The structures and morphologies of polymers have been under investigation for more than 60 years. Scanning electron microscopy (SEM) was introduced in the 1960´s, and has been used to investigate fracture of surfaces, phase separation in polymer blends and crystallization of spherulites. Several improvements have been made in electron generation and electron optics that have enhanced the resolution power. Also, computerized SEM led to the introduction of the digital scanning generator for digital image recording and processing. Consequently, SEM is at present the most popular of the microscopic techniques. This is because of the user-friendliness of the apparatus, the ease specimen preparation, and the general simplicity of image interpretation. The limitation is that only surface features are easily accessible. Furthermore, chemical analysis of different elements is usually possible with SEM (energy dispersive or wavelength dispersive analysis of X-rays, EDXA, WDXA) (Michler, 2008). According to Sawyer (2008), SEM is used to evaluate features as the size, distribution, and adhesion of the fibers or particles, their adhesion to the matrix play a major role in revealing the strength and toughness enhancement. Regarding to the studied material, coarse structures such as larger particles in a matrix can easily be studied at low temperature (brittle) fracture surfaces in the SEM, since the fracture path follows the shape of the particle (Michler, 2008) considering that from a practical point of view, a good indicator of the degree of compatibility is the heterogeneity of the polymer system (e.g how the sizes of the dispersed domains depend on the processing conditions). The objective of this chapter is to present the effect of ionizing radiation on the morphology of PLLA:PCL blend and composites containing coconut fiber. SEM and field emission (FE) SEM micrographs were taken of non irradiated and irradiated samples with gamma rays

Coconut fibers were from three different origins: Embrapa- Empresa Brasileira de Pesquisa Agropecuária, Paraipaba region, Ceará; Projeto Coco Verde – Quissamã, Rio de Janeiro;

Size reduction of the coconut fibers was carried out using helix mill Marconi – model MA 680, from Laboratório de Matéria-prima Particulados e Sólidos Não Metálicos – LMPSol,

The fiber size distribution was measured using sieves of the Tyler series 16, 20, 35 and 48, fiber sizes of 1.0mm, 0.84mm, 0.417mm, and 0.297mm, respectively. The 0.417mm fiber size was used for the assays. The triturated material was separated using a sieve shaker

Departamento de Engenharia de Materiais of Escola Politécnica/USP.

without loss of the original characteristics of polymeric components.

2005).

and electron beam.

**2. Experimental**

Produtest, for 1 min.

Poematec – Ananindeua, Pará.

*Coconut fiber*

PLLA pellets were dried in a vacuum oven at 90°C and PCL pellets were dried at 40°C overnight to avoid hydrolysis of polymers during the melt-processing. Sheets of PCL and PLLA homo-polymers and blends with PCL:PLLA weight ratio of 25:75, 50:50 and 75:25 were prepared using a twin screw extruder (Labo Plastomill Model 150C, Toyoseki, Japan) equipped with a T-die (60mm width and 1.05mm thickness). T-die temperature was set at 205°C for PLLA homo-polymer and its blends, and at 90°C for PCL. Extruded sheets were quenched using a water bath set at room temperature. The take up speed was selected at 0.35 m min-1. As the take up speed was set at slightly higher than the extrusion out-put speed, finally obtained thickness of films was around 1 mm.

#### *Preparation of composite pellets and sheets*

PCL (pellets, ܯഥௐ=2.14 105 g mol-1; ܯഥௐ/ܯഥ= 1.423), PLLA (pellets, ܯഥௐ=2.64 105 g mol-1 ܯഥௐ/ܯഥ= 1.518 – Gel Permeation Chromatographic values) and dry coconut fiber (from Embrapa, Ceará, Brazil) were used to prepare blends and composites. A Labo Plastomil model 50C 150 of Toyoseiki twin screw extruder was used for pellets preparation. Pellets of PLLA:PCL 80:20 (w:w) blend and composites containing 5 and 10% of untreated and chemically treated coconut fiber were prepared at AIST.

Sheets (150mm x 150mm x 0.5mm) of PCL, PLLA, PLLA:PCL 80:20 (w:w) blend and composites containing 5 and 10% untreated and chemically treated coconut fiber were prepared using Ikeda hot press equipment of JAEA– Japan Atomic Energy Agency. Mixed pellets of samples were preheated at 195°C for 3 min and then pressed by under heating at the same temperature for another 3 min under pressure of 150 kgf cm-2. The sample was then cooled in the cold press using water as a coolant for 3 min.

#### *Gamma irradiation*

Samples were irradiated at IPEN–CNEN/SP (Brazil) using a Co-60 irradiator Gammacell model 220, series 142 from Atomic Energy of Canada Limited. Doses of 25, 50, 75, 100 and

Ionizing Radiation Effect on Morphology of PLLA:

and chemical properties (Tomczak et al., 2007).

treated with Na2SO3.

chemically treated with Na2SO3 are shown in Figure 2.

PCL Blends and on Their Composite with Coconut Fiber 249

There are some studies involving chemical treatment of vegetal fibers to improve its compatibility to polymers (Kapulskis et al, 2006; Abdul-Khalil & Rozman, 2000; Lee et al., 2004). Cell wall of a plant in the dry state consist mainly of carbohydrates combined to lignin, and few quantities of protein, starch and inorganic compounds, chemical composition varies from plant to plant and through different parts of the same plant (Rowell et al., 2000). The lignocellulosic fibers characteristics vary considerably with the place where they are produced, and possess different chemical compositions that affect their physical

According to Calado et al. (2000), coconut coir fiber treated with Na2SO3 suffers lignin removal from surface and its roughness increases. This increase of surface roughness improves their adhesion due to promotion of mechanical interaction between fibers and polymeric matrix. In this sense, scanning electron micrographs of coconut fibers surface,

Acceleration voltage: 20.0 kV Magnification 600× Acceleration voltage: 20.0 kV Magnification: 350×

Fig. 2. Scanning electron micrographs of coconut fibers surface from Embrapa , chemically

Chemical treatment with anhydride acetic reduces the number of hydroxyl radicals, as shown in Figure 3. This treatment would reduce cellulose molecules polarity and then

would improve the compatibility with thermo rigid matrix used in composites.

Fig. 3. Cellulose acetylation reaction (Calado et al., 2000).

500 kGy were applied at a dose rate of 4.3 kGy h-1. Samples were cut 10 × 100 cm2, and irradiated at room temperature in air.

#### *Electron beam irradiation*

Hot pressed sheets were irradiated using a electron beam accelerator (E=2 MeV, 2 mA) applying radiation doses from 10 kGy to 500 kGy with a dose rate of 0.6 kGy s-1, at JAEA, Takasaki, Japan.

#### *Scanning Electron Microscopy (SEM)*

Morphology of the fractured surfaces of the non-irradiated homopolymers and blends was examined by a scanning electron microscope (SEM) DS-720 TOPCON Co. The photomicrographs of the cryogenic fractured surface of the blends sheet were taken after 4- 5 min gold coating.

SEM micrographs of the irradiated homopolymers and blends sheets; and coconut fibers surfaces from cryogenic fractured samples were obtained using a scanning electron microscope model JXA-6400 (JEOL) at Centro Tecnológico da Marinha em São Paulo.

#### *Field Emission Scanning Electron Microscopy (FE-SEM)*

Photomicrographs of the, cryogenic fractured, non-irradiated and irradiated samples were taken using a field emission scanning electron microscope, JEOL, JSM – 7401F, acceleration voltage 1.0 kV at Central Analítica IQ-USP.

## **3. Results and discussion**

Figure 1 shows scanning electron micrographs of coconut fiber surface, as received samples from Embrapa.

Acceleration voltage: 20.0 kV Magnification: 700× Acceleration voltage: 20.0 kV Magnification: 350×

Fig. 1. Scanning electron micrographs of coconut fiber surface, as received from Embrapa.

500 kGy were applied at a dose rate of 4.3 kGy h-1. Samples were cut 10 × 100 cm2, and

Hot pressed sheets were irradiated using a electron beam accelerator (E=2 MeV, 2 mA) applying radiation doses from 10 kGy to 500 kGy with a dose rate of 0.6 kGy s-1, at JAEA,

Morphology of the fractured surfaces of the non-irradiated homopolymers and blends was examined by a scanning electron microscope (SEM) DS-720 TOPCON Co. The photomicrographs of the cryogenic fractured surface of the blends sheet were taken after 4-

SEM micrographs of the irradiated homopolymers and blends sheets; and coconut fibers surfaces from cryogenic fractured samples were obtained using a scanning electron

Photomicrographs of the, cryogenic fractured, non-irradiated and irradiated samples were taken using a field emission scanning electron microscope, JEOL, JSM – 7401F, acceleration

Figure 1 shows scanning electron micrographs of coconut fiber surface, as received samples

Acceleration voltage: 20.0 kV Magnification: 700× Acceleration voltage: 20.0 kV Magnification: 350×

Fig. 1. Scanning electron micrographs of coconut fiber surface, as received from Embrapa.

microscope model JXA-6400 (JEOL) at Centro Tecnológico da Marinha em São Paulo.

irradiated at room temperature in air.

*Scanning Electron Microscopy (SEM)*

*Field Emission Scanning Electron Microscopy (FE-SEM)*

voltage 1.0 kV at Central Analítica IQ-USP.

**3. Results and discussion**

from Embrapa.

*Electron beam irradiation*

Takasaki, Japan.

5 min gold coating.

There are some studies involving chemical treatment of vegetal fibers to improve its compatibility to polymers (Kapulskis et al, 2006; Abdul-Khalil & Rozman, 2000; Lee et al., 2004). Cell wall of a plant in the dry state consist mainly of carbohydrates combined to lignin, and few quantities of protein, starch and inorganic compounds, chemical composition varies from plant to plant and through different parts of the same plant (Rowell et al., 2000). The lignocellulosic fibers characteristics vary considerably with the place where they are produced, and possess different chemical compositions that affect their physical and chemical properties (Tomczak et al., 2007).

According to Calado et al. (2000), coconut coir fiber treated with Na2SO3 suffers lignin removal from surface and its roughness increases. This increase of surface roughness improves their adhesion due to promotion of mechanical interaction between fibers and polymeric matrix. In this sense, scanning electron micrographs of coconut fibers surface, chemically treated with Na2SO3 are shown in Figure 2.

Acceleration voltage: 20.0 kV Magnification 600× Acceleration voltage: 20.0 kV Magnification: 350×

Fig. 2. Scanning electron micrographs of coconut fibers surface from Embrapa , chemically treated with Na2SO3.

Chemical treatment with anhydride acetic reduces the number of hydroxyl radicals, as shown in Figure 3. This treatment would reduce cellulose molecules polarity and then would improve the compatibility with thermo rigid matrix used in composites.

Fig. 3. Cellulose acetylation reaction (Calado et al., 2000).

Ionizing Radiation Effect on Morphology of PLLA:

PCL Blends and on Their Composite with Coconut Fiber 251

(spherulites and sheaf-like boundless of lamellae) come in a variety of forms depending on the polymer and its crystalline structure. These superstructures generally form a texture consisting of one or more spherulite types with a characteristic spherulite size distribution.

Energy (keV)

Counts Counts

Energy (keV)

Calado et al. (2000) observed clear difference on the morphologies of non treated and chemical treated surfaces of coir fibers. Same behavior was observed on the fibers studied in this chapter.

It was possible to observe roughness increase on the surface treated with Na2SO3 and anhydride acetic, as shown in Figure 4.

Acceleration voltage: 20.0 kV Magnification: 600× Acceleration voltage: 20.0 kV Magnification: 350×

Fig. 4. Scanning electron micrographs of coconut fibers surface from Embrapa, chemically treated with Na2SO3 and anhydride acetic, respectively.

X rays spectra by EDS of Embrapa coconut fiber, non irradiated with 0.297 mm up to 0.417 mm size, are shown in Figure 5. It was possible to identify that chemical composition of the residue varied for the same fiber. It can be explained by the fact that chemical composition varies from plant to plant and also through different parts of the same plant (Rowell et al., 2000; Severiano et al., 2010).

In the residue of thermal decomposition in air of fibers from three different origins, most common elements found were K, Na, Si, Ca and, in some cases, Fe. In the literature, they found P, Mg and , in small concentrations, Cu, Zn, Mn (Rosa et al., 2001), in addition to Cl, S, Br e Rb (Mothé & Miranda, 2009) that were not observed in samples in this study. This variation can be attributed to soil where coconut tree was grown. Peak that had attributed to Zr, in fact is due to P, as they appear at the same channel of energy and, it is more probable to find P in higher quantity in soil than Zr. Peak that was attributed to As observed in some spectra was due to coating process used to allow image formation.

