Anthelmintic-Like Activity and Ultrastructure Changes Produced by Two Polyphenolic Combinations against Cooperia punctata Adult Worms and Infective Larvae

Cooperia punctata is one of the most prevalent gastrointestinal nematodes affecting cattle under grazing conditions, and the increasing reports of anthelmintic resistance forces researchers to look for novel control measures. Previous reports have proposed the use of polyphenolic compound (PC) combinations (Coumarin:Quercetin (CuQ) and Caffeic-acid:Rutin (CaR)) against free-living stages (L3) of C. punctata. The objective of this study was to assess the in vitro motility inhibition of C. punctata adult worms and infective larvae using the Larval Motility Inhibition Assay (LMIA) and Adult Motility Inhibition Assay (AMIA), and to assess the structural and ultrastructural changes induced by both treatments using Scanning and Transmission Electron Microscopy. For the LMIA, infective larvae were incubated for 3 h in 0.8 mg mL−1 and 0.84 mg mL−1 of CuQ and CaR, respectively. For AMIA, six concentrations and five incubation periods (2, 4, 6, 12 and 24 h) were assessed using each PC combination. Cooperia punctata motility was calculated as a percentage and corrected using control motility percentages. A multiple comparisons Brown–Forsythe and Welch ANOVA test was used to compare larval motility; and to fit the dose–response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism® V.9.2.0. Although larval motility was barely affected by both treatments (p > 0.05), adult worm motility was inhibited 100% and 86.9% after 24 h incubation with CuQ and CaR, respectively (p < 0.05). The best fit EC50 for adult worm motility inhibition were 0.073 ± 0.071 mg mL−1 and 0.051 ± 0.164 mg mL−1 for CuQ and CaR, respectively. Main structural and ultrastructural lesions observed in both biological stages were: (i) L3 sheath–cuticle complex disruption, (ii) collagen fibers degradation; (iii) hypodermic detachment, (iv) seam cell apoptosis and (v) mitochondrial swelling. The alterations observed suggest that the PC combinations interfere with the anatomy and physiology of the locomotive apparatus of the nematodes.


Introduction
Cooperia punctata represents one of the most prevalent and pathogenic species among gastrointestinal nematodes (GIN) affecting grazing cattle in the tropics [1]. Gastrointestinal nematodes are one of the weakest links of animal health in grazing cattle industry; not only due to their negative impact upon health, welfare, and producer's economy, but The LMIA was used to evaluate the effect of each polyphenolic compound combination on the mobility of four-weeks-old ensheathed C. punctata L 3 [10]. A thousand ensheathed L 3 mL −1 were placed in 15 mL falcon tubes and incubated on each combination to be tested (0.8 mg mL −1 and 0.84 mg mL −1 of CuQ and CaR, respectively) and in ethanol at 2.5% (negative control). Thiabendazole (TBZ) 99% (Sigma ® , St. Louis, MO, USA) was used as positive control at a concentration of 10 mg/mL −1 . All incubations were carried out for 3 h at 21.3 • C. Afterwards, L 3 from each tube were immediately washed with distilled water and centrifuged (908× g) three times. Larvae were then transferred to sieves (inserts equipped with a 20 µm mesh positioned in a conical tube). After 3 h at room temperature (26 • C), the number of L 3 migrated through the mesh were counted. The percentage of migration was calculated as %M = (M/T) × 100 (where T represents the total number of L 3 deposited on the sieve and M stands for the number of L 3 that had migrated). Four replicates were run for each combination and control.

