**5. Conclusion**

Reports of *P. vivax* resistance to primaquine and chloroquine have been well documented. Nevertheless, attempts to validate the resistance status of primaquine rendered an equivocal results. With the current limitation in testing platform both *in vivo* and *in vitro*, the use of primaquine as anti-relaps compound is still recommended. Therefore, factors that may limit its use in *P. vivax* endemic setting such as G5PD deficiency should be excluded by deploying a cheap, easy to use Point-of-Care (PoC) G6PD test.

*Plasmodium vivax* resistance to chloroquine present different burden to each geographic areas. Therefore, the use of alternative drug ACTs should be tailored following the degree of resistance to chloroquine, as well as therapeutic response to any available ACTs.

*Plasmodium vivax* resistance to artemisinin has never been found in any of the *P. vivax* isolates examined from different geographic regions but resistance to partner drug such as amodiaquine, piperaquine, lumefantrine, mefloquine and pyronaridine should be regularly monitored to safeguard our arsenal for achieving malaria elimination by 2030.


**HERBIVORY**

Edited by **Breno Barros** and **Marcus E. B. Fernandes**

http://dx.doi.org/10.5772/2718 Edited by Breno Barros and Marcus E. B. Fernandes

#### **Contributors**

Marcus Emanuel Barroncas Fernandes, Allan Sebata, Michael Joseph Stout, Breno Barros, Yoichi Sakai, Hiroaki Hashimoto, Kenji Gushima, Marcelo Vallinoto, Yrlan Oliveira, Fernando Araújo Abrunhosa, Glenn Nader, Roger Ingram, Morgan Doran

#### **© The Editor(s) and the Author(s) 2013**

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First published in Croatia, 2013 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

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**7. Conclusions**

Based on the HYCOM reanalysis results, the NIWs generated by typhoon Megi in 2010 in the SCS were investigated in this study. Through a comparison with in situ observations at mooring UIB6, we first showed that the HYCOM reanalysis results can reasonably reproduce typhoon-induced NIWs, which can act as a supplement to in situ observations and provide us with an opportunity to better understand NIWs.

The results indicate that Megi-induced NIWs showed temporal and spatial variations in the SCS. The NIKE in the SCS was rapidly enhancedin response to typhoon Megi. However, the strongest NIKE appeared several days after the passage of Megi, rather than under its influence. Moreover, it is interesting to note that Megi-induced NIKE was mainly concentrated in the deep SCS basin where the water depth is greater than 1000 m, although typhoon Megi passed over both the deep SCS basin and shallow continental shelf and slope in the northern SCS. Through analysis, it was found that the continental slope in the northern SCS is supercritical to Megi-induced NIWs. In other words, when Megi-induced NIWs impinged on the continental slope, they were reflected and trapped in the deep SCS basin. This conclusion was validated by comparing the northward–downward and southward–downward components of Megi-induced NIWs. Moreover, it is found that Megi-induced NIWs could reach 1000 m depth in the vertical direction.

In situ observations have shown that Megi-induced NIWs dampened quickly at mooring UIB6 [32,37]. However, the HYCOM reanalysis results indicate that only in the region near Megi's track did the NIWs exhibit a similar feature, with the e-folding time generally being smaller than one week; whereas in two zones to the west of Luzon Island and the Luzon Strait, which are far away from Megi's track, the e-folding time of Megiinduced NIWs could be longer than 20 days. It was found that the NIWs generated at other sites could propagate to the two zones and influence the local NIKE, which accounted for the long e-folding time.

The modal content of Megi-induced NIWs was also explored in this study. Near Megi's track, Megi-induced NIWs were dominated by mode-2, which was followed by mode-3 and mode-1. The three modes accounted for 76% of the total NIKE. However, at the region far away from Megi's track, the NIKE of higher modes (mode-4 to mode-7) was enhanced several days after the passage of typhoon Megi. The cause of these higher modes still needs further exploration.

**Author Contributions:** Conceptualization, A.C.; methodology, A.C. and Z.G.; formal analysis, A.C., Z.G. and Y.P.; writing—original draft preparation, A.C.; writing—review and editing, A.C., Z.G., J.S., H.H. and P.L.; supervision, A.C. and J.S.; funding acquisition, A.C. and H.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Foundation of Zhejiang Province through grant LY21D060005, the Joint Project of Zhoushan Municipality and Zhejiang University through grant 2019C81060, and the National Natural Science Foundation of China through grant 41621064. This research was also supported by the Laboratory for Regional Oceanography and Numerical Modeling, Qingdao National Laboratory for Marine Science and Technology through grant 2019A01.

