study, the most abundant phytosterols identified in S. crispus were β-sitosterol and stigmasterol, both of which were reported to have an antioxidant effect [t effect . Phytostt effect . The cheS. crispus was also investigated in this study; 2-hexen-1-ol 2-hexenal 1-octen-3-ol linalool, and benzaldehyde constituted the major compounds of S. crispus volatile content. However, the antioxidant activity of S. crispus is more likely to be contributed by terpenoids present in S. crispus, such as linalool, p-cymene, limonene, and isopulegol. S. crispus, is a monoterpene alcohol reported to have an antioxidant effect [+ to free radicals produced during oxidation, becoming radicals themselves. The resonance delocalization of electrons in the aromatic ring will act to stabilize these radical intermediates [+-donating abilities are predicted to contribute to the structure–activity relationship [S. crispus, such as terpenoids and phytosterols, the overall antioxidant activity may have been contributed by the synergistic interactions of these compounds. Synergism between antioxidants involves several mechanisms. Synergism occurs when there is a combination of two or more antioxidants with different antioxidant mechanisms, or when there is a combination of two or more different free-radical scavengers, in which an antioxidant is found to be regenerated by other antioxidants. More specifically, an antioxidant is characterized as a sacrificial antioxidant which is oxidized to exert protection on another antioxidant. Furthermore, regeneration of a primary antioxidant, with higher reduction potential by a secondary antioxidant (co-antioxidant), with less reducing power, is able to contribute to a higher net interactive antioxidant effect compared to the total individual antioxidant effects [The volatile composition of t effect . A vast mediates . Therefotionship . With th effects . Synergi effects .Drying intensity slightly influenced the antioxidant activity and TPC. However, CD at a low temperature of 40 °C produced a sample with the highest antioxidant activity among the convective-dried samples (50 °C and 60 °C). The antioxidant activity obtained with a moderate VMD treatment intensity of 9 W/g was relatively better than that found with VMD at 6 and 12 W/g.S. crispus leaves extracted using headspace solid-phase microextraction (HS-SPME). A typical chromatogram showing the volatile compounds of S. crispus is shown in −1 db (dry basis). The most abundant volatiles in fresh S. crispus were 2-hexen-1-ol (84.11 mg 100 g−1 db), 2-hexenal (51.18 mg 100 g−1 db), 1-octen-3-ol (36.10 mg 100 g−1 db), linalool (34.91 mg 100 g−1 db), and benzaldehyde (27.61 mg 100 g−1 db). The volatiles present in S. crispus could be categorized under the following chemical groups: alcohol (50.2%), aldehyde (24.3%), monoterpenoid (10.9%), ketone (4.1%), ester (4.0%), pyridine (3.4%), monoterpene (1.4%), and enone (0.9%). Other compounds were also found, such as cycloalkenes (0.5%), cyclohexenones (0.3%), and unidentified volatiles, represented 0.1% of the total volatiles.S. crispus in terms of retaining volatiles. However, we assumed that VMD and CPD-VMFD would retain a higher concentration of volatiles because the drying rate could be increased by the volumetric heating of microwaves. Microwave treatment could also cause a large disruption of the orderly tissue structure to improve mass and heat transfer, thereby increasing the drying rate. However, in this study, the increasing microwave power of VMD led to high losses in volatile content possibly caused by the high intensity of the drying treatment. Similar results were reported in the drying of basil [All of the drying methods resulted in considerable losses in volatile content . The volof basil , marjoraof basil , rosemarof basil , and thyof basil .−1 db). At a high temperature of 60 °C, the concentration of volatiles was reduced by 13.8%.Although CD had few disadvantages, such as a long drying time and being prone to volatile oxidation, convective-dried leaves are known to develop a partially dried layer on the surface following CD ,26. This−1 dw) than CD (87.2 g kg−1 dw) [Our results also agreed with a previous study which reported that the VMD of rosemary leads to higher losses in volatile content (61.9 g kgkg−1 dw) . It is pkg−1 dw) ,29. The kg−1 dw) . Therefokg−1 dw) . Drying kg−1 dw) . Higher kg−1 dw) .w of 0.0245. Moreover, the porous structure of freeze-dried leaves may have facilitated the diffusion and escape of volatiles [Since water acts as a solvent in which volatiles are dissolved; when a high amount of water is removed from the sample, a high volatile content could be lost. Therefore, the low total volatile concentration of the freeze-dried samples could be attributed to the high removal of water during FD, considering that the freeze-dried samples had the lowest aolatiles ,27. The olatiles ,32. The olatiles .S. crispus were β-sitosterol, stigmasterol, and campesterol, with concentrations of 1476.67, 1207.96, and 791.57 mg 100 g−1 db, respectively. Considerable losses of phytosterol