Adult Motility Inhibition Assay (AMIA)
Immediately after recovery, 13-15 motile C. punctata adult males were gently washed three times in a physiological saline solution (PSS 0.09%, Pisa ® JAL, MX) previously heated at 37 • C, and placed in a 6-well cell culture cluster, with their corresponding treatment. Polyphenolic combinations proposed by Escareño et al. (2019) [7]: Coumarin:Quercetin (CuQ) and Caffeic acid:Rutin (CaR) in an 8:2 ratio were used to obtain the mean effective concentration (EC 50 ) against adult worms. A total of 3920 ± 10.4 (Mean ± SE) C. punctata adult worms of were used to assess the effect of the PCs-combinations on C. punctata adult motility over the time (2, 4, 6, 12 and 24 h). For the CuQ combination, C. punctata adult worms were incubated in multiple 6-well cell culture clusters at increasing concentrations of 0.022, 0.043, 0.087, 0.175, 0.35, and 0.8 mg/mL −1 of physiological saline solution (PSS 0.09%, Pisa ® ). For the CaR combination, adult worms were incubated at increasing concentrations of 0.026, 0.052, 0.105, 0.21, 0.42, and 0.84 mg/mL −1 of physiological saline solution (PSS 0.09%, Pisa ® ). Wells containing 2.5% ethanol diluted in physiological saline solution (0.09% NaCl, Pisa ® ) were used as negative control. Cell culture clusters were incubated in a CO 2 incubator (37 • C with a 5% CO 2 inclusion) (CO 2 -Incubator C-150 Binder ® ). Four replicates were run for each concentration, time lapse and control. Worm motility was examined at 2, 4, 6, 12 and 24 h after incubation using an inverted microscope (Nikon Eclipse Ts2 ® ), and the number of motile and non-motile C. punctata (no movement detected in 10 s) was recorded [11]. Individual adult motility per well was calculated as: Worm motility % = 100 × (number of motile worms in the concentration/total number of worms in the same concentration). Finally, motility was adjusted using control motility percentages. Worm motility percentages correction per concentration was calculated using the following formula: Adult motility corrected % (AMc) = 100 − (100 × (1 − MwTx/MwC)). Where MwTX represents the motility percentage of worms incubated in the treatment, and MwC represents the motility percentage of worms in the control group. to assess structural and ultrastructure changes in both C. punctata stages. For the control groups L 3 were incubated in 2.5% ethanol diluted in distilled water and adult worms in 2.5% ethanol diluted in physiological saline solution (0.09% NaCl, Pisa ® ). Four replicates were assessed for each combination and control. After incubation, L 3 were washed and centrifuged (908× g) three times in DW (pH 7.2), placed in the fixing solution (see Section 2.5) and stored at 4 • C. Adult worms were individually washed in physiological saline solution (0.09% NaCl, Pisa ® ), fixed and stored at 4 • C until processed for ultrastructure analysis (see Section 2.5).

Scanning and Transmission Electron Microscopy (TEM and SEM)
Immediately after incubation (see Section 2.4.1 and 2.4.2) C. punctata L 3 and adult worms (12,000 and 360, respectively) were placed in 15 mL falcon tubes, fixated with paraformaldehyde, and transported at 4 • C for further processing [12]. Scanning and Transmission Electron Microscopy samples were processed at the Electron Microscopy laboratory of the biology area and cellular biology department, respectively, from the Science Faculty UNAM, Mexico. At the laboratory, samples were washed with PBS to remove the fixer, followed by a second fixation with osmium tetroxide 2%; L 3 were fixated for 1 h while adult worms were kept overnight. After fixation, samples were dehydrated using increasing ethanol series [13]. Finally, samples were equally divided for further SEM and TEM processing.
Samples for SEM (6000 L 3 and 180 adult worms) were dried with CO 2 in a criticalpoint dryer (BAL-TEC, CPD 030 ® ) and coated with gold for 5 min at an ionizer (DESK II DENTON, VACUUM ® ). Finally, both L 3 and adult worms were observed under a Scanning Electron Microscope (JSM-5310LV, JEOL ® TKY, JP) at an accelerating voltage of 15 and 20 kV.
Samples destinated for TEM (6000 L 3 and 180 adult worms) were pre-embedded with propylene's oxide and Epon 812 resin (1:1) for 24 h; afterwards, samples were embedded in polymerized epoxy resin at 60 • C for 24 h. Semi-thin sections (250 nm) were performed and stained with toluidine blue to obtain a general description under an optic microscope. Ultrathin sections (60 nm) were made using a microtome (Ultracut R, Leica ® WETZLAR, DE) and samples were placed on formvar-coated cooper grids. Afterwards, samples were contrasted first with uranyl acetate 4% (20 min and 30 min for L 3 and adult worms, respectively) and then with lead citrate 0.3% for 10 min [9]. Finally, observation of the ultra-thin sections was performed using a Transmission Electron Microscope (JEM 1010, JEOL ® ) operating at 80 kV. The Digital Micrograph V.2.32.888.0 software was used to obtain the microphotographs.
The alterations observed in the specimens were counted and expressed as percentages. Percentages were obtained after being corrected with the control group using the following formula: Percentage = ((100 − nC)/N) × nTx. Where nC stands for the number of specimens presenting the alteration, N is the total number of specimens assessed, and nTx stands for the number of specimens presenting the alteration in the treated group.