**Conflicts of Interest:** The authors declare no conflict of interest.


*2.2. Sample Preparation*

As part of the health examination while under general anesthesia, blood samples were collected from the jugular, cephalic or saphenous vein of each animal. Blood was collected in plain tubes (Sistema BD Vacutainer ®, Becton Dickinson Italia SPA, Italy) and serum was obtained by centrifugation at 10 min at 2500× *g*. Written owner consent for use of surplus blood samples, and use of data for scientific purposes, was obtained during consultations. The study design was approved by University of Milan Animal Welfare Bioethical Committee (Approval number OPBA 31/2019).

#### *2.3. Total Serum Protein*

Total serum protein concentration was measured by spectrophotometry using the colorimetric biuret method (Hagen Diagnostica S.R.L., Via Pratese 13 Firenze) on a Cobas Mira Classics Roche automated chemistry analyzer (Roche S.p.A., Mannheim, Germany).

#### *2.4. Agarose Gel Electrophoresis*

Sera samples were refrigerated at 4 ◦C and were analyzed within 8 hours of sampling. Protein fractions were analyzed using a semiautomated agarose gel electrophoresis (AGE) system (with HYDRAGEL Kit β1-β2 (SEBIA, Issy-les-Moulineaux, France). Serum was electrophoresed for 7 minutes at 33 volts hours and stained with diluted Amidoschwarz dye at pH 2 (4 g/L Amidoschwarz dye and 6.7% ethylene glycol). The AGE procedure was conducted according to the manufacturer's instructions, and commercial human serum was used as the control (normal control serum, Sebia, Evry, France). Using the computer software Phoresis for Windows 2000 or XP Pro (Sebia), the electrophoretic curve for each sample was displayed. Protein fractions were determined as the percentage optical absorbance, and the absolute concentration in g/dL was automatically calculated from the total serum protein concentration. Albumin to globulin (A/G) ratios were also calculated. The same operator analyzed all samples. To establish the inter-assay-accuracy of agarose gel electrophoresis on tiger serum, sera from two healthy tigers, one Bengal Tiger, 9A, F and one Siberian Tiger 1,1A, F, were tested 3 times on the same day, in the same laboratory and interpreted in duplicate by two operators. Coe fficient of variability (CV) computed as SD/mean × 100 was calculated for each protein fraction.


*3.10. Statistical Analysis*

SPSS software was used to perform an analysis of variance (ANOVA) on the obtained experimental results. 'Duncan's test was employed to conduct a mean comparison at a 0.05 level of significance (*p* ≤ 0.05).

#### **4. Results and Discussion**

#### *4.1. Density*

Figure 2 illustrates the density (*ρcomp*. *exp*.) value measurement for non-hybrid and hybrid SPF/GF/PLA composites. The density result showed that the density was improved by adding GF to the SP/PLA composites from 1.21 to 1.32 gm/cm3 . Comparing S1, –S3 composites, the density of S3 exhibited the highest value of 1.32 gm/cm3 , concluding that the hybridization of SPF/PLA composite increases the density of the composite. A similar finding was also revealed from a previous work done by Afzaluddin et al. [36] that density increment resulted from the addition of glass fiber into SPF/GF/TPU hybrid composites. This was due to the fact that GF has a higher density than SPF. The densities of untreated S3 and treated S5, S8 SP/GF hybrid composites were 1.32, 1.31, and 1.19 gm/cm3 . The density slightly decreased after treatment of SPF. In previous studies by Atiqah et al. [38] and Merlini et al. [50], density reductions of treated fibers were also recorded. Comparing alkaline treated S4–S6, and BC treated S7–S9 hybrid composites, density results showed that the density decreased slightly as the SPF content was increased, irrespective of whether the SPF has been alkaline or BC treated. As SPF wt % increased, the hydrophilic nature of the SPF made it more difficult to develop the composite properly. This led to voids in the hybrid composites, decreasing the composite density. A similar finding was supported by Safri et al. [51], where the density was measured for SPF/GF/epoxy hybrid composites.

**Figure 2.** Composite experimental density *ρcomp*. *exp*. vs. samples for non-hybrid and hybrid SPF/GF/PLA composites. \* Values with different letters in the figures are significantly different (*p* < 0.05).