Scanning electron micrographs of surfaces of cryogenic fractured non irradiated as extruded blend samples were taken. As extruded PCL micrograph shows a homogeneous morphology, as shown in Figure 6. The as extruded PCL:PLLA 50:50 micrograph shows spheres with different sizes and shapes, as shown in Figure 7. According to Michler (2008), when a polymer is cooled down from the melt, the (primary) crystallization starts from initial points that are randomly distributed in the volume. Such starting points are either homogeneous or heterogeneous nuclei (i.e. nucleating agents, impurities, or filler particles). This radial growth results in a characteristic arrangement of lamellae. The superstructures

Calado et al. (2000) observed clear difference on the morphologies of non treated and chemical treated surfaces of coir fibers. Same behavior was observed on the fibers studied in

It was possible to observe roughness increase on the surface treated with Na2SO3 and

Acceleration voltage: 20.0 kV Magnification: 600× Acceleration voltage: 20.0 kV Magnification: 350×

Fig. 4. Scanning electron micrographs of coconut fibers surface from Embrapa, chemically

X rays spectra by EDS of Embrapa coconut fiber, non irradiated with 0.297 mm up to 0.417 mm size, are shown in Figure 5. It was possible to identify that chemical composition of the residue varied for the same fiber. It can be explained by the fact that chemical composition varies from plant to plant and also through different parts of the same plant

In the residue of thermal decomposition in air of fibers from three different origins, most common elements found were K, Na, Si, Ca and, in some cases, Fe. In the literature, they found P, Mg and , in small concentrations, Cu, Zn, Mn (Rosa et al., 2001), in addition to Cl, S, Br e Rb (Mothé & Miranda, 2009) that were not observed in samples in this study. This variation can be attributed to soil where coconut tree was grown. Peak that had attributed to Zr, in fact is due to P, as they appear at the same channel of energy and, it is more probable to find P in higher quantity in soil than Zr. Peak that was attributed to As observed in some

Scanning electron micrographs of surfaces of cryogenic fractured non irradiated as extruded blend samples were taken. As extruded PCL micrograph shows a homogeneous morphology, as shown in Figure 6. The as extruded PCL:PLLA 50:50 micrograph shows spheres with different sizes and shapes, as shown in Figure 7. According to Michler (2008), when a polymer is cooled down from the melt, the (primary) crystallization starts from initial points that are randomly distributed in the volume. Such starting points are either homogeneous or heterogeneous nuclei (i.e. nucleating agents, impurities, or filler particles). This radial growth results in a characteristic arrangement of lamellae. The superstructures

treated with Na2SO3 and anhydride acetic, respectively.

spectra was due to coating process used to allow image formation.

(Rowell et al., 2000; Severiano et al., 2010).

this chapter.

anhydride acetic, as shown in Figure 4.

(spherulites and sheaf-like boundless of lamellae) come in a variety of forms depending on the polymer and its crystalline structure. These superstructures generally form a texture consisting of one or more spherulite types with a characteristic spherulite size distribution.

Ionizing Radiation Effect on Morphology of PLLA:

fractured sample.

PCL Blends and on Their Composite with Coconut Fiber 253

Fig. 7. Scanning electron micrograph of as extruded PCL:PLLA 50:50 (w:w), cryogenic

15 kV 1000× 10,00 μm

Fig. 8. Scanning electron micrograph of as extruded PLLA, cryogenic fractured sample.

15 kV 2500× 4,00 μm

which proceeds through melt, nucleation, lamellar growth and, spherulitic growth.

Micrographs of annealed PLLA (Figure 9) and PCL:PLLA 50:50 (w:w) (Figure 10) shows changes in the morphology due to the crystallization of PLLA. Utracki (1989) explained that depending on the crystallization conditions various types of morphology can be obtained,

Energy (keV)

Fig. 5. Scanning electron micrograph of non irradiated Embrapa coconut fiber (0.297 and 0.417 mm) residue and X ray spectra by EDS (points 1, 2 and 3).

Fig. 6. Scanning electron micrograph of as extruded PCL, cryogenic fractured sample.

It is possible to observe the continuous PLLA-rich phase and the PCL-rich dispersed phase with a maximum domain size of 1.5 μm, as visualized before by Tsuji et al. (2001) in PCL:PLLA solution-cast blends. The PCL-rich phase is homogeneously dispersed in the PLLA matrix. One can observe some cracks or voids in the PLLA film probably caused by the temperature of processing, as shown in Figure 8. It has mentioned before that the degradation of aliphatic polyesters can occur because of melting at high temperatures (Yoshii et al., 2000; Kodama et al., 1997).

Fig. 5. Scanning electron micrograph of non irradiated Embrapa coconut fiber (0.297 and

Energy (keV)

Fig. 6. Scanning electron micrograph of as extruded PCL, cryogenic fractured sample.

15kV 2500X 4,00 μm

(Yoshii et al., 2000; Kodama et al., 1997).

It is possible to observe the continuous PLLA-rich phase and the PCL-rich dispersed phase with a maximum domain size of 1.5 μm, as visualized before by Tsuji et al. (2001) in PCL:PLLA solution-cast blends. The PCL-rich phase is homogeneously dispersed in the PLLA matrix. One can observe some cracks or voids in the PLLA film probably caused by the temperature of processing, as shown in Figure 8. It has mentioned before that the degradation of aliphatic polyesters can occur because of melting at high temperatures

0.417 mm) residue and X ray spectra by EDS (points 1, 2 and 3).

Counts

Fig. 7. Scanning electron micrograph of as extruded PCL:PLLA 50:50 (w:w), cryogenic fractured sample.

Fig. 8. Scanning electron micrograph of as extruded PLLA, cryogenic fractured sample.

Micrographs of annealed PLLA (Figure 9) and PCL:PLLA 50:50 (w:w) (Figure 10) shows changes in the morphology due to the crystallization of PLLA. Utracki (1989) explained that depending on the crystallization conditions various types of morphology can be obtained, which proceeds through melt, nucleation, lamellar growth and, spherulitic growth.

Ionizing Radiation Effect on Morphology of PLLA:

by a thin layer of the polymeric matrix of the blends.

in the half time of crystallization (Dell'Erba et al., 2001).

transition temperature (Tg) (IAEA-TECDOC-1420, 2004).

morphology.

PCL Blends and on Their Composite with Coconut Fiber 255

morphological changes. The spheres are apart from the matrix, and in addition the matrix was changed due to the crystallization of PLLA. The blends are not miscible and the after extrusion cooling from the melt to room temperature causes the phase separation due to the difference between the melting temperatures of both blend components, PCL and PLLA.

In preliminary studies by differential scanning calorimetry (DSC) no change in the PLLA melting temperature was observed by increasing the PCL content in the blends. It was observed that, as PCL amount increased, PCL Tm peak increased in the region of the glass transition temperature of PLLA (Kodama et al., 1997). Although the immiscibility occurs, it is possible to observe by SEM some interfacial interaction, as the spheres seem to be covered

It should be noted that the blends were well mixed during extrusion as shown by the distribution and the size of the spherulites in the matrix. Dell'Erba et al. (2001) have found that it was reasonable to assume that low interfacial tensions were obtained in PLLA:PCL blends because of their similar chemical nature of the blends components, which allows interpolymer polar interactions across phase boundaries, thus favoring a well-dispersed

Preliminary studies have shown that although both are semi-crystalline polymers, only PCL crystallizes during extrusion. PLLA is amorphous and crystallizes after annealing, which was observed by x-ray diffraction of the non-irradiated samples (Broz et al., 2003). Also the orientation of crystallites in the blends was observed by x-ray diffraction, PLLA crystallizes in the α form with 103 helical conformation (Zhang et al., 2005). The thermal treatment increases the quantity of spheres, it was possible to notice some ellipsoids. This suggests that the new spherulites were formed due to the crystallization of amorphous PLLA, as observed previously (Broz et al., 2003). In this case, the segregation is also clear. It is possible to observe the separated spheres from the matrix and the cavities. It was discussed in literature (Dell'Erba et al., 2001) that the PLLA spherulites growth mechanism does not change when different amounts of PCL are present in the blend. Additionally, the presence of PCL enhances the PLLA crystallization rate, suggested to likely occur through the increase in the nucleation rate, it was observed that the presence of PCL domains in the PLLA matrix causes a small lowering

Furthermore, the results indicated that even though PLLA and PCL are immiscible, revealed by the presence of two glass transition temperatures for the blends very close to those found for pure PLLA and PCL, they are not highly incompatible (Dell'Erba et al., 2001). The binary mixture of (two) polymers is considered a compatible blend, when a homogeneous solid system is formed, without phase separations. It means a complete mutual solubility of the two polymers in molten state as well. This compatibility is reflected in, among other physical and mechanical properties, the fact that the system will have one single glass

According to Michler (2008), γ or electron irradiation initiates pronounced crosslink in the amorphous parts of semi-crystalline polymers, whereas the structure inside the lamellae (the crystallinity) is not destroyed, so long as critical doses are not used. In this study, micrographs of PCL and PLLA homopolymers and PCL:PLLA 50:50 (w:w) blend irradiated

with 100 kGy and 500 kGy, respectively, are shown in Figure 11 up to Figure 16.

Fig. 9. Scanning electron micrograph of *annealed* PLLA, cryogenic fractured sample.

Fig. 10. Scanning electron micrograph of *annealed* PCL:PLLA 50:50 (w:w), cryogenic fractured sample.

Although the temperature of annealing was lower than the PLLA melting temperature (Tm), the thermal treatment allowed the crystallization of PLLA. Moreover, even though as extruded and annealed samples temperatures of processing were the same, one can observe the reduction of the cracks on the PLLA annealed sample. It is also possible to verify some

Fig. 9. Scanning electron micrograph of *annealed* PLLA, cryogenic fractured sample.

15 kV 2500× 4,00 μm

Fig. 10. Scanning electron micrograph of *annealed* PCL:PLLA 50:50 (w:w), cryogenic

15 kV 2500× 4,00 μm

Although the temperature of annealing was lower than the PLLA melting temperature (Tm), the thermal treatment allowed the crystallization of PLLA. Moreover, even though as extruded and annealed samples temperatures of processing were the same, one can observe the reduction of the cracks on the PLLA annealed sample. It is also possible to verify some

fractured sample.

morphological changes. The spheres are apart from the matrix, and in addition the matrix was changed due to the crystallization of PLLA. The blends are not miscible and the after extrusion cooling from the melt to room temperature causes the phase separation due to the difference between the melting temperatures of both blend components, PCL and PLLA.

In preliminary studies by differential scanning calorimetry (DSC) no change in the PLLA melting temperature was observed by increasing the PCL content in the blends. It was observed that, as PCL amount increased, PCL Tm peak increased in the region of the glass transition temperature of PLLA (Kodama et al., 1997). Although the immiscibility occurs, it is possible to observe by SEM some interfacial interaction, as the spheres seem to be covered by a thin layer of the polymeric matrix of the blends.

It should be noted that the blends were well mixed during extrusion as shown by the distribution and the size of the spherulites in the matrix. Dell'Erba et al. (2001) have found that it was reasonable to assume that low interfacial tensions were obtained in PLLA:PCL blends because of their similar chemical nature of the blends components, which allows interpolymer polar interactions across phase boundaries, thus favoring a well-dispersed morphology.

Preliminary studies have shown that although both are semi-crystalline polymers, only PCL crystallizes during extrusion. PLLA is amorphous and crystallizes after annealing, which was observed by x-ray diffraction of the non-irradiated samples (Broz et al., 2003). Also the orientation of crystallites in the blends was observed by x-ray diffraction, PLLA crystallizes in the α form with 103 helical conformation (Zhang et al., 2005). The thermal treatment increases the quantity of spheres, it was possible to notice some ellipsoids. This suggests that the new spherulites were formed due to the crystallization of amorphous PLLA, as observed previously (Broz et al., 2003). In this case, the segregation is also clear. It is possible to observe the separated spheres from the matrix and the cavities. It was discussed in literature (Dell'Erba et al., 2001) that the PLLA spherulites growth mechanism does not change when different amounts of PCL are present in the blend. Additionally, the presence of PCL enhances the PLLA crystallization rate, suggested to likely occur through the increase in the nucleation rate, it was observed that the presence of PCL domains in the PLLA matrix causes a small lowering in the half time of crystallization (Dell'Erba et al., 2001).

Furthermore, the results indicated that even though PLLA and PCL are immiscible, revealed by the presence of two glass transition temperatures for the blends very close to those found for pure PLLA and PCL, they are not highly incompatible (Dell'Erba et al., 2001). The binary mixture of (two) polymers is considered a compatible blend, when a homogeneous solid system is formed, without phase separations. It means a complete mutual solubility of the two polymers in molten state as well. This compatibility is reflected in, among other physical and mechanical properties, the fact that the system will have one single glass transition temperature (Tg) (IAEA-TECDOC-1420, 2004).

According to Michler (2008), γ or electron irradiation initiates pronounced crosslink in the amorphous parts of semi-crystalline polymers, whereas the structure inside the lamellae (the crystallinity) is not destroyed, so long as critical doses are not used. In this study, micrographs of PCL and PLLA homopolymers and PCL:PLLA 50:50 (w:w) blend irradiated with 100 kGy and 500 kGy, respectively, are shown in Figure 11 up to Figure 16.

Ionizing Radiation Effect on Morphology of PLLA:

Acceleration voltage: 10.0 kV Magnification: 2000×

Acceleration voltage: 20.0 kV Magnification: 2500×

A B

non irradiated and B) irradiated with 100 kGy.

irradiated with 500 kGy.

al., 2000; Nugroho et al., 2001).

PCL Blends and on Their Composite with Coconut Fiber 257

changes are visible by SEM on the irradiated PLLA with 500 kGy radiation dose, in contrast to other properties observed previously in the literature (Södergard & Stolt, 2002; Ohrlander et

Fig. 12. Scanning electron micrograph of as extruded PCL, cryogenic fractured sample

Fig. 13. Scanning electron micrographs of as extruded PLLA, cryogenic fractured sample: A)

Acceleration voltage: 20.0 kV Magnification: 2500

Fig. 11. Scanning electron micrographs of as extruded PCL, cryogenic fractured sample: A) non irradiated and B) irradiated with 100 kGy.

In Figure 12 it is possible to observe that lamellar structures increase on the fractured surface of PCL irradiated with 500 kGy, indicating that possibly significant crosslinking occurred.

Comparing the scanning electron micrographs, it is observed very few changes on the surface of ruptured samples. In earlier studies by size exclusion chromatography of electron beam irradiated PCL in air, it was observed a small increase followed by a decrease of crosslinking degree up to 5 kGy and after that an increase of gel-content of 15 wt %, indicating the enhance of crosslinking degree up to 200 kGy radiation dose (Södergard & Stolt, 2002). Even though PCL crosslinking predominates at radiation doses higher than 5 kGy and random chainscission at lower doses (Södergard & Stolt, 2002; Ohrlander et al.,2000). In this chapter, only few changes could be seen by SEM for the irradiated PCL up to 100 kGy. However, the ruptured sample surface of irradiated PCL with 500 kGy became full of scales suggesting that the increase of crosslinking density induced by the ionizing radiation caused this alteration.