Statistical Analyses
A multiple comparisons Brown-Forsythe and Welch ANOVA test was used to compare larval motility (GraphPad Prism ® , LLC V.9.2.0.). To fit the dose-response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism ® V.9.2.0. All analyses were performed after transforming the data into logarithms (X = logX) and constraining the bottom and top values to 0 and 100, respectively. Finally, EC 50 , 95% confidence limits and R 2 values were also calculated.

Effect of Polyphenolic Combinations on the Motility of C. punctata Infective Larvae
Larval migration in the negative control was 86.44 ± 3.22%; while the positive control (Thiabendazole) migrated 9.02 ± 5.42% of C. punctata larval motility. Both PC combinations showed a low motility inhibition (p > 0.05); thus, CaR had a higher inhibitory activity (13.95 ± 4.84) than the CuQ combination (Table 1; p < 0.05).

Effect of PCs-Combinations on the Motility of C. punctata Adult Worms
Adult worms from the negative control showed a motility of 100 ± 0.00%, 83.41 ± 1.59% and 74.54 ± 6.17% at 6, 12 and 24 h post-treatment, respectively. The motility of adult worms using both PC combinations was not affected after 2 h, 4 h and 6 h post-exposure to the CaR; however, after a 6 h incubation with CuQ, the motility was inhibited in 27.3%. The adult motility at 12 and 24 h after incubation in CaR was 87.01% and 86.9%, respectively; conversely to the inhibition observed with CuQ, where at the maximal concentration, motility inhibition reached 73.81% and 100% after 12 and 24 h, respectively. The best-fit EC 50 showing a higher coefficient of determination (R 2 ) was CuQ after 24 h (0.073 ± 0.071 mg mL −1 ; R 2 0.94) and CaR after 12 h exposure (0.192 ± 0.061 mg mL −1 ; R 2 0.95) ( Figure 1). Mean effective concentrations, 95% confidence intervals and R 2 are presented in Table 2.

Effect of Polyphenolic Combinations on the Motility of C. punctata Infective Larvae
Larval migration in the negative control was 86.44 ± 3.22%; while the positive control (Thiabendazole) migrated 9.02 ± 5.42% of C. punctata larval motility. Both PC combinations showed a low motility inhibition (p > 0.05); thus, CaR had a higher inhibitory activity (13.95 ± 4.84) than the CuQ combination (Table 1; p < 0.05).

Effect of PCs-Combinations on the Motility of C. punctata Adult Worms
Adult worms from the negative control showed a motility of 100 ± 0.00%, 83.41 ± 1.59% and 74.54 ± 6.17% at 6, 12 and 24 h post-treatment, respectively. The motility of adult worms using both PC combinations was not affected after 2 h, 4 h and 6 h postexposure to the CaR; however, after a 6 h incubation with CuQ, the motility was inhibited in 27.3%. The adult motility at 12 and 24 h after incubation in CaR was 87.01% and 86.9%, respectively; conversely to the inhibition observed with CuQ, where at the maximal concentration, motility inhibition reached 73.81% and 100% after 12 and 24 h, respectively. The best-fit EC50 showing a higher coefficient of determination (R 2 ) was CuQ after 24 h (0.073 ± 0.071 mg mL −1 ; R 2 0.94) and CaR after 12 h exposure (0.192 ± 0.061 mg mL −1 ; R 2 0.95) ( Figure 1). Mean effective concentrations, 95% confidence intervals and R 2 are presented in Table 2.