#### *4.2. Moisture and Void Contents*

Table 4 shows moisture contents, theoretical/experimental composites density, and void contents of non-hybrid and hybrid SPF/GF/PLA composites. In general, the experimental and theoretical densities differed from each other due to a significant influence of voids and pores in the composite towards the behavior of the composite [40]. It is clear from Table 4 that, with the help of glass hybridization, the percentage of voids decreased, as confirmed by SEM images in the morphological investigation. The finding was also supported by the results from work by Radzi et al. [20]. The S4–S6 (alkaline treated) hybrid composites exhibited lower %voids than S7–S9 (benzoyl treated) hybrid composites. This was due to the good compatibility of SPF and GF with the PLA matrix. According to Jawaid et al. [36], voids formation is due to the incompatibility of natural fiber with matrix to displace all the trapped air which is entrained during fabrication of hybrid composite and incomplete wetting out of the fibers by the matrix. This research also reported that after Brabender mixing, the composite samples need to be placed in an electric oven for 24 h at 60 ◦C before the hot press. Otherwise, more voids are visible due to their moist content.

**Table 4.** Moisture contents, densities, and void contents of non-hybrid and hybrid SPF/GF/PLA composites.

\* Values with different letters in the same column are significantly different (*p* < 0.05).

#### *4.3. Water Absorption Analysis*

Figure 3 shows the effect of treatments on the values of water absorption (*WA*) of non-hybrid and hybrid SP/GF/PLA composites. The higher SPF content (S1 composite) resulted in a higher *WA* value. The S2 (GF/PLA) composite was the lowest *WA* compared to other non-hybrid or hybrid composites. Among the SPF/GF/PLA hybrid composites, S5 and S8 composites had the lowest *WA* values as both composites exhibited the same 15 wt % SPF. S5 composite was alkaline treated, indicating that alkali hydroxyl (—OH) groups inside the molecules were broken down and when reacted with H2O molecules and left the fiber structure. The other reactive molecules produce fiber-cell-O-Na groups in the molecular cellulose chains. This reduced hydrophilic hydroxyl groups and increased the water absorption resistance of the fiber. A considerable number of hemicelluloses, lignin, pectin, wax, and oil were also extracted after alkaline treatment [25], while BC treated S8 composite indicated that the fibers were treated with BC after pre-alkaline treatment. The —OH fiber groups were substituted by the benzoyl group and bonded to the backbone of the cellulose. This resulted in the increased hydrophobicity of the fiber and enhanced matrix adhesion. The following is the order of the decreasing value of hybrid and non-hybrid SPF/GF/PLA composites' water absorption S2< S8< S5< S7< S9< S6< S4< S3< S1. After the 6th day, no change was observed in *WA*. For hybrid composite, the higher loading of natural fibers was considered to have more *WA*, whereas the higher GF loading was related to lower *WA*. This was also consistent with the past work of Afzaluddin et al. [36], who found that water resistance increased with the addition of GF in the SPF/TPU composites.

**Figure 3.** % Water absorption (*WA*) versus samples for non-hybrid and hybrid SPF/GF/PLA composites.

#### *4.4. Thickness Swelling*

Figure 4 presents the effect of treatments on the values of thickness swelling (*TS*) of non-hybrid and hybrid SP/GF/PLA composites. With the increase in *WA*, the thickness swellings of all the composites were increased. The higher the percentage of GF, the lower the *TS* value, while the higher the SPF content, the higher the *TS* value. This was due to the fact that GF possessed water resistance ability while SPF has hydrophilic nature [52]. The highest *TS* value was shown by untreated S1 (SPF/PLA) composite, having maximum content of SPF. In the S2 composite, no *TS* value was observed since it contained only

GF and PLA. Among the hybrid composite, the maximum *TS* value was observed in the S3 (untreated SPF/GF/PLA) hybrid composite. The thickness swelling was due to the prolonged immersion duration, where more water molecules were bonded to the hydrogen bonds of fiber. This *TS* value was randomly decreased after alkaline treatment due to the reduction in the micropores and collapsing capillaries, as well as the removal of wax and impurities at the fiber surface after the treatment. This predicted reduced water retention, as indicated by lessening the amount of water absorbed by the fiber. This reduced the *TS* value of the hybrid composite. Due to this cause, as the alkaline treated SPF content was increased, the *TS* value decreased. A similar finding was reported by N.B.M. Hafidz et al. [53] after alkaline treatment of palm oil fiber composites as well as kenaf fiber composites.

**Figure 4.** % Thickness swelling (*TS*) versus samples for non-hybrid and hybrid SPF/GF/PLA composites.

The reduction of *TS* was also observed after BC treatment of SPF for hybrid composite. S8 composite showed the lowest *TS* value among all hybrid composite. For the S9 composite, as the benzoyl treated SPF content was increased to 20 wt %, the *TS* value increased due to the disruption of lignin and polysaccharides during treatment that enhanced cellulose concentration. The cellulose chemical structure is composed of hydroxyl that is accessible to water. This directly stimulated the *WA*, thus increased the *TS* [51].