Some differences observed on micrographs A and B on Figure 13 probably are related to different regions analyzed. Apparently, an increase of granulation occurred on the polymeric matrix for 100 kGy irradiated sample.

Comparing Figures 13 and 14, some cracks appear, and polymeric surface seems to become smoother, probably due to significant scission of polymeric chains.

The surface of PLLA sample became rough. This fact is correlated to chain scissions promoted by gamma radiation. In the literature it was observed that PLA mainly undergoes chain-scissions at doses below 250 kGy. At higher doses of radiation, crosslinking reactions increase as a function of the increasing radiation dose. The reactions occur in the amorphous phase of the polymer (Södergard & Stolt, 2002). Samples submitted to doses in the range of 30 up to 100 kGy showed a marked depression in mechanical properties attributed to oxidative chain-scissions in amorphous region (Nugroho et al., 2001). Apparently no

Fig. 11. Scanning electron micrographs of as extruded PCL, cryogenic fractured sample: A)

In Figure 12 it is possible to observe that lamellar structures increase on the fractured surface of PCL irradiated with 500 kGy, indicating that possibly significant crosslinking

Comparing the scanning electron micrographs, it is observed very few changes on the surface of ruptured samples. In earlier studies by size exclusion chromatography of electron beam irradiated PCL in air, it was observed a small increase followed by a decrease of crosslinking degree up to 5 kGy and after that an increase of gel-content of 15 wt %, indicating the enhance of crosslinking degree up to 200 kGy radiation dose (Södergard & Stolt, 2002). Even though PCL crosslinking predominates at radiation doses higher than 5 kGy and random chainscission at lower doses (Södergard & Stolt, 2002; Ohrlander et al.,2000). In this chapter, only few changes could be seen by SEM for the irradiated PCL up to 100 kGy. However, the ruptured sample surface of irradiated PCL with 500 kGy became full of scales suggesting that the increase of crosslinking density induced by the ionizing radiation caused this alteration.

Some differences observed on micrographs A and B on Figure 13 probably are related to different regions analyzed. Apparently, an increase of granulation occurred on the

Comparing Figures 13 and 14, some cracks appear, and polymeric surface seems to become

The surface of PLLA sample became rough. This fact is correlated to chain scissions promoted by gamma radiation. In the literature it was observed that PLA mainly undergoes chain-scissions at doses below 250 kGy. At higher doses of radiation, crosslinking reactions increase as a function of the increasing radiation dose. The reactions occur in the amorphous phase of the polymer (Södergard & Stolt, 2002). Samples submitted to doses in the range of 30 up to 100 kGy showed a marked depression in mechanical properties attributed to oxidative chain-scissions in amorphous region (Nugroho et al., 2001). Apparently no

Acceleration voltage: 20.0 kV Magnification: 2500

A B

non irradiated and B) irradiated with 100 kGy.

polymeric matrix for 100 kGy irradiated sample.

smoother, probably due to significant scission of polymeric chains.

occurred.

changes are visible by SEM on the irradiated PLLA with 500 kGy radiation dose, in contrast to other properties observed previously in the literature (Södergard & Stolt, 2002; Ohrlander et al., 2000; Nugroho et al., 2001).

Acceleration voltage: 10.0 kV Magnification: 2000×

Fig. 12. Scanning electron micrograph of as extruded PCL, cryogenic fractured sample irradiated with 500 kGy.

Acceleration voltage: 20.0 kV Magnification: 2500×

Fig. 13. Scanning electron micrographs of as extruded PLLA, cryogenic fractured sample: A) non irradiated and B) irradiated with 100 kGy.

Ionizing Radiation Effect on Morphology of PLLA:

had been stated as miscible in the molten state.

Acceleration voltage: 10.0 kV Magnification: 2000××

cryogenic fractured sample irradiated with 500 kGy.

Fig. 16. Scanning electron micrograph of as extruded PCL:PLLA 50:50 (w:w),

It seems that the ionizing radiation induced some shape alteration in the PCL dispersed phase in blends that were irradiated with 100 kGy. Likewise, samples submitted to irradiation processing up to dose of 500 kGy present the matrix with decreased PCL spherulites disperse phase, suggesting that some interaction between both polymeric phases had been promoted by the ionizing radiation. Previous studies demonstrated that gamma radiation does not affect significantly thermal properties of the blends when doses were kept bellow 75 kGy. A small decrease of PLLA Tm occurred probably due to PLLA main chain-scission. Thermal treatment induces PLLA Tm variation on irradiated blends with high concentration of PLLA. The crystallinity of PCL homopolymer and PLLA homopolymer as well as in the studied blends was not significantly affected by irradiation up to 100 kGy (Kodama et al., 2005). On the other hand, after irradiation with higher doses, PCL samples were more thermally stable than PLLA and blend (Nugroho et al., 2001). Thermal properties of PLLA were not affected by gamma radiation up to 100 kGy (Kodama et al., 2006a). Other results obtained previously by Kodama et al.(2006b) have shown that both, gamma and EB radiation, at doses up to 500 kGy, do not cause sample degradation to any significant extent to be detectable by FTIR (Fourier Transform Infrared Spectroscopy). As well, the miscibility of the polymeric blends was not affected by the irradiation process (Kodama et al., 2006b).

It can be observed in Figure 17A and B that fractured surface of non irradiated blend presented several needles like structures orthogonal to the surface, apparently related to PCL, due to the

PCL Blends and on Their Composite with Coconut Fiber 259

spherical inclusions and polymeric matrix seemed to be clean, suggesting that exists a weak adhesion between two phases, in consonance with absence of thermal transitions dislodgement observed by DSC. This lack of adhesion is unexpected since both polymers

Acceleration voltage: 10.0 kV Magnification: 2000×

Fig. 14. Scanning electron micrograph of as extruded PLLA, cryogenic fractured sample irradiated with 500 kGy.

Acceleration voltage: 20.0 kV Magnification: 2500

Fig. 15. Scanning electron micrographs of as extruded PCL:PLLA 50:50 (w:w), cryogenic fractured sample: A) non irradiated and B) irradiated with 100 kGy.

Broz et al. (2003) observed that microstructure of PLLA 0.4 in PCL was characterized by relatively wide quantity of spherical inclusions of PLLA on PCL matrix. Particles had sizes varying from 5 μm to 100 μm, apparently isolated on the matrix. Similar micrograph was observed in Figure 15A. Irradiated blends micrographs, shown in Figures 15B and 16, suffered scales formation similar to that observed for irradiated PCL. The interface between

Fig. 14. Scanning electron micrograph of as extruded PLLA, cryogenic fractured sample

Fig. 15. Scanning electron micrographs of as extruded PCL:PLLA 50:50 (w:w), cryogenic

Broz et al. (2003) observed that microstructure of PLLA 0.4 in PCL was characterized by relatively wide quantity of spherical inclusions of PLLA on PCL matrix. Particles had sizes varying from 5 μm to 100 μm, apparently isolated on the matrix. Similar micrograph was observed in Figure 15A. Irradiated blends micrographs, shown in Figures 15B and 16, suffered scales formation similar to that observed for irradiated PCL. The interface between

fractured sample: A) non irradiated and B) irradiated with 100 kGy.

Acceleration voltage: 10.0 kV Magnification: 2000×

Acceleration voltage: 20.0 kV Magnification: 2500

A B

irradiated with 500 kGy.

spherical inclusions and polymeric matrix seemed to be clean, suggesting that exists a weak adhesion between two phases, in consonance with absence of thermal transitions dislodgement observed by DSC. This lack of adhesion is unexpected since both polymers had been stated as miscible in the molten state.

Acceleration voltage: 10.0 kV Magnification: 2000××

Fig. 16. Scanning electron micrograph of as extruded PCL:PLLA 50:50 (w:w), cryogenic fractured sample irradiated with 500 kGy.

It seems that the ionizing radiation induced some shape alteration in the PCL dispersed phase in blends that were irradiated with 100 kGy. Likewise, samples submitted to irradiation processing up to dose of 500 kGy present the matrix with decreased PCL spherulites disperse phase, suggesting that some interaction between both polymeric phases had been promoted by the ionizing radiation. Previous studies demonstrated that gamma radiation does not affect significantly thermal properties of the blends when doses were kept bellow 75 kGy. A small decrease of PLLA Tm occurred probably due to PLLA main chain-scission. Thermal treatment induces PLLA Tm variation on irradiated blends with high concentration of PLLA. The crystallinity of PCL homopolymer and PLLA homopolymer as well as in the studied blends was not significantly affected by irradiation up to 100 kGy (Kodama et al., 2005). On the other hand, after irradiation with higher doses, PCL samples were more thermally stable than PLLA and blend (Nugroho et al., 2001). Thermal properties of PLLA were not affected by gamma radiation up to 100 kGy (Kodama et al., 2006a). Other results obtained previously by Kodama et al.(2006b) have shown that both, gamma and EB radiation, at doses up to 500 kGy, do not cause sample degradation to any significant extent to be detectable by FTIR (Fourier Transform Infrared Spectroscopy). As well, the miscibility of the polymeric blends was not affected by the irradiation process (Kodama et al., 2006b).

It can be observed in Figure 17A and B that fractured surface of non irradiated blend presented several needles like structures orthogonal to the surface, apparently related to PCL, due to the

Ionizing Radiation Effect on Morphology of PLLA:

blend morphology sterilized by ionizing radiation.

blends: A) non irradiated and B) irradiated with 20 kGy.

A B

A B

PCL Blends and on Their Composite with Coconut Fiber 261

In Figure 19 is observed surface micrographs of samples of PCL:PLLA 20:80 (w:w) prepared by hot press process , non irradiated and irradiated with 20 kGy radiation dose. This dose look as if do not affect significantly blend surface. Considering that conventional radiation dose for sterilization is 25 kGy, it is not expected that significant alteration occurs on the

Fig. 19. Field emission scanning electron micrographs of hot pressed PCL:PLLA 20:80 (w:w)

Arbelaiz et al. (2006) studied composites of linen fiber and PCL, and observed by SEM that fibers were clean and almost without adhesion points with PCL polymeric matrix, that indicated low wettability of fibers and lack of adhesion between phases. In Figure 20 it was no possible to observe in this sample regions containing fibers as observed by the authors mentioned before, probably it is related to its low concentration on the composite (5 or 10%)

Fig. 20. Field emission scanning electron micrographs of composite with 10% non chemically treated coconut fiber: A) non irradiated; B) irradiated with 100 kGy, cryogenic fractured.

studied in this chapter. It was just observed what looks like a fragment of fiber.

proportion o this component in the blend. Radiation dose seemed to induce these structures to diminish, probably related to PCL crosslinking that occurred with 100 kGy radiation dose.

Acceleration voltage: 20.0 kV Magnification: 2500×

Fig. 17. Scanning electron micrographs of as extruded PCL:PLLA 75:25 (w:w), cryogenic fractured sample: A) non irradiated and B) irradiated with 100 kGy.

Following micrographs were obtained using field emission scanning electron microscopy (FE-SEM) that dispense the use of Au0 coating and energy for image obtaining is lower. Micrograph of PCL:PLLA 20:80 (w:w), non irradiated is shown in Figure 18. It was observed a rough surface.

Fig. 18. Field emission scanning electron micrograph of as extruded PCL:PLLA 20:80 (w:w), cryogenic fractured sample, non irradiated.

proportion o this component in the blend. Radiation dose seemed to induce these structures to diminish, probably related to PCL crosslinking that occurred with 100 kGy radiation dose.

Fig. 17. Scanning electron micrographs of as extruded PCL:PLLA 75:25 (w:w), cryogenic

Following micrographs were obtained using field emission scanning electron microscopy (FE-SEM) that dispense the use of Au0 coating and energy for image obtaining is lower. Micrograph of PCL:PLLA 20:80 (w:w), non irradiated is shown in Figure 18. It was observed

Fig. 18. Field emission scanning electron micrograph of as extruded PCL:PLLA 20:80 (w:w),

fractured sample: A) non irradiated and B) irradiated with 100 kGy.

Acceleration voltage: 20.0 kV Magnification: 2500×

A B

cryogenic fractured sample, non irradiated.

a rough surface.

In Figure 19 is observed surface micrographs of samples of PCL:PLLA 20:80 (w:w) prepared by hot press process , non irradiated and irradiated with 20 kGy radiation dose. This dose look as if do not affect significantly blend surface. Considering that conventional radiation dose for sterilization is 25 kGy, it is not expected that significant alteration occurs on the blend morphology sterilized by ionizing radiation.

Fig. 19. Field emission scanning electron micrographs of hot pressed PCL:PLLA 20:80 (w:w) blends: A) non irradiated and B) irradiated with 20 kGy.

Arbelaiz et al. (2006) studied composites of linen fiber and PCL, and observed by SEM that fibers were clean and almost without adhesion points with PCL polymeric matrix, that indicated low wettability of fibers and lack of adhesion between phases. In Figure 20 it was no possible to observe in this sample regions containing fibers as observed by the authors mentioned before, probably it is related to its low concentration on the composite (5 or 10%) studied in this chapter. It was just observed what looks like a fragment of fiber.

Fig. 20. Field emission scanning electron micrographs of composite with 10% non chemically treated coconut fiber: A) non irradiated; B) irradiated with 100 kGy, cryogenic fractured.