Scanning Electron Microscopy (SEM)
The 80% of infective larvae analyzed from the control group (ethanol 2.5%) maintained the structural integrity of the sheath with a normal transversal and longitudinal ridges pattern (black asterisk; trp). The cylindric shape of the body was kept in 80% of the L 3 , however, a slight loss of turgor observed in all specimens (Figure 2A-C). Conversely, 96% of C. punctata L 3 exposed to PCs-combinations and thiabendazole evidenced a loss of structural integrity of the sheath ( Figure 2D-L). In a generalized manner, 77.4% and 88.2% of the L 3 incubated in CuQ and CaR, respectively, lost their cylindric shape and presented an irregular surface of both the cephalic and body regions ( Figure 2D,G). Furthermore, over 80% of the L 3 incubated both PC combinations and TBZ showed a shrunken appearance with a loss of body turgor (bt) and multiple furrows (f) and depressions (d) along the sheath, and a loss of definition of the transversal ridges pattern in a diffuse focal pattern (trp).

Scanning Electron Microscopy (SEM)
The 80% of infective larvae analyzed from the control group (ethanol 2.5%) maintained the structural integrity of the sheath with a normal transversal and longitudinal ridges pattern (black asterisk; trp). The cylindric shape of the body was kept in 80% of the L3, however, a slight loss of turgor observed in all specimens (Figure 2A-C). Conversely, 96% of C. punctata L3 exposed to PCs-combinations and thiabendazole evidenced a loss of structural integrity of the sheath ( Figure 2D-L). In a generalized manner, 77.4% and 88.2% of the L3 incubated in CuQ and CaR, respectively, lost their cylindric shape and presented an irregular surface of both the cephalic and body regions ( Figure 2D,G). Furthermore, over 80% of the L3 incubated both PC combinations and TBZ showed a shrunken appearance with a loss of body turgor (bt) and multiple furrows (f) and depressions (d) along the sheath, and a loss of definition of the transversal ridges pattern in a diffuse focal pattern (trp).  Scanning Electron Microscopy of adult worms showed that 86.67% of the specimens incubated for 24 h in ethanol 2.5% had a regular cylindric shape with a slight loss of body turgor (bt) and with a normal pattern of both longitudinal and transverse ridges (LRP and TRP, respectively) (negative control; Figure 3A-D). However, 89.18% to 91% of C. punctata worms exposed to the PCs-combinations had an increased number of cuticular furrows (f) and depressions (d); a marked loss of body turgor (bt) giving the appearance of cuticular stiffness, and a loss of the typical cylindric shape of nematodes ( Figure 3D-L). Furthermore, over 90% of the adult worms exposed to the PCs-combinations showed a loss of the transversal ridges pattern definition (trp) and partial deformation of the longitudinal ridges pattern ( Figure 3H; black arrow).
Pathogens 2023, 12, 744 7 of 18 TRP, respectively) (negative control; Figure 3A-D). However, 89.18% to 91% of C. punctata worms exposed to the PCs-combinations had an increased number of cuticular furrows (f) and depressions (d); a marked loss of body turgor (bt) giving the appearance of cuticular stiffness, and a loss of the typical cylindric shape of nematodes ( Figure 3D-L). Furthermore, over 90% of the adult worms exposed to the PCs-combinations showed a loss of the transversal ridges pattern definition (red arrow) and partial deformation of the longitudinal ridges pattern ( Figure 3H; black arrow).