#### *4.5. Tensile Testing*

Figure 5a,b shows the effect of treatment on tensile strength and modulus of nonhybrid and hybrid SPF/GF/PLA composites. Hybrid composites were used for various weight percentages of SPFs and GFs. Hybrid composites had a combined weight percentage concentration of fibers (SPF/GF) fixed at 30%, whereas PLA was at 70%. Figure 5a confirmed that by incorporating the GFs, the tensile strength of the PLA composite was

increased significantly. From Table 1, the tensile strength of GF was more than SPF, while the tensile strengths of S1 (SPF only) and S2 (GF only) reinforced PLA composite were 16.0 MPa and 23.7 MPa, which indicated that the tensile strength on the addition of GFs was better than untreated SPF. It was observed that the addition of alkali-treated SPF increased the tensile strength of the hybrid SPF/GF reinforced PLA composites. The highest tensile strength of 26.3 MPa was shown by the S6 composite among all hybrid composites. According to Afzaluddin et al. [36], the tensile strength of SPF/GF reinforced TPU hybrid composites could be increased by adding SPF. Among S4–S6 (alkaline treated) hybrid composites, S6 exhibited a maximum tensile strength of 26.3 MPa, while S5 and S4 demonstrated only 14 MPa and 18.7 MPa, respectively. This proves that the interfacial bonding between SPF and PLA matrix had improved after alkaline treatment. S6 hybrid composite exhibited maximum tensile strength that might be due to GF (10% loaded); therefore, SPF can effectively transfer the load from the GF on this particular composition [30]. When alkaline treated SPF/GF/PLA hybrid composites were compared with benzoyl treated SPF/GF/PLA hybrid composites, it is clearly shown in Figure 5a that the alkaline treatment was the best treatment approach used for the improvement of tensile strength. This was due to the improvement in the interface bonding by giving rise to additional sites of mechanical interlocking, facilitating the interpretation of fiber-matrix at the interface. The alkaline treatment of fiber increased the binding properties of the surface by eliminating natural and artificial impurities, which created rough surface topography [30]. This finding was also supported by the morphological investigation of this research.

S3, S5, and S8 had the same composition of 15/15/70 wt % SPF/GF/PLA. The only difference they possessed was the untreated or treated SPF. The analysis showed that the tensile strength of S5 was improved by 27% compared to the S3 hybrid composite. The tensile strength of the alkaline treated SPF/GF/PLA was higher than untreated hybrid composites due to the strong interfacial bonding between treated fiber with PLA matrix. A similar study was carried out by Atiqah et al. [3], that the surface treatment of fiber improved the tensile properties of SP/GF reinforced TPU hybrid composites. The minimum value of tensile strength was 9.3 MPa, which was recorded for S8. The inclusion of BC treated SPF in SPF/GF/PLA hybrid composites caused a remarkable decrease in tensile strength. One of the investigations by Swain et al. [54] demonstrated that the tensile strength only decreased when there was weak bonding between fiber and the matrix.

In general, the stiffness property of the SPF/GF reinforced PLA hybrid composites was also determined. Material stiffness property is mainly indicated by tensile modulus. In general, the value of tensile modulus was increased with an increase in wt % of GFs. This statement was valid for both untreated as well as treated SPF/GF reinforced PLA hybrid composite. The tensile moduli for BC treated SPF/GF/PLA hybrid composites were increased by the GF percentage, i.e., for 10, 15, and 20 wt % GF in S9, S8, and S7 composites, the value of tensile moduli were 500, 505, and 607 MPa.

Comparing untreated SPF of S1–S3 composites indicated increasing percentage of GFs improved the tensile modulus of composites. The highest tensile modulus was shown by S2, having 30 wt % of GFs as depicts in Figure 5b. This is since the tensile modulus and strength of GF was higher than SPF. The increment of tensile modulus denoted the improvement of load-bearing capacity.

**Figure 5.** (**a**) Tensile strength versus samples of non-hybrid and hybrid SPF/GF/PLA composite. (**b**) Tensile modulus versus samples of non-hybrid and hybrid SPF/GF/PLA composite. \* Values with different letters in the figures are significantly different (*p* < 0.05).