were noted in dried samples. Rudzinska et al. (2009) reported that the stability of phytosterols is affected by chemical structure, processing temperature, and time [−1 db) because of the favorable drying condition of the reduced oxygen and the accelerated drying process. The freeze-dried samples had the lowest phytosterol content (462.07 mg 100 g−1 db). Overall, moderate hot air temperature (CD at 50 °C) and microwave power (9 W/g) retained higher phytosterol contents. Soupas et al. (2004) reported that several factors, such as temperature and heating duration, phytosterol structure, and lipid matrix composition, affect the oxidative stability of phytosterols [GC–MS analysis identified nine phytosterols, and the respective concentrations influenced by the different drying methods and drying intensities are shown in and time . In our osterols . Gawrysiosterols . With reosterols . With thBarriuso et al. (2012) indicated that the degradation level of phytosterols depends on their chemical structure  and inveS. crispus is shown in S. crispus. α-Linolenic acid is essential for overall well-being, as the human body does not have the required enzyme for the synthesis of this compound, which must be acquired from dietary sources [The fatty-acid profile of  sources .w) is a measure of the degree of water bound to a food product, and it is a key parameter that influences not only microbial systems, but also the chemical and physical stability of food products. The measured aw of fresh and dried leaves is shown in w of the leaves dried using the drying treatments ranged from 0.0245 to 0.1577. The FD samples showed the lowest aw. The aw of the samples subjected to CD at 50 °C and 60 °C and CPD-VMFD was lower than that of the samples subjected to CD at 40 °C and VMD (6 and 12 W/g). A high applied temperature coupled with a long drying duration in CD ensured the low residual moisture in the leaves. Increasing temperature in CD resulted in a low aw. Applying different levels of microwave power exhibited no evident trends. Dried products should have a low aw, ranging from 0.60 to 0.80, because spoilage bacteria and most molds are inhibited below aw of 0.91 and 0.80, respectively [w of the dried leaf samples shows that all of the drying treatments were effective in ensuring microbiologically safe products. The same conclusion concerns the chemical and physical stability of the dried product. The aw is a good indicator of biochemical degradation reactions that may take place during storage. For instance, it is known that Maillard reaction occurs at an optimum aw of 0.65. Therefore, the determination of aw is not only crucial in the prediction of shelf-life, but is also informative in terms of understanding biochemical reactions that may take place in relation to the aw value [Water activity  and greenest  color. The convective-dried samples at 40 °C and the VMD-treated samples  also exhibited lightness and greenness values comparable with those of the freeze-dried samples. The freeze-dried samples effectively preserved the color of herbs because heat was not involved in dehydration, in addition to a reduced oxygen condition. This finding was possible because the most widely occurring process in plants is the conversion of chlorophyll a and b to pheophytins (olive green) during heat treatment. Drying inherently led to the mechanical disruption of the cytoplasmic membrane, thereby allowing membrane permeability to increase, causing cellular acids to be released and degrading chlorophylls to pheophytins [Color is often perceived as an index of freshness and is associated with a nutritional value. Therefore, color is an important cue used by consumers to evaluate the quality of a certain product. Among factors contributing to the changes in the color profile of dried herbs are hydrolysis, caramelization, and nonenzymatic and enzymatic reactions. Chlorophylls are unstable and may undergo transformation or degradation, forming olive-brownish and greenish derivatives or colorless substances. of color . In our b*, a high b* (yellow) was obtained for the freeze-dried samples, indicating that carotenoids were preserved well during FD, followed by VMD at 12, 6, and 9 W/g, and all of these processes exposed the samples to a reduced oxygen condition. Carotenoids are susceptible to oxidation. As such, this result was expected. Carotenoids were preserved to a greater extent with CPD-VMFD at low hot air temperature (40 °C) and exposure to oxygen than with CD at 50 °C and 60 °C. For the fresh samples, the period between harvest and analysis could have allowed an extensive chlorophyll degradation to occur because of the high water content in the samples (aw of 0.9879). Hence, fresh leaves appeared dark, and their green and yellow intensity decreased.In p-methoxyphenol, chloroform/(folsch), BF3/MeOH, potassium hydroxide, hexane, sodium chloride, methanol, hydrochloric acid, magnesium sulfate, pyridine, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were purchased from Sigma-Aldrich .Folin–Ciocalteu reagent, sodium carbonate, gallic