Transmission Electron Microscopy (TEM)
3.4.1. Cooperia punctata Infective Larvae L3 Figure 4A-D shows the cuticle-sheath complex and internal ultrastructure of C. punctata L3 incubated in the control group (ethanol 2.5%), CuQ, CaR and TBZ, respectively. In the control group, only 26.67% of the L3 presented a detachment of the sheath, while 96%    (Figure 6B,D) and union plates (UP) were also observed ( Figure 6C).
The somatic cell muscle (ScM) of larvae exposed to CaR displayed a myofilament disorganization on the A band of some sarcomeres with an apparent reduction in the number of TnM ( Figure 6C, oval shape) due to an increased electron lucency of TnM (white asterisk) and increased electron density of TkM (black asterisk). The alterations previously described were also observed for the positive control, although in the TBZ treatment, L3 showed a stronger degeneration of TkM ( Figure 6D, black asterisk).  (Figure 6B,D) and union plates (UP) were also observed ( Figure 6C).
The somatic cell muscle (ScM) of larvae exposed to CaR displayed a myofilament disorganization on the A band of some sarcomeres with an apparent reduction in the number of TnM ( Figure 6C, oval shape) due to an increased electron lucency of TnM (white asterisk) and increased electron density of TkM (black asterisk). The alterations previously described were also observed for the positive control, although in the TBZ treatment, L 3 showed a stronger degeneration of TkM ( Figure 6D, black asterisk).
Finally, micrographs of the lateral end of Cooperia punctata L 3 allowed the visualization of the cuticle-sheath complex and epithelial specializations ( Figure 7A-D). Up to 100% of specimens exposed to the treatments lost the ILSp transverse band pattern and the cilia of the deirid-like neuron tip (Dn) and deirid-like neuron base ( Figure 7B Finally, micrographs of the lateral end of Cooperia punctata L3 allowed the visualization of the cuticle-sheath complex and epithelial specializations ( Figure 7A-D). Up to 100% of specimens exposed to the treatments lost the ILSp transverse band pattern and the cilia of the deirid-like neuron tip (Dn) and deirid-like neuron base ( Figure 5B-D; black star).
The seam cell (SC) of treated L3 was also affected within all treatments, and the main lesions observed were: loss of nuclear envelope ( Figure 7C,D; NcE); and vacuolization of the cytoplasm in 93.33%, 53% and 100% of L3 incubated in CuQ, CaR and TBZ, respectively (Va; Figure 7B-D). Condensation and margination of the heterochromatin within the nucleus (white asterisk) was registered in 33.3% of the L3 incubated in CuQ and 100% of the L3 incubated CaR and TBZ. Finally, electron densification of the nucleus cytoplasm was evident in 66.67% and 7% of L3 exposed to CuQ and CaR, respectively. The seam cell (SC) of treated L 3 was also affected within all treatments, and the main lesions observed were: loss of nuclear envelope ( Figure 7C,D; NcE); and vacuolization of the cytoplasm in 93.33%, 53% and 100% of L 3 incubated in CuQ, CaR and TBZ, respectively (Va; Figure 7B-D). Condensation and margination of the heterochromatin within the nucleus (white asterisk) was registered in 33.3% of the L 3 incubated in CuQ and 100% of the L 3 incubated CaR and TBZ. Finally, electron densification of the nucleus cytoplasm was evident in 66.67% and 7% of L 3 exposed to CuQ and CaR, respectively.  Figure 8B). The hypodermis (Hy) presents a homogeneous electron density and several mitochondria ( Figure 8A, white squares). In the cuticle, we can distinguish the medial zone (MZ), the fibrous tri-layer from the basal zone (FTL), and the hemidesmosomes projected from the basal zone and connected with epithelial cells (Hd; Figure 8C). Figure 8C allows visualization of the FTL present in the basal zone with two well-defined external layers in a spiral disposition (black asterisks) that are crossed by a third layer of fibers that are longitudinally disposed (white asterisk).  Figure 8B). The hypodermis (Hy) presents a homogeneous electron density and several mitochondria ( Figure 8A, white squares). In the cuticle, we can distinguish the medial zone (MZ), the fibrous tri-layer from the basal zone (FTL), and the hemidesmosomes projected from the basal zone and connected with epithelial cells (Hd; Figure 8C). Figure 8C allows visualization of the FTL present in the basal zone with two well-defined external layers in a spiral disposition (black asterisks) that are crossed by a third layer of fibers that are longitudinally disposed (white asterisk).