Ionizing Radiation Effect on Morphology of PLLA:

B

PCL Blends and on Their Composite with Coconut Fiber 263

Fig. 22. Field emission scanning electron micrograph surface of hot pressed sheet of composite containing 10% non chemically treated coconut fiber: A) non irradiated; B) EB

Due to coalescence effect it was possible to observe spherical inclusions of PLLA in PCL:PLLA blend. Increasing radiation dose induced elongation of inclusions, as well, lamellar structures increase in the PCL matrix. Radiation doses higher than 100 kGy altered morphologies of samples surfaces, that became smoother, attributed to the presence of smaller fractions of PLLA, as a result of long chain scission, and high crosslinking reaction in PCL phase. Radiation processing and chemical acetylating did not promote measurable

The SEM micrographs of the fractured homopolymers and blends have shown their immiscibility. The crystallization of PLLA could be observed on the annealed samples. Samples irradiated with 100 kGy presented little variation on the morphology, even

irradiated with 50 kGy, and C) EB irradiated with 100 kGy.

C

interaction between fibers with polymeric matrix.

**4. Conclusion**

It was no possible to observe in Figure 21 significant alteration between structure existent and blend matrix. Apparently, acetylating process was not effective referring to the adhesion. However, it seems that ellipsoidal structures apart from polymeric matrix increased. Visually, structures suffered elongation and size reduction. Irradiated sample have smoother surface, probably related to scission process prevail of PLLA with doses above 100 kGy.

Fig. 21. Field emission scanning electron micrographs of composite with 10% acetylated coconut fiber: A) non irradiated; B) irradiated with 100 kGy, cryogenic fractured.

Micrographs of hot pressed sheets surface of composites containing 10% non chemically treated coconut fiber, non irradiated, and EB irradiated with 50 kGy and 100 kGy radiation doses, respectively, are shown in Figure 21. It was not possible to observe fiber presence on the surface analyzed. Neither any significant alteration on the irradiated surface, in the dose range studied. It suggests that cryogenic fractured surfaces allows the observation of irradiation effect on the polymeric bulk that otherwise could not be observed on sample surface. Probably it occurs due to the fact that species formed by energy deposition of radiation through polymeric matrix reacts mainly on the bulk than on the surface.

It was no possible to observe in Figure 21 significant alteration between structure existent and blend matrix. Apparently, acetylating process was not effective referring to the adhesion. However, it seems that ellipsoidal structures apart from polymeric matrix increased. Visually, structures suffered elongation and size reduction. Irradiated sample have smoother surface,

Fig. 21. Field emission scanning electron micrographs of composite with 10% acetylated coconut fiber: A) non irradiated; B) irradiated with 100 kGy, cryogenic fractured.

radiation through polymeric matrix reacts mainly on the bulk than on the surface.

A

A B

Micrographs of hot pressed sheets surface of composites containing 10% non chemically treated coconut fiber, non irradiated, and EB irradiated with 50 kGy and 100 kGy radiation doses, respectively, are shown in Figure 21. It was not possible to observe fiber presence on the surface analyzed. Neither any significant alteration on the irradiated surface, in the dose range studied. It suggests that cryogenic fractured surfaces allows the observation of irradiation effect on the polymeric bulk that otherwise could not be observed on sample surface. Probably it occurs due to the fact that species formed by energy deposition of

probably related to scission process prevail of PLLA with doses above 100 kGy.

Fig. 22. Field emission scanning electron micrograph surface of hot pressed sheet of composite containing 10% non chemically treated coconut fiber: A) non irradiated; B) EB irradiated with 50 kGy, and C) EB irradiated with 100 kGy.

## **4. Conclusion**

Due to coalescence effect it was possible to observe spherical inclusions of PLLA in PCL:PLLA blend. Increasing radiation dose induced elongation of inclusions, as well, lamellar structures increase in the PCL matrix. Radiation doses higher than 100 kGy altered morphologies of samples surfaces, that became smoother, attributed to the presence of smaller fractions of PLLA, as a result of long chain scission, and high crosslinking reaction in PCL phase. Radiation processing and chemical acetylating did not promote measurable interaction between fibers with polymeric matrix.

The SEM micrographs of the fractured homopolymers and blends have shown their immiscibility. The crystallization of PLLA could be observed on the annealed samples. Samples irradiated with 100 kGy presented little variation on the morphology, even

Ionizing Radiation Effect on Morphology of PLLA:

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supposing that the structural modifications induced by ionizing radiation may alter the morphology of the samples. It seems that some shape alteration in the PCL dispersed phase in blends occurred. Likewise, samples submitted to irradiation processing up to dose of 500 kGy presented the matrix with decreased PCL spherulites disperse phase, suggesting that some interaction between both polymeric phases had been promoted by the ionizing radiation. However, in PCL homopolymer and PCL:PLLA 50:50 irradiated with 500 kGy samples it was possible to observe significant alteration. The ruptured sample surface of irradiated PCL with 500 kGy became full of scales probably due to an increase of crosslinking density induced by the ionizing radiation. The surface of PLLA sample became rough with 100 kGy radiation dose correlated to chain scissions promoted by gamma radiation. On the other hand, apparently no changes are visible by SEM on the irradiated PLLA with 500 kGy radiation dose, in contrast to the observed previously in the literature. It was also studied blends and composites based on PCL, PLLA, and coconut fiber. Acetylation of fibers was not effective in order to induce any interaction between fibers and polymeric matrix, as expected. Ionizing radiation neither promoted detectable interaction between polymeric matrix and fibers.

### **5. Acknowledgements**

We are grateful to the financial support from JICA and IAEA. Additionally, to Dr. Akihiro Oishi and Dr. Kazuo Nakayama, from National Institute of Advanced Industrial Science and Technology – AIST, Japan, for samples preparation and valuable discussion; to Dr. Naotsugu Nagasawa and Dr. Masao Tamada , from Japan Atomic Energy Agency – JAEA, Japan, for samples preparation and irradiation. We also would like to thank Dr. Morsyleide Freitas Rosa from Embrapa for providing coconut fiber; to Prof. Dr. Hélio Wiebeck, and Mr. Wilson Maia from Laboratório de Matérias-Primas Particuladas e Sólidos Não Metálicos – LMPSol, Departamento de Engenharia de Materiais, Escola Politécnica da USP (EPUSP) for coconut fiber size reduction and segregation; also to Eng. Elisabeth S.R. Somessari, Eng. Carlos G. da Silveira, and Mr. Paulo de Souza Santos, from IPEN, for blends and composites irradiation. In addition, to Centro Tecnológico da Marinha em São Paulo – CTMSP, for SEM and SEM EDS micrographs. We would like also to thank Dr. Luci Diva Brocardo Machado for helpful discussion.

#### **6. References**

supposing that the structural modifications induced by ionizing radiation may alter the morphology of the samples. It seems that some shape alteration in the PCL dispersed phase in blends occurred. Likewise, samples submitted to irradiation processing up to dose of 500 kGy presented the matrix with decreased PCL spherulites disperse phase, suggesting that some interaction between both polymeric phases had been promoted by the ionizing radiation. However, in PCL homopolymer and PCL:PLLA 50:50 irradiated with 500 kGy samples it was possible to observe significant alteration. The ruptured sample surface of irradiated PCL with 500 kGy became full of scales probably due to an increase of crosslinking density induced by the ionizing radiation. The surface of PLLA sample became rough with 100 kGy radiation dose correlated to chain scissions promoted by gamma radiation. On the other hand, apparently no changes are visible by SEM on the irradiated PLLA with 500 kGy radiation dose, in contrast to the observed previously in the literature. It was also studied blends and composites based on PCL, PLLA, and coconut fiber. Acetylation of fibers was not effective in order to induce any interaction between fibers and polymeric matrix, as expected. Ionizing radiation neither promoted detectable interaction

We are grateful to the financial support from JICA and IAEA. Additionally, to Dr. Akihiro Oishi and Dr. Kazuo Nakayama, from National Institute of Advanced Industrial Science and Technology – AIST, Japan, for samples preparation and valuable discussion; to Dr. Naotsugu Nagasawa and Dr. Masao Tamada , from Japan Atomic Energy Agency – JAEA, Japan, for samples preparation and irradiation. We also would like to thank Dr. Morsyleide Freitas Rosa from Embrapa for providing coconut fiber; to Prof. Dr. Hélio Wiebeck, and Mr. Wilson Maia from Laboratório de Matérias-Primas Particuladas e Sólidos Não Metálicos – LMPSol, Departamento de Engenharia de Materiais, Escola Politécnica da USP (EPUSP) for coconut fiber size reduction and segregation; also to Eng. Elisabeth S.R. Somessari, Eng. Carlos G. da Silveira, and Mr. Paulo de Souza Santos, from IPEN, for blends and composites irradiation. In addition, to Centro Tecnológico da Marinha em São Paulo – CTMSP, for SEM and SEM EDS micrographs. We would like also to thank Dr. Luci Diva Brocardo Machado

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**14**

*Argentina*

**Study of Helminth Parasites of Amphibians**

Cynthya Elizabeth González1, Monika Inés Hamann1 and Cristina Salgado2

Amphibians, like all other animals, are subject to a variety of parasites and diseases, including viral, bacterial and fungal infections as well as some forms of cancer and tuberculosis (Hoff et al., 1984). Various viruses and bacteria such as *Pseudomonas* or *Salmonella*, and fungi such as *Candida*, are recorded as common infectious agents in amphibians, but currently the focus of studies are the fungi of genus *Batrachochytrium*, agents of the disease known as chytridiomycosis, which is considered as one of the factors responsible for the decline of amphibian populations in many parts of the world (Berger and Speare, 1998). In addition, protozoans of the genera *Opalina* and *Entamoeba* in the digestive tract, and trypanosomes in the circulatory system, as well as coccidian protozoa have been

However, helminths are the most common invertebrate parasites of amphibians. One well known example among trematodes is the monogenean genus *Polystoma*, which infects the urinary bladder of adult amphibians around the world. Parasitic digenean trematodes include both larval stages (metacercariae) and adults. Cestodes are not common parasites in the gastrointestinal tract of amphibians, but when present may persist for a long time. Adult acanthocephalans adhere to the mucosa of the stomach or intestine. Finally, nematodes are particularly abundant in the digestive tract, lungs and blood vessels of these vertebrates (Pough et al., 2001). Amphibians are also hosts to other groups of less common parasitic invertebrates, such as annelids, pentastomids and arthropods (copepods, ticks, insects)

Of these, analyses using scanning electron microscopy techniques have been mainly applied to digenean trematodes (flatworms), nematodes (roundworms) and acanthocephalans

The study of parasitic nematodes and trematodes by scanning electron microscopy began in the 1970s and involved mainly those organisms that produced diseases in humans and livestock, as well as parasites of crops (Halton, 2004). In particular, studies in amphibian hosts were first made by Nollen and Nadakavukaren (1974) and Nadakavukaren and

recorded in amphibians (Duellman and Trueb, 1986; Duszynski et al., 2007).

(thorny or spiny headed), particularly to their adult stages (Fig. 1-3).

**1. Introduction**

(Tinsley, 1995).

**by Scanning Electron Microscopy**

*Consejo Nacional de Investigaciones Científicas y Técnicas,*

*Universidad Nacional del Nordeste (UNNE), Corrientes,*

*1Centro de Ecología Aplicada del Litoral,*

*2Servicio de Microscopia Electrónica de Barrido,*

## **Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy**

Cynthya Elizabeth González1, Monika Inés Hamann1 and Cristina Salgado2 *1Centro de Ecología Aplicada del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, 2Servicio de Microscopia Electrónica de Barrido, Universidad Nacional del Nordeste (UNNE), Corrientes, Argentina*

## **1. Introduction**

266 Scanning Electron Microscopy

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caprolactone) by radiation technique and its biodegradability, *Radiation Physics and*

modifications and thermal behavior of poly (L-lactic acid) revealed by infrared

Amphibians, like all other animals, are subject to a variety of parasites and diseases, including viral, bacterial and fungal infections as well as some forms of cancer and tuberculosis (Hoff et al., 1984). Various viruses and bacteria such as *Pseudomonas* or *Salmonella*, and fungi such as *Candida*, are recorded as common infectious agents in amphibians, but currently the focus of studies are the fungi of genus *Batrachochytrium*, agents of the disease known as chytridiomycosis, which is considered as one of the factors responsible for the decline of amphibian populations in many parts of the world (Berger and Speare, 1998). In addition, protozoans of the genera *Opalina* and *Entamoeba* in the digestive tract, and trypanosomes in the circulatory system, as well as coccidian protozoa have been recorded in amphibians (Duellman and Trueb, 1986; Duszynski et al., 2007).

However, helminths are the most common invertebrate parasites of amphibians. One well known example among trematodes is the monogenean genus *Polystoma*, which infects the urinary bladder of adult amphibians around the world. Parasitic digenean trematodes include both larval stages (metacercariae) and adults. Cestodes are not common parasites in the gastrointestinal tract of amphibians, but when present may persist for a long time. Adult acanthocephalans adhere to the mucosa of the stomach or intestine. Finally, nematodes are particularly abundant in the digestive tract, lungs and blood vessels of these vertebrates (Pough et al., 2001). Amphibians are also hosts to other groups of less common parasitic invertebrates, such as annelids, pentastomids and arthropods (copepods, ticks, insects) (Tinsley, 1995).

Of these, analyses using scanning electron microscopy techniques have been mainly applied to digenean trematodes (flatworms), nematodes (roundworms) and acanthocephalans (thorny or spiny headed), particularly to their adult stages (Fig. 1-3).