acid, FRAP reagent, acetate buffer, acetic acid, 2,4,6-tripyridyl-1,3,5-triazine (TPTZ), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulfate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), S. cripus leaves were purchased from TKC nursery  and verified at the Forest Research Institute of Malaysia (voucher specimen: 023/18). The moisture content of the fresh samples was gravimetrically determined by drying the leaves to a constant mass through vacuum-oven drying at 60 °C for 24 h. The leaves were divided into 40 g of samples and dried using four drying protocols. Drying was stopped when the mass loss of the consecutive samples was 0.05 g or less. For VMD and VMFD, the leaf samples were periodically removed from the vacuum microwave dryer to measure the temperature using an infrared thermometer . The obtained temperature was assumed to represent the maximal temperature of the sample.The samples were spread on trays and placed on top of the drying chambers of a convective hot air oven at 40 °C, 50 °C, and 60 °C.−1 and a temperature of 22 °C and, thus, prevent local overheating.The samples were dried in a vacuum microwave dryer  at 6, 9, and 12 W/g and placed in a glass container connected to a vacuum system with pressure ranging from 4 kPa to 6 kPa. The glass container was rotated at 6 rpm throughout the drying process and equipped with a fan to create an air stream with a velocity of 1 m s−1 dw was achieved. The pre-dried leaves were then dried with VMD  at 360 W [The samples were pre-dried at 50 °C in a convective hot air oven constructed in the Institute of Agriculture Engineering . Drying was stopped after 90 min, and a moisture content of 0.2666 kg water kgat 360 W .FD was performed in a freeze dryer  at a pressure of 65 Pa and a temperature of −60 °C. The temperature of the heating plate reached 30 °C. The freeze-dried samples were used as control samples.S. crispus leaves through CD, VMD, and CPD-VMFD were based on the mass losses of samples. 2O/kg dry matter (dm)) after drying time t, 2O/kg dm), and 2O/kg dm). At the final stage of drying, the equilibrium moisture content  and (8) show the specific energy consumptions of VMD, namely, Equations (9) and (10) present the value of the specific energy consumptions of CPD-VMFD, namely Approximately 0.3 g of ground samples was measured, placed in test tubes, and added with 80% aqueous methanol (0.7 mL) and 1% HCl. The suspension in the test tubes was stirred, sonicated twice for 15 min and left to stand at 4 °C for 24 h. The extract was centrifuged at 15,000 rpm  for 10 min, and the resultant supernatants were collected.●+). This mixture was left to stand in the dark at room temperature for 12–16 h before it was used in the assay. This solution was diluted with redistilled water to obtain an absorbance of 0.700 ± 0.02 at 734 nm. For the antioxidant activity analysis, 300 µL of the diluted radical solution was mixed with 20 µL of the extracted supernatant. Absorbance was read at 734 nm after 6 min. Determinations were performed in triplicate. The obtained results were expressed relative to micromolar Trolox per 100 g of dry weight in terms of Trolox equivalent antioxidant capacity.An ABTS radical-scavenging assay was conducted to evaluate the free-radical-scavenging activity as previously described . ABTS wa3 at a ratio of 10:1:1 (v/v/v). Subsequently, 300 µL of FRAP reagent was thoroughly mixed with 10 µL of the sample solution. Absorbance was read at 593 nm after 10 min. All of the prepared solutions were used on the same day of the preparation. A standard curve with different concentrations of Trolox was constructed. Determinations were performed in triplicates.FRAP assay, previously described by Benzie and Strain (1996), was used as a measure of antioxidant potential . For FRAThe total phenolic content was estimated by using the Folin–Ciocalteu method as described by Gao et al. (2000) . A leaf p-methoxyphenol as an internal standard. Glass vials were covered with a polytetrafluoroethylene (PTFE)/silicone septum and subsequently placed in a water bath at 60 °C for equilibration for 10 min. Extraction was performed using SPME fiber  with a vivinylbenzene/carboxen/polydimethylsiloxane fiber (divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS), 50/30 μm, coating 2 cm) (Supelco). The SPME fiber was conditioned by inserting it into a gas chromatography injection port at 220 °C for 1 h before extraction was conducted. Then, the SPME fiber was inserted into the vial containing the sample that was kept in a water bath at 60 °C for 30 min. The fiber was withdrawn into the needle and injected into the injection port of GC–MS at an injection temperature of 220 °C for 3 min. GC–MS analysis was conducted on a Varian CP-3800/Saturn 2000  equipped with a ZB-5MS capillary column (30 m × 0.25 mm inner diameter (i.d.) × 0.25 μm film thickness).Volatiles were analyzed using HS-SPME method. Crushed, dried, and fresh leaf samples of approximately 0.25 g were added in glass vials with 2 µg of −1, subsequently increased to 180 °C at 10 °C min−1, and finally increased