Adult Worms
Parasites incubated in PC combinations showed a slight electron densification of the CL, however, an increased electron lucency in the MZ was evident in all treated worms. Parasites incubated in PC combinations showed a slight electron densification of the CL, however, an increased electron lucency in the MZ was evident in all treated worms. Furthermore, parasites exposed to CuQ combination presented higher electron lucency of the three-layer fibers from the basal zone with a marked loss in the fiber's definition, apparent length reduction, and an increased angle of the spiral fibers with respect to the longitudinal layer ( Figure 8F; black asterisk and white asterisk, respectively). Hemidesmosomes (Hd) showed a reduced electron lucency and definition ( Figure 8F), and the hypodermis reveals cell derangement and electron densification ( Figure 8D; black square) with electron-lucent zones suggesting hypodermic detachment ( Figure 8D; black triangles). Moreover, 75% of parasites exposed to CuQ combination presented mitochondrion swelling and electron density alterations in both mitochondrial cristae and matrix ( Figure 8E; white squares). gles). Moreover, 75% of parasites exposed to CuQ combination presented mitochondrion swelling and electron density alterations in both mitochondrial cristae and matrix ( Figure  8E; white squares).
Lastly, in all analyzed C. punctata worms incubated in the CaR combination, the FTL had a significant increase in electron density in both spiral layers and an increased electron lucency of the longitudinal layer ( Figure 8I; black asterisk and white asterisk, respectively). Electron-lucent spaces through the layers were also observed ( Figure 8I, black triangles) suggesting detachment between fibrous layers. Finally, an apparent shortening of the fibers length that could be associated to an increase in the spiral angle disposition with respect to the longitudinal fibrous layer.  Lastly, in all analyzed C. punctata worms incubated in the CaR combination, the FTL had a significant increase in electron density in both spiral layers and an increased electron lucency of the longitudinal layer ( Figure 8I; black asterisk and white asterisk, respectively). Electron-lucent spaces through the layers were also observed ( Figure 8I, black triangles) suggesting detachment between fibrous layers. Finally, an apparent shortening of the fibers length that could be associated to an increase in the spiral angle disposition with respect to the longitudinal fibrous layer.

Motility Inhibition of Polyphenolic Compounds Combinations (PCs-Combinations)
Development of new drugs is a time consuming and expensive industry, which can take up to 15 years, cost around $1.2 billion dollars and has only a 5% success rate [6]. Even though most drug-engineering technologies have rapidly improved over the years, there has been an anthelmintic drug-underdevelopment, which could be attributed to the poor knowledge of parasite biology [6]. In vitro assays as a tool for new drug screening is more expensive and time consuming; however, it allows us to target different biological stages of the most important parasites [6]. The molecules and combinations used in this study were selected from previous research reporting their AH-like activity against egg hatching and larval exsheathment of C. punctata [7]. In accordance with Paolini et al. (2003) [14], the results of this research showed that the concentration of PCs used have a higher activity against adult motility than ensheathed L 3 ; which could be associated with the differences in molecular binding sites of both biological stages of the nematode. Multiple studies have reported that the susceptibility of GIN to organic compounds with AH-like activity could be directly related to the parasitic genera, life stage and even their site of establishment within the host [13][14][15][16][17]. Furthermore, authors suggest that the cuticles of parasitic stages are more susceptible to anthelmintic compounds and affect the motility of adult worms; opposite to free-living stages, to whom their specialized structures provide protection towards environmental distress [15,18].
The screening for anthelmintic-like activity of plant secondary metabolites against adult nematodes has been mainly performed via assessing tannin-rich plant extracts [11,19]. Authors have reported total paralysis of C. oncophora at ≥ 500 µg extract mL 1 [11], results which are very similar to the EC 50 obtained with both combinations used in this study, CuQ and CaR: 0.073 ± 0.071 mg mL −1 and 0.051 ± 0.164 mg mL −1 , respectively. However, unlike Peña-Espinoza et al. (2017) [11], the EC 50 s were obtained after 24 h when the motility of the control group was still at 74.54 ± 6.17%. Observations performed after 48 h revealed a total death of the worms from the control group, which could be associated with the non-use of RPMI media as suggested by other authors [11,20].