The study of parasitic nematodes and trematodes by scanning electron microscopy began in the 1970s and involved mainly those organisms that produced diseases in humans and livestock, as well as parasites of crops (Halton, 2004). In particular, studies in amphibian hosts were first made by Nollen and Nadakavukaren (1974) and Nadakavukaren and

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 269

microscope (Fig. 4). Tadpoles were captured with a 45-cm-diameter dip net and kept alive in the laboratory until their dissection. They were killed using a chloroform solution, and

The analized amphibian species were: *Rhinella bergi, R. fernandezae, R. granulosa, R. schneideri* (Bufonidae), *Odontophrynus americanus* (Cycloramphidae), *Dendropsophus nanus, D. sanborni, Hypsiboas raniceps, Pseudis limellum, Phyllomedusa hypochondrialis, Scinax acuminatus, S. nasicus, Trachycephalus venulosus, Pseudis paradoxa* (Hylidae), *Physalaemus albonotatus, P. santafecinus, Pseudopaludicola boliviana, P. falcipes* (Leiuperidae), *Leptodactylus bufonius, L. chaquensis, L. elenae, L. latinasus, L. latrans, L. podicipinus* (Leptodactylidae), *Lepidobatrachus laevis* (Ceratophryidae). Anurans were identified using different guides and keys (Faivovich

In 1972, Allison et al. proposed a simplified four-step procedure that resulted in excellent preservation, support in the high vacuum and dissipation of surface charging of nematodes. The procedure involved: fixation, dehydration, treatment with an antistatic agent and goldpalladium coating. These authors obtained best results using fixation with 4% paraformaldehyde (phosphate-buffered) and AFA (acetic acid-formalin-alcohol); specimens were dehydrated in an ascending series of ethanol solutions to 70%, then transferred to 5% glycerine-95% ethanol from which the alcohol was allowed to evaporate, and cleared in 96.6% glycerol-0.05% potassium chloride-3.35% distilled water, 24 to 48 hours prior to examination. Subsequently, specimens were mounted on metal specimen stubs with Duco cement, outgassed in a vacuum evaporator for 1 hour or more and rotary-coated with gold-

In this study we basically followed the aforementioned procedure, with some modifications. Helminths were observed *in vivo*, counted, fixed and stored. Adult nematodes and trematodes were fixed in hot 4% formaldehyde; larval nematodes and trematodes were

subsequently all organs, musculature and body cavity were examined for parasites.

Fig. 4. Ventral view of adult amphibian showing all organ systems.

**2.2 Procedure applied to helminth. Complications**

et al., 2005; Frost et al., 2004).

palladium.

Nollen (1975) who provided details of the tegument of the trematodes *Megalodiscus temperatus* (Paramphistomatidae) and *Gorgoderina attenuata* (Gorgoderidae) from *Rana pipiens*. Regarding nematodes, Navarro et al. (1988) provided details of the cuticle of the species *Cosmocerca ornata*, *Oxysomatium brevicaudatum* (Cosmocercidae) and *Seuratascaris numidica* (Ascarididae) collected in ranid hosts from different geographical areas of the Iberian Peninsula.

Fig. 1-3. Helminth parasites found in amphibian hosts. 1. Flatworms (Trematoda), general view. 2. Roundworms (Nematoda) male and female mating. 3. Thorny or spine headed (Acanthocephala), general view.

This chapter presents scanning electron micrographs taken during diverse studies undertaken to determine the helminth fauna of Argentinean anurans, especially those living in Northeastern Argentina. The survey includes the classes Trematoda (specifically subclass Digenea) and Nematoda (specifically subclass Secernentea) and the phyllum Acanthocephala. A total of five families of amphibians (Bufonidae, Cycloramphidae, Hylidae, Leptodactylidae, Leiuperidae) were analyzed, both at larval (tadpole) and adult stages, to study their helminth parasites. At the end of the chapter we present a summary of the present-day advances in this topic, including new contributions presented in this work; finally, we discuss possible future lines of research in this field of Parasitology.

The classification of helminths follows Anderson et al. (2009) and Gibbons (2010) for class Nematoda; Gibson et al. (2002) and Jones et al. (2005) for class Trematoda and Amin (1985) for Acanthocephala.

## **2. Preparation of helminth parasites of amphibians for observation by scanning electron microscopy**

#### **2.1 Collection of hosts and obtaining of helminth parasites**

Adult frogs were hand captured, mainly at night, using the sampling technique defined as visual encounter survey (Crump and Scott, 1994). The individuals were transported live to the laboratory and killed in a chloroform solution (CHCl3). The abdominal cavity of each frog was opened and the oesophagus, stomach, gut, lungs, liver, urinary bladder, kidneys, body cavity, musculature, integument and brain examined for parasites under a dissecting

Nollen (1975) who provided details of the tegument of the trematodes *Megalodiscus temperatus* (Paramphistomatidae) and *Gorgoderina attenuata* (Gorgoderidae) from *Rana pipiens*. Regarding nematodes, Navarro et al. (1988) provided details of the cuticle of the species *Cosmocerca ornata*, *Oxysomatium brevicaudatum* (Cosmocercidae) and *Seuratascaris numidica* (Ascarididae) collected in ranid hosts from different geographical areas of the

Fig. 1-3. Helminth parasites found in amphibian hosts. 1. Flatworms (Trematoda), general view. 2. Roundworms (Nematoda) male and female mating. 3. Thorny or spine headed

This chapter presents scanning electron micrographs taken during diverse studies undertaken to determine the helminth fauna of Argentinean anurans, especially those living in Northeastern Argentina. The survey includes the classes Trematoda (specifically subclass Digenea) and Nematoda (specifically subclass Secernentea) and the phyllum Acanthocephala. A total of five families of amphibians (Bufonidae, Cycloramphidae, Hylidae, Leptodactylidae, Leiuperidae) were analyzed, both at larval (tadpole) and adult stages, to study their helminth parasites. At the end of the chapter we present a summary of the present-day advances in this topic, including new contributions presented in this work;

The classification of helminths follows Anderson et al. (2009) and Gibbons (2010) for class Nematoda; Gibson et al. (2002) and Jones et al. (2005) for class Trematoda and Amin (1985)

Adult frogs were hand captured, mainly at night, using the sampling technique defined as visual encounter survey (Crump and Scott, 1994). The individuals were transported live to the laboratory and killed in a chloroform solution (CHCl3). The abdominal cavity of each frog was opened and the oesophagus, stomach, gut, lungs, liver, urinary bladder, kidneys, body cavity, musculature, integument and brain examined for parasites under a dissecting

finally, we discuss possible future lines of research in this field of Parasitology.

**2. Preparation of helminth parasites of amphibians for observation by**

**2.1 Collection of hosts and obtaining of helminth parasites**

Iberian Peninsula.

(Acanthocephala), general view.

for Acanthocephala.

**scanning electron microscopy**

microscope (Fig. 4). Tadpoles were captured with a 45-cm-diameter dip net and kept alive in the laboratory until their dissection. They were killed using a chloroform solution, and subsequently all organs, musculature and body cavity were examined for parasites.

Fig. 4. Ventral view of adult amphibian showing all organ systems.

The analized amphibian species were: *Rhinella bergi, R. fernandezae, R. granulosa, R. schneideri* (Bufonidae), *Odontophrynus americanus* (Cycloramphidae), *Dendropsophus nanus, D. sanborni, Hypsiboas raniceps, Pseudis limellum, Phyllomedusa hypochondrialis, Scinax acuminatus, S. nasicus, Trachycephalus venulosus, Pseudis paradoxa* (Hylidae), *Physalaemus albonotatus, P. santafecinus, Pseudopaludicola boliviana, P. falcipes* (Leiuperidae), *Leptodactylus bufonius, L. chaquensis, L. elenae, L. latinasus, L. latrans, L. podicipinus* (Leptodactylidae), *Lepidobatrachus laevis* (Ceratophryidae). Anurans were identified using different guides and keys (Faivovich et al., 2005; Frost et al., 2004).

## **2.2 Procedure applied to helminth. Complications**

In 1972, Allison et al. proposed a simplified four-step procedure that resulted in excellent preservation, support in the high vacuum and dissipation of surface charging of nematodes. The procedure involved: fixation, dehydration, treatment with an antistatic agent and goldpalladium coating. These authors obtained best results using fixation with 4% paraformaldehyde (phosphate-buffered) and AFA (acetic acid-formalin-alcohol); specimens were dehydrated in an ascending series of ethanol solutions to 70%, then transferred to 5% glycerine-95% ethanol from which the alcohol was allowed to evaporate, and cleared in 96.6% glycerol-0.05% potassium chloride-3.35% distilled water, 24 to 48 hours prior to examination. Subsequently, specimens were mounted on metal specimen stubs with Duco cement, outgassed in a vacuum evaporator for 1 hour or more and rotary-coated with goldpalladium.

In this study we basically followed the aforementioned procedure, with some modifications. Helminths were observed *in vivo*, counted, fixed and stored. Adult nematodes and trematodes were fixed in hot 4% formaldehyde; larval nematodes and trematodes were

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 271

For examination by scanning electron microscop**y** (SEM), specimens were dehydrated through an alcohol 70° and acetone series (70%, 85% and 100%; 15 minutes in each solution) and then subjected to critical point drying using CO2; in the case of larval digenean trematodes, critical point drying time is shortened because these individuals are more fragile than adults. Samples were mounted on a metal sheet of copper or aluminum using double-sided tape. Then the specimens were sputter-coated with gold or gold-palladium for

Adequate fixation contributes to specimen preservation and stability within the microscope; dehydration allows outgassing and drying of specimens in the vaccum evaporator prior to coating without subsequent "bubbling" or shrinkage artifacts; glyceration provides an antistatic surface coating over all surface irregularities, even those which might not be adequately coated with 200 a of gold-palladium. The thin metal coating does not obscure fine structural detail but, in combination with the glycerol-KCl coating, ensures that the specimen can be exposed for extended periods of time to the electron beam without surface

**3. What can SEM studies tell us about helminth parasites of amphibians?**

The body surface of nematodes is covered by a truly inert cuticle of extracellular material in the form of cross-linked collagens and insoluble proteins that are synthesised and secreted by the underlying epidermis (= hypodermis). In addition to proteins and collagen, the cuticle possesses glycoproteins, fibrin and keratin. It consists of three main layers and the epicuticle. The external layer is divided into internal and external cortex; the middle layer varies from having a granular uniform structure to presenting skeletal rods, fibers or channels; the internal basal layer can be laminated or grooved. The thin epicuticle may have

The above discussion refers specifically to the cuticle of the external body surface. In other body parts, such as the buccal cavity, excretory pore, vagina, cloaca and rectum, as well as

The particular composition of the cuticle along the whole body of nematodes, and especially the ornamentations that they present in the anterior and posterior extremity, are the focus of

The nomenclature for the structures detailed here is based on Chitwood and Chitwood (1975), Gibbons (1986) and Willmott (2009). We describe the modifications of the cuticle along the body surface, at the anterior and posterior body ends, and finally the cuticle of the

The features analyzed along the body surface are the morphology and disposition of somatic papillae as well as the striation of the cuticle -longitudinal, transverse or oblique-, and the presence of annulations, punctuations, longitudinal ridges, alae -lateral, cervical or

the eggs and spicules in males, the cuticle is of a different nature.

primary study of these helminths by scanning electron microscopy.

three minutes. Helminth parasites were observed in high vacuum in all cases.

changes (Allison et al., 1972).

a coating of quinone (Lee, 2002).

eggs, vulva and spicules of these helminths.

**3.1.1 Cuticle of body**

caudal-, inflation and spination.

**3.1 Nematoda**

removed from cysts with the aid of preparation needlees and fixed in hot 4% formaldehyde; acanthocephalan larvae were placed in distilled water for 24 hours at 4° C for the proboscis to evert, and then fixed in hot 4% formaldehyde.

Another technique used for the study of nematodes and acanthocephalans by SEM consists of transferring specimens for 2 hours into 1% osmium tetroxide and dehydrate them in an ethanol series for 2 hours in each bath (Mafra and Lanfredi, 1998). In the case of trematodes, fixation can be made with paraformaldehyde, glutaraldehyde or a formaldehydeglutaraldehyde mix such as Karnovsky's fixative in phosphate or cacodylate buffer. Postfixation is usually done for 2 to 3 hours at 4°C with cacodylate– or phosphate- buffered 1% osmium tetroxide (Karnovsky, 1965).

For processing of helminths, it should be taken into account that a hypertonic solution will cause shrinkage and almost complete disappearance of inflation in specimens, whereas a hypotonic solution may produce artificial inflations. In some cases, in spite of careful processing of samples for SEM study, good results are not achieved. Very frequently, the samples contain bacteria or debris, or tears of the cuticle of nematodes or tegument of trematodes (Fig. 5-9).

Fig. 5-9. Some complications in samples for SEM studies. 5. 6. Bacteria in posterior end of *Skrjabinodon* sp*.* (5) and in ventral surface of *Cosmocerca parva* (6). 7. Broken cuticle in posterior end of female of *Aplectana* sp. 8. 9. Debris in spicules and adanal papillae of *Cosmocerca podicipinus* (8) and in posterior end of *Falcaustra* sp. (9).

#### **2.3 Characteristic of the scanning electron microscope used for this study**

The Microscopy department at Universidad Nacional del Nordeste possesses a Jeol 5800LV scanning electron microscopy. Specimens are critical-point dried using a Denton Vacuum DCP-1 critical point drying apparatus, and sputter-coating is made using a Denton Vacuum Desk II sputter-coating unit.

For examination by scanning electron microscop**y** (SEM), specimens were dehydrated through an alcohol 70° and acetone series (70%, 85% and 100%; 15 minutes in each solution) and then subjected to critical point drying using CO2; in the case of larval digenean trematodes, critical point drying time is shortened because these individuals are more fragile than adults. Samples were mounted on a metal sheet of copper or aluminum using double-sided tape. Then the specimens were sputter-coated with gold or gold-palladium for three minutes. Helminth parasites were observed in high vacuum in all cases.