On comparing the alkaline treated SPF/GF/PLA composites, the analysis showed that the S6 hybrid composite exhibited the maximum tensile modulus of 561 MPa. The S6 hybrid composite had a maximum of 20 wt % of SPF, indicating improvement in the interfacial bonding between SPF and PLA matrix after the alkaline treatment of SPF that consequently resulted in the improved tensile modulus [20]. In addition, after alkaline treatment, rougher topography might cause the qualitative interface between fiber and matrix due to the removal of waxy and impurity substances. A similar increment of tensile modulus after alkaline was revealed by Mukhtar et al. [18] for SPF/GF-reinforced polypropylene composites. Comparing the alkaline, BC treated, and untreated SPF/GF/PLA hybrid composites, it might be noted that BC treatment increased the tensile modulus of the same wt % composite, i.e., S3, S5, and S8. Among these three composites having 15/15/70 wt % (SPF/GF/PLA), the highest value of tensile modulus 505 MPa was shown by S8, followed by 435 MPa and 423 MPa, for S5 and S3 composites, respectively. Previous studies [29,51,55] indicated that the treatment of fibers with BC enhanced the mechanical contact surface with the matrix, which enhanced the interfacial bonding between fiber and matrix and increased stress transfer and tensile modulus of the entire composite. The maximum tensile modulus was shown by the S7 (BC treated) hybrid composite. The tensile properties in terms of the tensile stress–strain curve of various composites is shown in Figure 6.

**Figure 6.** Tensile stress (MPa) versus % tensile strain for various composites.


*3.2. Floquet Multiplier Estimation*

In Section 2.3, the matrix *∂P*(*q*∗)/*∂q* is provided by the monodromy matrix with restriction to the *n* − 1-dimensional hyperspace Ω. The *P*(*q*) can be expressed by Taylor series in the vicinity of the fixed point *q*∗. Assuming that periodic solution is stable, higher-order term can be negligible. Recalling that *P*(*q*∗) = *q*∗ at the fixed point, Equation (10) is linearized as

$$P(q) - q^\* = [\frac{\partial P(q^\*)}{\partial q}](q - q^\*)\tag{11}$$

For the specific vector *q*1, the corresponding critical eigenvalue *μ*1 is calculated using the linear approximated equation

$$P(q\_1) - q^\* = \mu\_1 (q\_1 - q^\*) \tag{12}$$

If we have series data *x*, the relationship between the mapping *P* of point *q* and point *q* can be followed by sequence *x*[*k*] and *x*[*k* + 1]. Hence, the computational form gives Equation (11) by

$$\mathbf{x}[k+1] - \mathbf{x}^\* = \mathfrak{A}(\mathbf{x}[k] - \mathbf{x}^\*) \tag{13}$$

Equation (12) is a typical form of linear regression, where *μ*ˆ is a constant to be determined by series data *x*.

For application to time series data, the equation should be modified to a scalar form. In that case, the calculated Floquet multiplier might not be the same as the actual value since the scalar value cannot reflect the direction. However, there will be negligible differences when selecting the acceptable component of the state variable. Using engineering judgment, it will be natural to select the component with the largest variance among the state variables *x*.

$$s[k+1] - s^\* = \pounds\_F(s[k] - s^\*) \tag{14}$$

The estimated Floquet multiplier *μ*ˆ *F* can be easily computed with Equation (10). The estimated value, however, is not the exact value of the calculated Floquet multiplier because it is estimated and projected in one dimension, regardless of the number of original system state variables it contains. But as long as the signal is periodic, the estimated Floquet multiplier gives information on stability even if we choose single measurement as a state variable.

For application to time series data, Equation (13) can be expressed after changing scalar *s* to voltage term *V*

$$\hat{\mu}\_{F,series} = \frac{1}{N} \sum\_{k=1}^{N} \frac{V[k+1] - (1 - \mu)V^\*}{V[k]} \tag{15}$$

Ironically, the linearized form of Equation (13) contradicts the stable orbits when *s*[*k* + 1] ≈ *s*[*k*] ≈ *s*∗. Hence, the *μ* term of Equation (14) should be 1 by applying the stable orbit condition.

$$\hat{\mu}\_{F,series} = \frac{1}{N} \sum\_{k=1}^{N} \frac{V[k+1]}{V[k]} \tag{16}$$

Then, the final estimation Equation (15) further simplifies the problem such that the estimated Floquet multiplier is only the average of the ratio of the previous and current local minima of the voltage at all peak data. In other words, we can set the fixed-point value to zero. It can be guaranteed that *μ*ˆ *F*,*series* = 1 is a sufficient condition for stable orbit. Using final Equation (15), other unstable cases are demonstrated by two examples of power systems in Section 4.