Structural and Ultrastructural Alterations Observed through SEM and TEM in Both L 3 and Adult Worms of C. punctata
In this study, the main alterations observed through SEM and TEM for both L 3 and adult worms were: (i) alterations of the sheath-cuticle complex of L 3 , with loss of epicuticle continuity, degradation of collagen bands of the basal zone and in the lateral specializations of the internal layer of the sheath; (ii) changes in hemidesmosomes; (iii) lateral hypodermic cord deirid-like neuron structure alterations; (iv) seam cell apoptosis; (v) mitochondrial swelling; (vi) electron densification, change of angle and length of the helicoidal fibers from the basal zone of adult worms; and (vii) mild and moderate degeneration of thin myofilaments in the sarcomeres of L 3 and adult worms, respectively.
The alterations observed in the sheath-cuticle complex of L 3 are suggestive of the protective effect of the sheath against chemical agents [21] and consistent with their reported capacity to inhibit larval exsheathment [7]. To our knowledge, this research paper is the first one to describe both the specializations present in the inner layer of the sheath and the deirid-like neuron structure observed in the cuticle of C. punctata L 3 . The deirid neuron has been widely studied in C. elegans larvae; and it has been categorized as a sensorial organ that allows the nematode to perceive environmental stimuli (oxygen levels, social feeding behavior, attraction/repulsion to chemicals, reproductive behavior, and temperature), allowing them to react toward those stimuli with actions such as molting and hypobiosis [22]. Thus, it could be suggested that the alterations observed in both the inner layer specializations and the deirid-like neuron of C. punctata L 3 could be responsible for the exsheathment blockage generated by these PCs-combinations and reported in previous research [7]. Further research is suggested to corroborate the nature and function of both structures observed in the sheath internal layer and in the lateral hypodermic cord of C. punctata L 3 .
Additionally, the seam cell has been described in C. elegans to have the ability to both self-renew and to give rise to differentiated cell types (hypodermal, glial and neuronal cells) [23]; thus, the alterations observed in both the seam cell and mitochondria of parasites exposed to the PC combinations could be considered to not be compatible with the development and survival of both infective larvae and adult worms.
Furthermore, alterations in cuticular collagen and structural collapse observed for both L 3 and adult worms incubated in both PC combinations, could be associated to the bioactive properties previously described for caffeic acid and coumarin, which include: (i) fibrinolytic activity and (ii) inhibition of the epidermic growth factor and metalloproteinases, which are expressed in the hypodermic tissue of nematodes and are necessary for normal collagen secretion [15,24].
Even though the anthelmintic-like mechanism of most phytochemicals is yet to be determined, it has been proposed that motility inhibition is a result of phytochemical blocking of sensory neurons, and such interference of nematodes' neurophysiology leads to their paralysis [25]. In that regard, recent studies have reported the acetylcholinesterase (AChE) inhibitory activity of polyphenolic compounds such as caffeic acid, coumarin and quercetin [2]. The enzyme that is essential for the regulation of cholinergic transmission in nematodes [26] and target for drug-engineering; Levamisole being one of the most known examples of cholinergic agonist drugs affecting the neuromuscular synapses of nematodes [27,28]. Caffeic acid, coumarin and its derivatives have been reported to be phytotoxins with a neurotoxic effect inhibiting the AChE activity in neuromuscular junctions [28,29]. Furthermore, coumarin has also been reported to block the octopamine receptor pathway [28]. Octopamine is a biogenic amine, which among others, is involved in the modulation of processes of pharyngeal pumping, muscle contractions and oviposition of female nematodes [30]. It is feasible that, one or the combination of both neurotoxic effects of coumarin and caffeic acid previously described are directly associated with the anthelmintic-like activity observed against adult worms of C. punctata; thus, further studies are required to corroborate this hypothesis.
However, mechanical alterations should also be addressed, as nematodes, as with most of the cylindrical-shaped animals, have a hydrostatic skeleton; in which movement depends on the internal fluids, the longitudinal muscle fibers and in the crossed-fiber helical array of connective tissue present in the basal zone of the cuticle [31,32]. It has been reported that the disposition of the different muscle fibers combined with the tri-layer connective tissue fibers (stiff and inextensible), and the pressurized internal fluid of nematodes allow cuticle deformation, therefore enabling locomotion. Thus, the coupling between body wall muscles and the cuticle implies that muscle contraction could dynamically affect the morphology of the nematode's cuticle [33]. Moreover, the cuticular ridge pattern (both transversal and longitudinal) confer the flexibility features of the cuticle, allowing nematodes to coil, assist locomotion and favor the attachment of nematodes to the host intestinal mucosa [34]; while lateral alae provide longitudinal stiffness and enable the diameter shift of nematodes' body shape [35]. Scanning and Transmission Electron Micrographs obtained through this investigation allowed the classification of specific treatment-induced lesions in both the cuticle and muscular tissue of L 3 and adult worms of C. punctata, involving the structures previously described, which could have led to the paralysis observed.