Adequate fixation contributes to specimen preservation and stability within the microscope; dehydration allows outgassing and drying of specimens in the vaccum evaporator prior to coating without subsequent "bubbling" or shrinkage artifacts; glyceration provides an antistatic surface coating over all surface irregularities, even those which might not be adequately coated with 200 a of gold-palladium. The thin metal coating does not obscure fine structural detail but, in combination with the glycerol-KCl coating, ensures that the specimen can be exposed for extended periods of time to the electron beam without surface changes (Allison et al., 1972).

## **3. What can SEM studies tell us about helminth parasites of amphibians?**

## **3.1 Nematoda**

270 Scanning Electron Microscopy

removed from cysts with the aid of preparation needlees and fixed in hot 4% formaldehyde; acanthocephalan larvae were placed in distilled water for 24 hours at 4° C for the proboscis

Another technique used for the study of nematodes and acanthocephalans by SEM consists of transferring specimens for 2 hours into 1% osmium tetroxide and dehydrate them in an ethanol series for 2 hours in each bath (Mafra and Lanfredi, 1998). In the case of trematodes, fixation can be made with paraformaldehyde, glutaraldehyde or a formaldehydeglutaraldehyde mix such as Karnovsky's fixative in phosphate or cacodylate buffer. Postfixation is usually done for 2 to 3 hours at 4°C with cacodylate– or phosphate- buffered

For processing of helminths, it should be taken into account that a hypertonic solution will cause shrinkage and almost complete disappearance of inflation in specimens, whereas a hypotonic solution may produce artificial inflations. In some cases, in spite of careful processing of samples for SEM study, good results are not achieved. Very frequently, the samples contain bacteria or debris, or tears of the cuticle of nematodes or tegument of

Fig. 5-9. Some complications in samples for SEM studies. 5. 6. Bacteria in posterior end of *Skrjabinodon* sp*.* (5) and in ventral surface of *Cosmocerca parva* (6). 7. Broken cuticle in posterior end of female of *Aplectana* sp. 8. 9. Debris in spicules and adanal papillae of

The Microscopy department at Universidad Nacional del Nordeste possesses a Jeol 5800LV scanning electron microscopy. Specimens are critical-point dried using a Denton Vacuum DCP-1 critical point drying apparatus, and sputter-coating is made using a Denton Vacuum

**2.3 Characteristic of the scanning electron microscope used for this study**

*Cosmocerca podicipinus* (8) and in posterior end of *Falcaustra* sp. (9).

to evert, and then fixed in hot 4% formaldehyde.

1% osmium tetroxide (Karnovsky, 1965).

trematodes (Fig. 5-9).

Desk II sputter-coating unit.

The body surface of nematodes is covered by a truly inert cuticle of extracellular material in the form of cross-linked collagens and insoluble proteins that are synthesised and secreted by the underlying epidermis (= hypodermis). In addition to proteins and collagen, the cuticle possesses glycoproteins, fibrin and keratin. It consists of three main layers and the epicuticle. The external layer is divided into internal and external cortex; the middle layer varies from having a granular uniform structure to presenting skeletal rods, fibers or channels; the internal basal layer can be laminated or grooved. The thin epicuticle may have a coating of quinone (Lee, 2002).

The above discussion refers specifically to the cuticle of the external body surface. In other body parts, such as the buccal cavity, excretory pore, vagina, cloaca and rectum, as well as the eggs and spicules in males, the cuticle is of a different nature.

The particular composition of the cuticle along the whole body of nematodes, and especially the ornamentations that they present in the anterior and posterior extremity, are the focus of primary study of these helminths by scanning electron microscopy.

The nomenclature for the structures detailed here is based on Chitwood and Chitwood (1975), Gibbons (1986) and Willmott (2009). We describe the modifications of the cuticle along the body surface, at the anterior and posterior body ends, and finally the cuticle of the eggs, vulva and spicules of these helminths.

#### **3.1.1 Cuticle of body**

The features analyzed along the body surface are the morphology and disposition of somatic papillae as well as the striation of the cuticle -longitudinal, transverse or oblique-, and the presence of annulations, punctuations, longitudinal ridges, alae -lateral, cervical or caudal-, inflation and spination.

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 273

Striations can be distributed uniformly along the whole body of the parasite (*Cosmocerca* spp.), or they may become less evident (*A. delirae*) or wider (*A. hylambatis*) at the body ends,

*Longitudinal markings*: these may take the form of ridges or alae, or they may be merely the

Fig. 16-21. Longitudinal markings in nematode parasites of amphibians. 16. 17. Lateral alae in anterior end of body in *Paraoxyascaris* sp. (16) and in *Porrocaecum* sp. (larvae) (17).

18. 19. Lateral alae in posterior end of body in *Cosmocerca* spp. 20. Cephalic cuticular vesicle

in *Oswaldocruzia* sp. 21. Longitudinal ridges in *Oswaldocruzia* sp. la: lateral alae; lr:

longitudinal ridges.

or more marked, as for example, in the posterior body (*Skrjabinodon*) (Figs. 10-15).

result of gaps in transverse markings.

There are several types of gross cuticular markings, namely, *transverse*, *longitudinal* and *oblique* markings. Of these, *transverse* and *longitudinal* markings are very common in nematode parasites of amphibians.

*Tranverse markings*: the superficial markings are grooves or ridges. *Striations* are defined as fine transverse grooves occurring at regular intervals; the distance between two striae is the intestrial region (*Aplectana hylambatis*, *Cosmocerca* spp.) (Figs. 10, 11). Deep striae are very commonly and are known as *annulations*; the distances between them are termed *annules* (*Aplectana delirae*, *Falcaustra* sp.) (Figs. 12, 13). In nematode parasites of the family Pharyngodonidae, annulations are much broader and more prominent (Figs. 14, 15, 34).

Fig. 10-15. Transverse marking in cuticle of nematode parasites of amphibians. 10. 11. Striations (*Aplectana hylambatis*, *Cosmocerca podicipinus*, respectively). 12. 13. Striations and annules (*A. delirae*, *Falcaustra* sp., respectively). 14. 15. Annulations (*Skrjabinodon* sp.).

There are several types of gross cuticular markings, namely, *transverse*, *longitudinal* and *oblique* markings. Of these, *transverse* and *longitudinal* markings are very common in

*Tranverse markings*: the superficial markings are grooves or ridges. *Striations* are defined as fine transverse grooves occurring at regular intervals; the distance between two striae is the intestrial region (*Aplectana hylambatis*, *Cosmocerca* spp.) (Figs. 10, 11). Deep striae are very commonly and are known as *annulations*; the distances between them are termed *annules* (*Aplectana delirae*, *Falcaustra* sp.) (Figs. 12, 13). In nematode parasites of the family Pharyngodonidae, annulations are much broader and more prominent (Figs. 14, 15, 34).

Fig. 10-15. Transverse marking in cuticle of nematode parasites of amphibians. 10. 11. Striations (*Aplectana hylambatis*, *Cosmocerca podicipinus*, respectively). 12. 13. Striations and annules (*A. delirae*, *Falcaustra* sp., respectively). 14. 15. Annulations (*Skrjabinodon* sp.).

nematode parasites of amphibians.

Striations can be distributed uniformly along the whole body of the parasite (*Cosmocerca* spp.), or they may become less evident (*A. delirae*) or wider (*A. hylambatis*) at the body ends, or more marked, as for example, in the posterior body (*Skrjabinodon*) (Figs. 10-15).

*Longitudinal markings*: these may take the form of ridges or alae, or they may be merely the result of gaps in transverse markings.

Fig. 16-21. Longitudinal markings in nematode parasites of amphibians. 16. 17. Lateral alae in anterior end of body in *Paraoxyascaris* sp. (16) and in *Porrocaecum* sp. (larvae) (17). 18. 19. Lateral alae in posterior end of body in *Cosmocerca* spp. 20. Cephalic cuticular vesicle in *Oswaldocruzia* sp. 21. Longitudinal ridges in *Oswaldocruzia* sp. la: lateral alae; lr: longitudinal ridges.

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 275

papillae. In this section, we deal with the distribution and structure of somatic papillae, i.e. those that are distributed throughout the body of the nematode. These papillae are generally

Finally, in some genera of nematode parasites of amphibians, the excretory pore has conspicuous cuticularized walls, surrounded by a rough cuticular area (Pharyngodonidae); in others, the excretory pore is located in a depression of the cuticula (*Falcaustra*) (Figs. 28, 29).

Fig. 25-27. Somatic papillae in nematode parasites of amphibians. 25. *Aplectana hylambatis*.

Fig. 28-29. Excretory pore in nematode parasites of amphibian. 28. *Falcaustra* sp. 29.

*Punctuations*: this type of marking is frequent and appears as minute round areas of the cuticle which are arranged in definite patterns for each species. In nematode parasites of amphibians, this type of marking appears in males of the family Cosmocercidae, specifically in the genus *Cosmocerca* as part of the rosette papillae (see below; Figs. 38-44) and in the

The cuticular modifications that occur in the anterior end of the body of nematode parasites of amphibians are: head papillae - cephalic papillae, externo-labial papillae, interno-labial papillae-, interlabia, deirids and amphids. These two latter structures are not modifications of the cuticle itself, but their opening in the cuticle can present diverse shape and structure. *Head papillae*: these are tactile sensory organs usually located on the lips or labial region, including two circles of six labial papillae and one circle of four cephalic papillae. This arrangement has been proposed for ancestral nematodes considering a radial type of symmetry (De Coninck, 1965). The head papillae are divided into: *cephalic papillae*: outer

26. *Aplectana delirae*. 27. *Cosmocerca podicipinus.* sp: somatic papillae.

*Skrjabinodon* sp.

genus Cosmocercella (Figs. 46, 48).

**3.1.2 Cuticle of anterior end of body**

arranged in two subdorsal and two subventral rows along the body of the nematode.

**Alae**: these are usually lateral or sublateral cuticular thickenings or projections. There are three types of alae: longitudinal, cervical and caudal.

*Longitudinal alae*: these are usually lateral or sublateral and occur in both sexes; extending along the length of the body. They occur in the families Cosmocercidae (*Aplectana*, *Cosmocerca*, *Cosmocercella*, *Paraoxyascaris*) (Figs. 16, 18, 19), Ascarididae (*Ortleppascaris*, *Porrocaecum*) (Fig. 17); in *Gyrinicola* sp. (Family Pharyngodonidae) lateral alae are present in males only.

*Cervical alae*: these structures are confined to the anterior part of the body. This modification of the cuticle does not occur in nematode species that parasitize amphibians.

In some parasitic nematodes, the cervical alae is modified as a *cephalic cuticular vesicle* as in the genus *Oswaldocruzia*; in some cases this cuticular vesicle is divided into a larger anterior part and a smaller posterior part (Fig. 20).

*Caudal alae*: these alae are confined to the caudal region of the body and limited to the males only; apparently they serve as clasping organs during copulation. Among nematodes that parasitize amphibians, they occur in the family Pharyngodonidae (*Parapharyngodon*). On the other hand, modified caudal alae occur in the males of some families such as the Molineidae, where they are called *bursa copulatrix* or *copulatory bursa* (see below).

**Longitudinal ridges:** these are raised areas that extend along the length of the body and are present on the submedian as well as on the lateral surfaces. *Oswaldocruzia* (Family: Molineidae) has longitudinal ridges throughout the body length that can disappear or appear along its body (Fig. 21). The system of longitudinal cuticular ridges or *synlophe* in this genus is very important as a taxonomic character.

*Inflation*: when the cuticle is swollen in a blister-like manner, this condition may be termed inflation (eg. *Rhabdias*; Figs. 22-24). Besides being inflated, the cuticle can be also striated. Inflation of the cuticle may be generalized over the whole body surface instead of restricted to certain areas.

Fig. 22-24. Inflation in the cuticle of nematode parasites of genus *Rhabdias*. 22. Cephalic end. 23. Vulvar region. 24. Anterior end of body.

*Papillae*: these structures are nerve endings, some of which have a tactile function and others are chemoreceptors; they appear as cuticular elevations of different shapes and sizes (Figs. 25-27). According to their position, they are divided into labial, cephalic, cervical and genital

**Alae**: these are usually lateral or sublateral cuticular thickenings or projections. There are

*Longitudinal alae*: these are usually lateral or sublateral and occur in both sexes; extending along the length of the body. They occur in the families Cosmocercidae (*Aplectana*, *Cosmocerca*, *Cosmocercella*, *Paraoxyascaris*) (Figs. 16, 18, 19), Ascarididae (*Ortleppascaris*, *Porrocaecum*) (Fig.

*Cervical alae*: these structures are confined to the anterior part of the body. This modification

In some parasitic nematodes, the cervical alae is modified as a *cephalic cuticular vesicle* as in the genus *Oswaldocruzia*; in some cases this cuticular vesicle is divided into a larger anterior

*Caudal alae*: these alae are confined to the caudal region of the body and limited to the males only; apparently they serve as clasping organs during copulation. Among nematodes that parasitize amphibians, they occur in the family Pharyngodonidae (*Parapharyngodon*). On the other hand, modified caudal alae occur in the males of some families such as the

**Longitudinal ridges:** these are raised areas that extend along the length of the body and are present on the submedian as well as on the lateral surfaces. *Oswaldocruzia* (Family: Molineidae) has longitudinal ridges throughout the body length that can disappear or appear along its body (Fig. 21). The system of longitudinal cuticular ridges or *synlophe* in this

*Inflation*: when the cuticle is swollen in a blister-like manner, this condition may be termed inflation (eg. *Rhabdias*; Figs. 22-24). Besides being inflated, the cuticle can be also striated. Inflation of the cuticle may be generalized over the whole body surface instead of restricted

Fig. 22-24. Inflation in the cuticle of nematode parasites of genus *Rhabdias*. 22. Cephalic end.