to 280 °C at a rate of 2 °C min−1. Helium was used as a carrier gas at a flow rate of 1.0 mL min−1. The samples were manually injected in a split mode (split mode 1:10). Mass spectra were obtained in an electronic ionization (EI) mode of 70 eV with a scan range of m/z 35–550. After each extraction was completed, the SPME fiber was placed in the injection port of GC–MS at 220 °C for 15 min to ensure that volatiles were completely eluted to prepare for the subsequent extraction. Volatiles were identified by comparing the mass spectra of the compounds obtained experimentally, and the mass spectra were available in the NIST14 database. The retention index (RI) experimentally determined by Kovats was compared with the RI in NIST WebBook and literature data [p-methoxyphenol, with a concentration of 1 mg/mL.The initial oven temperature was kept at 50 °C, raised to 130 °C at a rate of 4 °C minure data . Quantifv/v) for 24 h and filtered. The extraction solvent was removed from the filtrate with a vacuum rotary evaporator at 65 °C. The obtained crude lipids were initially weighed and saponified with 10 mL of 0.5 M KOH/MeOH at 80 °C for 10 min, boiled with reflux for 5–10 min, and left to cool at 12 °C. The sample was then placed in a separation funnel. Hexane (10 mL) and water (10 mL) were sequentially added twice and vigorously mixed. For phase separation, 5 mL of saturated NaCl solution was added to the mixture and vigorously mixed. Fatty acids that partitioned to the polar phase were separated and removed from the phytosterol fraction.Lipid fraction was extracted in accordance with a method described previously  with min3/MeOH (v/v) at 80 °C for 10 min. The formed fatty-acid methyl ester (FAME) was extracted with 2.5 mL of hexane, dried with MgSO4, and filtered with a cotton plug and silicate powder into a small container. The FAME profile was analyzed using a GC–MS . Separation was performed using a Zebron ZB-WAX capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness) . Scanning was conducted at a mass range of 50–400 m/z, at an EI of 70 eV, and at a 5 scan s−1 mode. The carrier gas was helium with a flow rate of 1.0 mL min−1 at a split ratio of 1:10. The following temperature gradient programs were used: (a) 45 °C for 3 min, (b) 45 °C to 220 °C at a rate of 5 °C min−1, and (c) 220 °C to 250 °C at a rate of 10 °C min−1 and maintained at 250 °C for 2 min. The injector was held at 260 °C.The fatty-acid fraction obtained in 4 and evaporated using a vacuum rotary evaporator to remove any solvent. The obtained residue was mixed with 0.2 mL of pyradine and 0.2 mL of BSTFA as a silylation agent in a tube. Then, approximately 1 mg of cholesterol was added as the internal standard. The mixture was heated in an incubator orbital shaker at 110 rpm and at 60 °C for 45 min. After derivitization was completed, the mixture was transferred to small capped tubes and analyzed with GC–MS. Phytosterols were identified by comparing their experimentally obtained mass spectra with the mass spectra available in literature, and by comparing the relative retention times of the standards. The concentrations of identified phytosterols were determined by comparing the peak area of each compound to the peak area of internal standard, cholesterol, with a concentration of 1 mg/mL.The phytosterol fraction obtained in w of S. crispus leaves was measured with a water activity meter  and analyzed at an average temperature of 24.9 ± 0.05 °C.The aL*, a*, and b* coordinates. Color measurements were repeated five times. Fragmented leaves were placed in different positions, and ground and dried leaves were mixed in each reading.The color of fresh and dried ground leaves was analyzed using a Minolta Chroma Meter CR-200 , and data were expressed using p ≤ 0.05) between mean values were evaluated with Tukey’s test. Mathematical modeling was conducted with Table Curve 2D Windows v2.03 . Root-mean-square error and coefficient of determination (2R) were determined to evaluate the goodness of fit of mathematical models.Statistical analysis was performed using SPSS version 20 . Results were expressed as means ± standard deviation. Significant differences (S. crispus with considerably high bioactivities. Suitable hot air temperature and microwave power are necessary to obtain high antioxidant activities, TPC, and total volatiles and phytosterols. The highest antioxidant activity was obtained for the samples dried with CD at 40 °C and VMD at 9 W/g, whereas the highest total phytosterols were achieved with VMD at 9 W/g. CD at 50 °C and 60 °C were the best options for volatile compounds and TPC, respectively. The drying kinetics of S. crispus for CD, VMD, and CPD-VMFD were best described by an exponential equation or the modified Page model. The specific energy consumption of VMD was the lowest among the methods because the total drying time was shortened throughout the heating duration of microwaves. The freeze-dried samples exhibited the lowest aw and better color preservation.This study showed that CD and VMD show potential for producing dried forms of