The structural alterations observed in the specimens are consistent with previous reports that correlate cuticular collagen alterations with the cylindric shape of nematodes. As collagen and other similar proteins constitute 80% of the structural components of parasites' cuticles [15]. Scanning Electron Micrographs of L 3 revealed loss of turgor and collapsed morphology; lesions that might be associated to the loss of cuticular ridges pattern and of the striated collagen layer observed in the cuticle basal zone; structures which according to Wharton (1986) confer nematodes with progressive resistance to deformation. Furthermore, hemidesmosomes alterations in both biological stages could also be involved in the structural changes observed in the nematode's morphology; as hemidesmosomes are trans-cellular protein complexes that promote the epidermal adherence, helping to maintain the tissue structure and integrity by providing resistance to mechanical stress [36]. Thus, changes in hemidesmosomes electron density could also be associated with the collagen degradation capacity of the PC combinations, and motif for the presence of electron-lucent zones suggesting hypodermic detachment with the cuticle.
Furthermore, and consistent with the lesions observed through SEM, Petzold (2011) [33] reported that treatments inducing rigid paralysis, such as ACHs inhibitors, provoke the head and tail region to collapse more frequently than the midbody of nematodes; while the changes regarding body length, diameter, and body stiffness are representative of alterations of the body wall muscle tone.
Moreover, TEM microphotographs of specimens treated with caffeic acid evidenced an apparent loss of thin myofilaments and the electro-densification of thick myofilaments in both L 3 and adult worms' muscular cells. Findings consistent with previous studies reporting that caffeic acid, rutin and quercetin affect myofibrillar proteins of muscular tissue [37]. Cheng et al. (2020) [37] reported caffeic acid as the polyphenolic compound that affects muscular tissue the most via inhibiting collagen production, decreasing the alphahelix structure of thin myofilaments and increasing beta-sheets and beta-turns of thick myofilaments, alterations that trigger coiling of muscular tissue. The changes observed through TEM in C. punctata myofilaments and in the crossed-fiber helical connective tissue of the basal zone are consistent with previous reports affirming that muscle contraction shortens the body shape and increases the body stiffness of nematodes [33], showing a consistency in the lesions observed through both SEM and TEM techniques.
Moreover, it has been reported that the shortening of the nematodes' body shape compels an increase in diameter, which is resisted by the crossed-fiber helical connective tissue array [31]. Such alterations could be directly associated with the turgor loss in the case of L 3 , and in the case of adult worms, it could be related to the apparent thin myofilament degeneration within the sarcomere and to the reduction in length and angle disposition of the tri-layered helicoidal fibers from the basal zone of the cuticle. Concluding that, the possible mechanism of action in adult worms is a rigid paralysis and muscular tone alterations.
Lastly, apoptotic cells observed through TEM micrographs with both treatments are consistent with previous reports describing caffeic acid as a time and concentration dependent proapoptotic molecule, which promotes chromatin condensation [38] and induces mitochondria-mediated apoptosis [39]. Similarly, coumarin has been reported with antileishmania activity causing both cytoplasm vacuolization and mitochondrial swelling [40]; lesions that were observed in this study after incubation of both L 3 and adult worms in both PC combinations.
Combination of in vitro bioassays and Electron Microscopy techniques allowed us to conclude that although some enzymatic inhibition could impair C. punctata viability, mechanical alterations are also involved in the PC combinations bioactivity and are conclusive for the effectiveness of both PC combinations to affect collagen and muscular tissue of C. punctata adult worms causing muscular contraction, leading to cuticular collapse, loss of turgor pressure and cellular damage. The motility of infective larvae was less affected, most likely because of the resistance conferred by the sheath; however, mild damage was observed in the muscle tissue. Nevertheless, the sheath, cuticle and cellular alterations are consistent with the exsheathment impairment of both PC combinations that was previously reported by Escareño-Díaz et al. (2019) [7]. Further studies assessing the effect of PC combinations on mortality rates and over the motility of exsheathed larvae are suggested.

Conclusions
The results of the present study led us to conclude that both PC combinations have anthelmintic-like activity against both biological stages of C. punctata; thus, motility inhibition could only be stated against adult worms. After addressing their potential toxicity and pharmacokinetics, both combinations could be considered for in vivo field trials.  Informed Consent Statement: Not applicable.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.