*Papillae*: these structures are nerve endings, some of which have a tactile function and others are chemoreceptors; they appear as cuticular elevations of different shapes and sizes (Figs. 25-27). According to their position, they are divided into labial, cephalic, cervical and genital

17); in *Gyrinicola* sp. (Family Pharyngodonidae) lateral alae are present in males only.

of the cuticle does not occur in nematode species that parasitize amphibians.

Molineidae, where they are called *bursa copulatrix* or *copulatory bursa* (see below).

three types of alae: longitudinal, cervical and caudal.

part and a smaller posterior part (Fig. 20).

genus is very important as a taxonomic character.

23. Vulvar region. 24. Anterior end of body.

to certain areas.

papillae. In this section, we deal with the distribution and structure of somatic papillae, i.e. those that are distributed throughout the body of the nematode. These papillae are generally arranged in two subdorsal and two subventral rows along the body of the nematode.

Finally, in some genera of nematode parasites of amphibians, the excretory pore has conspicuous cuticularized walls, surrounded by a rough cuticular area (Pharyngodonidae); in others, the excretory pore is located in a depression of the cuticula (*Falcaustra*) (Figs. 28, 29).

Fig. 25-27. Somatic papillae in nematode parasites of amphibians. 25. *Aplectana hylambatis*. 26. *Aplectana delirae*. 27. *Cosmocerca podicipinus.* sp: somatic papillae.

Fig. 28-29. Excretory pore in nematode parasites of amphibian. 28. *Falcaustra* sp. 29. *Skrjabinodon* sp.

*Punctuations*: this type of marking is frequent and appears as minute round areas of the cuticle which are arranged in definite patterns for each species. In nematode parasites of amphibians, this type of marking appears in males of the family Cosmocercidae, specifically in the genus *Cosmocerca* as part of the rosette papillae (see below; Figs. 38-44) and in the genus Cosmocercella (Figs. 46, 48).

## **3.1.2 Cuticle of anterior end of body**

The cuticular modifications that occur in the anterior end of the body of nematode parasites of amphibians are: head papillae - cephalic papillae, externo-labial papillae, interno-labial papillae-, interlabia, deirids and amphids. These two latter structures are not modifications of the cuticle itself, but their opening in the cuticle can present diverse shape and structure.

*Head papillae*: these are tactile sensory organs usually located on the lips or labial region, including two circles of six labial papillae and one circle of four cephalic papillae. This arrangement has been proposed for ancestral nematodes considering a radial type of symmetry (De Coninck, 1965). The head papillae are divided into: *cephalic papillae*: outer

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 277

Fig. 30-37. Modifications of the cuticle in the anterior end of body. 30. *Aplectana* sp. 31. *Cosmocerca* sp. 32. *Schrankiana* sp. 33. *Rhabdias* sp. 34. *Pharyngodon* sp. 35. *Falcaustra* sp. 36. *Porrocaecum* sp. (larvae). 37. *Brevimulticaecum* sp. (larvae). am: amphids; cf: cuticular flap; lp: labial papillae; one asterisk: external labial papillae; two asterisk: internal labial papillae.

one amphid on each ventrolateral lip (Fig. 35)

**3.1.3 Cuticle of posterior end of body**

different shapes and structures.

and 1 ventral (Fig. 37).

externolateral tooth.

In the lung nematodes of genus *Rhabdias*, which have an inflated cuticle, the cephalic structures are in most cases very difficult to observe (Fig. 33). In oxyurid nematodes, eg. *Parapharyngodon*, there are three lips, each one bilobed with one small papillae (Fig. 34). In genus *Falcaustra* the mouth is surrounded by 3 large lips, each with 2 forked papillae, and

On the other hand, in nematode parasites found in larval stage, the cephalic structures of adult forms are generally not present. For example, *Porrocaecum*, a genus found in the liver of the host, has a very little developed lip anlagen (Fig. 36); *Brevimulticaecum*, found encapsulated in various organs of the hosts, presents two toothlike prominences, 1 dorsal

*Interlabia*: these are cuticular outgrowths (neoformations) originating at the base of the lips or pseudolabia and extending between them, occurring in some ascarids and spirurids. These modifications of the cuticle are present in the larval stage of genus *Physaloptera*; this genus is characterized by the presence of four teeth on the internolateral face of each pseudolabium, an internal group of three teeth, two sublateral and one lateral, and a single

*Deirids*: a pair of sensory organs found laterally in the cervical region and usually protruding above the surface of the cuticle. That may be simple or claw-like and forked, and are situated laterally in the vicinity of the nerve ring in most species (Rhabdochonidae).

*Amphids*: a pair of glandular sensory organs situated laterally in the cephalic region and opening through the cuticle; according to some authors, they are chemoreceptors. They have

In the case of nematode parasites of amphibians, the special modifications of the cuticle in the posterior end of males, which are generally associated with the copula, are particularly important. These comprise: plectanes, rosettes papillae, vesiculated rosette, papillae, bursa, phasmids, spines suckers and caudal lateral alae. Phasmids are not modifications of the cuticle such as the deirids and the amphids, but their opening onto the cuticle can present

various shapes and sizes but usually occur as two lateral pores (Figs. 30, 31).

circle of four head papillae (or latero-ventral and latero-dorsal papillae); e*xterno-labial papillae*: median circle of six head papillae, and *interno-labial papillae*: inner circle of six head papillae.

The basic structure proposed by De Coninck (1965) shows modifications in the nematodes that parasitize amphibians. For example, cosmocercid nematodes present three lips. The genus *Aplectana* has a circle of four internal labial papillae, 1 on each subventral lip and 2 on the dorsal lip, and a circle of six external labial papillae; the amphids are large, one on each subventral lip. The anterior end of the oesophagus presents three tooth-like projections covered with a thick cuticle, also called cuticular flap (Fig. 30). The genus *Cosmocerca* presents the same arrangement in the internal and external labial papillae and, as in the previous genus, the anterior end of the oesophagus bears three tooth-like projections (cuticular flap); the amphids are very prominent in some cases (Fig. 31). In the nematodes of family Atractidae, specifically in the genus *Schrankiana*, each lip has a cuticular flange overhanging the mouth opening (Fig. 32).

circle of four head papillae (or latero-ventral and latero-dorsal papillae); e*xterno-labial papillae*: median circle of six head papillae, and *interno-labial papillae*: inner circle of six head

The basic structure proposed by De Coninck (1965) shows modifications in the nematodes that parasitize amphibians. For example, cosmocercid nematodes present three lips. The genus *Aplectana* has a circle of four internal labial papillae, 1 on each subventral lip and 2 on the dorsal lip, and a circle of six external labial papillae; the amphids are large, one on each subventral lip. The anterior end of the oesophagus presents three tooth-like projections covered with a thick cuticle, also called cuticular flap (Fig. 30). The genus *Cosmocerca* presents the same arrangement in the internal and external labial papillae and, as in the previous genus, the anterior end of the oesophagus bears three tooth-like projections (cuticular flap); the amphids are very prominent in some cases (Fig. 31). In the nematodes of family Atractidae, specifically in the genus *Schrankiana*, each lip has a cuticular flange

papillae.

overhanging the mouth opening (Fig. 32).

Fig. 30-37. Modifications of the cuticle in the anterior end of body. 30. *Aplectana* sp. 31. *Cosmocerca* sp. 32. *Schrankiana* sp. 33. *Rhabdias* sp. 34. *Pharyngodon* sp. 35. *Falcaustra* sp. 36. *Porrocaecum* sp. (larvae). 37. *Brevimulticaecum* sp. (larvae). am: amphids; cf: cuticular flap; lp: labial papillae; one asterisk: external labial papillae; two asterisk: internal labial papillae.

In the lung nematodes of genus *Rhabdias*, which have an inflated cuticle, the cephalic structures are in most cases very difficult to observe (Fig. 33). In oxyurid nematodes, eg. *Parapharyngodon*, there are three lips, each one bilobed with one small papillae (Fig. 34). In genus *Falcaustra* the mouth is surrounded by 3 large lips, each with 2 forked papillae, and one amphid on each ventrolateral lip (Fig. 35)

On the other hand, in nematode parasites found in larval stage, the cephalic structures of adult forms are generally not present. For example, *Porrocaecum*, a genus found in the liver of the host, has a very little developed lip anlagen (Fig. 36); *Brevimulticaecum*, found encapsulated in various organs of the hosts, presents two toothlike prominences, 1 dorsal and 1 ventral (Fig. 37).

*Interlabia*: these are cuticular outgrowths (neoformations) originating at the base of the lips or pseudolabia and extending between them, occurring in some ascarids and spirurids. These modifications of the cuticle are present in the larval stage of genus *Physaloptera*; this genus is characterized by the presence of four teeth on the internolateral face of each pseudolabium, an internal group of three teeth, two sublateral and one lateral, and a single externolateral tooth.

*Deirids*: a pair of sensory organs found laterally in the cervical region and usually protruding above the surface of the cuticle. That may be simple or claw-like and forked, and are situated laterally in the vicinity of the nerve ring in most species (Rhabdochonidae).

*Amphids*: a pair of glandular sensory organs situated laterally in the cephalic region and opening through the cuticle; according to some authors, they are chemoreceptors. They have various shapes and sizes but usually occur as two lateral pores (Figs. 30, 31).

#### **3.1.3 Cuticle of posterior end of body**

In the case of nematode parasites of amphibians, the special modifications of the cuticle in the posterior end of males, which are generally associated with the copula, are particularly important. These comprise: plectanes, rosettes papillae, vesiculated rosette, papillae, bursa, phasmids, spines suckers and caudal lateral alae. Phasmids are not modifications of the cuticle such as the deirids and the amphids, but their opening onto the cuticle can present different shapes and structures.

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 279

Fig. 38. Plectanes and rosette papillae in nematode parasites of genus *Cosmocerca*. pl:

*Rosette papillae*: these structures consist of papillae surrounded by punctuations. This modification is present in the genus *Cosmocerca*. In this genus, the plectanes are located outside the rosette papillae and are directed toward the anterior and posterior end (Fig. 38). These rosette papillae are formed by two circles of punctuations, one internal and one external. The number of punctuations in each circle varies among species and within the same species for individuals collected from different hosts (González and Hamann, 2008; 2010b) (Figs. 38-44). In the genus *Cosmocercoides*, the caudal rosette papillae are not raised

plectanes; ip: internal circle of punctations; ep: external circle of punctations.

above the cuticular surface.

*Plectanes*: these are cross striated cuticular plates functioning as supports for the genital papillae in some males (Fig. 38). This structure is very common in the genus *Cosmocerca*, which is widely distributed in amphibians*.* In some species, these supports are very marked, as in *C. podicipinus*, in this case, the plectanes of each row are even fused to each other (Fig. 39). In other species, this support is not so developed (eg. *C. parva*); on the other hand, the same species in different hosts can present different degree of development of these structures (Figs. 40-43); indeed, in some immature specimens, the plectanes are imperceptible (Fig. 44).

These structures are arranged in two longitudinal rows on the ventral surface of the males; the number of pairs of plectanes varies among species (*C. podicipinus, C. cruzi*, *C. travassossi*: 5 pairs; *C. chilensis* and *C. rara*: 6 pairs; *C. longispicula*, *C. uruguayensis*, *C. vrcibradici*: 7 pairs). Furthermore, some species show wide intraspecific variation regarding the number of pairs of plectanes, even in the same host species (*C. parva*: 5-7 pairs; *C. brasiliensis*: 8-11 pairs; *C. paraguayensis*: 4-5 pairs) (Figs. 18, 19) (González and Hamann, 2010b).

Table 1 shows characteristics of the posterior end of males for species of *Cosmocerca* that parasitize amphibians, observed under SEM.

Table 1. Characteristics of the caudal region of males of *Cosmocerca* spp. that parasitize amphibian hosts, studied under SEM. \*Arrangement of adanal papillae: unpaired papillae anterior to anus + pairs of adanal papillae.

*Plectanes*: these are cross striated cuticular plates functioning as supports for the genital papillae in some males (Fig. 38). This structure is very common in the genus *Cosmocerca*, which is widely distributed in amphibians*.* In some species, these supports are very marked, as in *C. podicipinus*, in this case, the plectanes of each row are even fused to each other (Fig. 39). In other species, this support is not so developed (eg. *C. parva*); on the other hand, the same species in different hosts can present different degree of development of these structures (Figs.

These structures are arranged in two longitudinal rows on the ventral surface of the males; the number of pairs of plectanes varies among species (*C. podicipinus, C. cruzi*, *C. travassossi*: 5 pairs; *C. chilensis* and *C. rara*: 6 pairs; *C. longispicula*, *C. uruguayensis*, *C. vrcibradici*: 7 pairs). Furthermore, some species show wide intraspecific variation regarding the number of pairs of plectanes, even in the same host species (*C. parva*: 5-7 pairs; *C. brasiliensis*: 8-11 pairs; *C.*

Table 1 shows characteristics of the posterior end of males for species of *Cosmocerca* that

Caudal papillae

Not stablished

Not stablished

1 + 2-4 3 pairs Interior and

stablished

stablished

stablished

Table 1. Characteristics of the caudal region of males of *Cosmocerca* spp. that parasitize amphibian hosts, studied under SEM. \*Arrangement of adanal papillae: unpaired papillae

Puncatations of rosette papillae

6-7 pad-like protuberances around the posterior border

exterior rosette:

exterior rosette:

Interior rosette: 11-12; exterior rosette: 12-15

Interior rosette: 10-11; exterior rosette: 12-14

only

15

12-16

Interior rosette: 12-15; exterior rosette: 12-15

Several pairs Interior and

References

(1988)

Grabda-Kazubska and Tenora (1991)

Grabda-Kazubska and Tenora (1991)

Mordeglia and Digiani (1998)

González and Hamann (2010b)

González and Hamann (2010b)

González and Hamann (2008)

Not stablished Navarro et al.

40-43); indeed, in some immature specimens, the plectanes are imperceptible (Fig. 44).

*paraguayensis*: 4-5 pairs) (Figs. 18, 19) (González and Hamann, 2010b).

Adanal papillae\*

stablished

stablished

stablished

parasitize amphibians, observed under SEM.

Plectanes + rosette papillae

*C. ornata* 5 pairs Not

*C. ornata* 5 pairs Not

*C. commutata* 7 pairs Not

unpaired

*C. podicipinus* 5 pairs 3 pairs Not

*C. parva* 5-7 pairs 1 + 3 pairs Not

*C. parva* 4-5 pairs 1 + 3 pairs Not

anterior to anus + pairs of adanal papillae.

*C. parva* 5-7 pairs + 1

*Cosmocerca* spp.

Fig. 38. Plectanes and rosette papillae in nematode parasites of genus *Cosmocerca*. pl: plectanes; ip: internal circle of punctations; ep: external circle of punctations.

*Rosette papillae*: these structures consist of papillae surrounded by punctuations. This modification is present in the genus *Cosmocerca*. In this genus, the plectanes are located outside the rosette papillae and are directed toward the anterior and posterior end (Fig. 38).

These rosette papillae are formed by two circles of punctuations, one internal and one external. The number of punctuations in each circle varies among species and within the same species for individuals collected from different hosts (González and Hamann, 2008; 2010b) (Figs. 38-44). In the genus *Cosmocercoides*, the caudal rosette papillae are not raised above the cuticular surface.

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 281

*Vesiculated rosette*: caudal rosette papillae raised on the surface of a clear vesicle. These modifications are present in the genus *Cosmocercella*. In *C. minor*, for example, there are 4 pairs of vesiculated papillae (Fig. 45) and in *C. phyllomedusae* there is a combination of different structures; this species has small rosette papillae in the preanal subventral surface (Figs. 46, 47), rosette papillae surrounded by wide areas of cuticular punctuations (Fig. 46, 48), and large unpaired vesiculated papillae extending almost to the level of the

*Papillae*: some genera of nematode parasites, such as *Aplectana*, *Raillietnema*, *Falcaustra* or *Paraoxyascaris* do not possess conspicuous structures in the posterior end such as plectanes, rosette papillae or vesiculated papillae; these genera have simple papillae with variable number and arrangement (Figs. 49-56). These structures can be divided into caudal papillae (located in the tail, posteriorly to the anus) and cloacal papillae (surrounding the cloaca; these can be precloacal, postcloacal and adcloacal). These papillae may be sessile (*Aplectana*, *Schrankiana*, *Falcaustra*) (Figs. 49-52, 54-56) or pedunculate (*Parapharyngodon*) (Fig. 53). On the other hand, other genera such as *Cosmocercella* and *Cosmocerca* have this type of papillae in addition to plectanes, rosettes and vesiculated rosette. These papillae are commonly surrounded by one or two small rosettes of punctuations (Fig. 51). In some cases they

oesophagus.

protrude above the surface of the cuticle (Fig. 52).

Fig. 39-44. Plectanes and rosette papillae in nematode parasites of genus *Cosmocerca*. 39. *Cosmocerca podicipinus* collected in *Pseudopaludicola falcipes* with fused plectanes. 40. *Cosmocerca parva* collected in *Rhinella granulosa* with very marked plectanes. 41. 42. *Cosmocerca parva* collected in *Rhinella schneideri*. 43. *Cosmocerca parva* collected in *Leptodactylus bufonius.* 44. *Cosmocerca parva*, immature specimen, collected in *Rhinella fernandezae* with imperceptible plectanes*.* sp: somatic papillae; pl: plectanes; rs: rosette papillae; ip: internal circle of punctations; ep: external circle of punctations.

Fig. 45-48. Modifications of the cuticle in the posterior end of males of the genus *Cosmocercella.* 45. Vesiculated rosette in *C. minor.* 46. Combination of different structures in *C. phyllomedusae*. 47. Detail of small rosette papillae. 48. Detail of rosette papillae surrounded by wide areas of cuticular punctations. vr: vesiculated rosette; rp: rosette papillae; cp: cuticular punctation; rp+cp: rosette papillae surrounded by areas of cuticular punctations.

Fig. 39-44. Plectanes and rosette papillae in nematode parasites of genus *Cosmocerca*. 39. *Cosmocerca podicipinus* collected in *Pseudopaludicola falcipes* with fused plectanes. 40. *Cosmocerca parva* collected in *Rhinella granulosa* with very marked plectanes.

41. 42. *Cosmocerca parva* collected in *Rhinella schneideri*. 43. *Cosmocerca parva* collected in *Leptodactylus bufonius.* 44. *Cosmocerca parva*, immature specimen, collected in *Rhinella fernandezae* with imperceptible plectanes*.* sp: somatic papillae; pl: plectanes; rs: rosette

papillae; ip: internal circle of punctations; ep: external circle of punctations.

Fig. 45-48. Modifications of the cuticle in the posterior end of males of the genus

*Cosmocercella.* 45. Vesiculated rosette in *C. minor.* 46. Combination of different structures in *C. phyllomedusae*. 47. Detail of small rosette papillae. 48. Detail of rosette papillae surrounded by wide areas of cuticular punctations. vr: vesiculated rosette; rp: rosette papillae; cp: cuticular punctation; rp+cp: rosette papillae surrounded by areas of cuticular punctations.

*Vesiculated rosette*: caudal rosette papillae raised on the surface of a clear vesicle. These modifications are present in the genus *Cosmocercella*. In *C. minor*, for example, there are 4 pairs of vesiculated papillae (Fig. 45) and in *C. phyllomedusae* there is a combination of different structures; this species has small rosette papillae in the preanal subventral surface (Figs. 46, 47), rosette papillae surrounded by wide areas of cuticular punctuations (Fig. 46, 48), and large unpaired vesiculated papillae extending almost to the level of the oesophagus.

*Papillae*: some genera of nematode parasites, such as *Aplectana*, *Raillietnema*, *Falcaustra* or *Paraoxyascaris* do not possess conspicuous structures in the posterior end such as plectanes, rosette papillae or vesiculated papillae; these genera have simple papillae with variable number and arrangement (Figs. 49-56). These structures can be divided into caudal papillae (located in the tail, posteriorly to the anus) and cloacal papillae (surrounding the cloaca; these can be precloacal, postcloacal and adcloacal). These papillae may be sessile (*Aplectana*, *Schrankiana*, *Falcaustra*) (Figs. 49-52, 54-56) or pedunculate (*Parapharyngodon*) (Fig. 53). On the other hand, other genera such as *Cosmocercella* and *Cosmocerca* have this type of papillae in addition to plectanes, rosettes and vesiculated rosette. These papillae are commonly surrounded by one or two small rosettes of punctuations (Fig. 51). In some cases they protrude above the surface of the cuticle (Fig. 52).

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 283

muscles in this area which later becomes a sucker through modification of the cuticle. This

*Phasmids*: these are paired glandular sensory organs situated laterally in the caudal region

As in the previous case, the structures and modifications of the cuticle that are observed in

Fig. 57-59. Modifications of the cuticle in posterior end of body of nematode parasites. 57. Spines in the tail of *Skrjabinodon* sp. 58. Phasmid in *Rhabdias* sp. 59. Posterior end of

thin membrane that has no special features, as in the Cosmocercidae.

The eggs are variable in size, shape and structure; they usually have a many-layered shell with either smooth or rough, sometimes sculptured, external surface, and their poles may bear a characteristic operculum or plug. Among the nematode parasites of amphibians, eggs may present punctuations (Pharyngodonidae) (Figs. 60, 61), an operculum (*Gyrinicola*) or a

Fig. 60-61. Egg cuticle of nematode parasites of amphibians. 60. General view. 61. Detail of

modification of the cuticle is found in some species of genus *Falcaustra.*

the posterior body end of adult specimens do not occur in larvae (Fig. 59).

and opening to the surface by a slit or pore (Fig. 58).

*Physaloptera* sp. spi: spines; ph: phasmid.

**3.1.4 Eggs**

punctations.

Fig. 49-56. Papillae in the posterior end of body of males nematode parasites of amphibians. 49. 50. Arrangement of caudal papillae in *Aplectana hylambatis*. 51. Arrangement of precloacal, adcloacal and postcloacal papillae in genus *Cosmocerca*. 52. Detail of precloacal, adcloacal and postcloacal papillae in *Cosmocerca.* 53. Pedunculate papillae in tail of *Parapharyngodon*. 54-56. Sessile papillae in *Schrankiana* (54), *Falcaustra* (55) and *Cosmocerca*  (56). sp: somatic papillae; pl: plectane; pap: preanal papillae; ap: adanal papillae; cp: caudal papillae.

*Bursa*: this structure is present in nematodes of the genus *Oswaldocruzia.* This structure may be circular or oval, often divided into two symmetrical or asymmetrical lateral lobes, separated by a dorsal lobe and supported by rays or papillae. The rays of the bursa are visualized well under light microscope because they are not a part of the cuticle but embedded in the lobes.

*Caudal lateral alae*: these are sublateral or lateral longitudinal wings of the cuticle that occur on the male tail. Among nematode parasites of amphibians, they occur in the genus *Physaloptera*; however, these caudal alae develop in the adult stage, while it is typically the larval stage of *Physaloptera* that occurs as a parasite of amphibians.

*Spines*: some male and female nematode parasites, such as those of the genus *Skrjabinodon*, have cuticular spines on the tail filament (Fig. 57).

*Suckers*: this is a sucker-like pre-cloacal structure. A series of stages in sucker development occurs in some kathlaniids, indicating that there is first a concentration of copulatory muscles in this area which later becomes a sucker through modification of the cuticle. This modification of the cuticle is found in some species of genus *Falcaustra.*

*Phasmids*: these are paired glandular sensory organs situated laterally in the caudal region and opening to the surface by a slit or pore (Fig. 58).

As in the previous case, the structures and modifications of the cuticle that are observed in the posterior body end of adult specimens do not occur in larvae (Fig. 59).

Fig. 57-59. Modifications of the cuticle in posterior end of body of nematode parasites. 57. Spines in the tail of *Skrjabinodon* sp. 58. Phasmid in *Rhabdias* sp. 59. Posterior end of *Physaloptera* sp. spi: spines; ph: phasmid.

## **3.1.4 Eggs**

282 Scanning Electron Microscopy

Fig. 49-56. Papillae in the posterior end of body of males nematode parasites of amphibians.

precloacal, adcloacal and postcloacal papillae in genus *Cosmocerca*. 52. Detail of precloacal,

*Bursa*: this structure is present in nematodes of the genus *Oswaldocruzia.* This structure may be circular or oval, often divided into two symmetrical or asymmetrical lateral lobes, separated by a dorsal lobe and supported by rays or papillae. The rays of the bursa are visualized well under light microscope because they are not a part of the cuticle but

*Caudal lateral alae*: these are sublateral or lateral longitudinal wings of the cuticle that occur on the male tail. Among nematode parasites of amphibians, they occur in the genus *Physaloptera*; however, these caudal alae develop in the adult stage, while it is typically the

*Spines*: some male and female nematode parasites, such as those of the genus *Skrjabinodon*,

*Suckers*: this is a sucker-like pre-cloacal structure. A series of stages in sucker development occurs in some kathlaniids, indicating that there is first a concentration of copulatory

larval stage of *Physaloptera* that occurs as a parasite of amphibians.

have cuticular spines on the tail filament (Fig. 57).

49. 50. Arrangement of caudal papillae in *Aplectana hylambatis*. 51. Arrangement of

adcloacal and postcloacal papillae in *Cosmocerca.* 53. Pedunculate papillae in tail of *Parapharyngodon*. 54-56. Sessile papillae in *Schrankiana* (54), *Falcaustra* (55) and *Cosmocerca*  (56). sp: somatic papillae; pl: plectane; pap: preanal papillae; ap: adanal papillae; cp: caudal

papillae.

embedded in the lobes.

The eggs are variable in size, shape and structure; they usually have a many-layered shell with either smooth or rough, sometimes sculptured, external surface, and their poles may bear a characteristic operculum or plug. Among the nematode parasites of amphibians, eggs may present punctuations (Pharyngodonidae) (Figs. 60, 61), an operculum (*Gyrinicola*) or a thin membrane that has no special features, as in the Cosmocercidae.

Fig. 60-61. Egg cuticle of nematode parasites of amphibians. 60. General view. 61. Detail of punctations.

Study of Helminth Parasites of Amphibians by Scanning Electron Microscopy 285

often present in different areas of the body; there are also numerous sensory papillae, pits and ridges of various configurations (Fried, 1997; Schmidt and Roberts, 2000). The surface topography of the cirrus of digenetic trematodes also shows spine-shape protrusions and

The rim of the oral and ventral suckers in some species of digenean parasites of amphibians (both larval and adult) shows sensory papillae with variable morphology and distribution. Thus, papillae may appear as button-like structures and can be distributed as single and double papillae on the oral and ventral surfaces of the sucker (Hamann and González, 2009; Mata-López, 2006; Nadakavukaren and Nollen, 1975). In other digeneans, the oral sucker has a spongy surface with numerous pores (Whitehouse, 2002). Figures 68-70 show some characteristics of the tegument of the oral and ventral suckers of digenean trematodes found

Fig. 68-70. Tegument of suckers of digenean parasites of amphibians. 68. Papillae on oral sucker of Macroderoididae. 69. Detail of papillae on oral sucker. 70. Detail of papillae on

The tegument of digenean trematodes (larval and adults) that occur in amphibian hosts shows spines with varied morphology (e.g. scale-like spines) and variable distribution; they generally extend from the anterior end to variable levels of the posterior region (Hamann and González, 2009; Razo-Mendivil et al., 2006). In other digeneans, the surface of the tegument possesses regular ridges and interspersed protuberances (Nadakavukaren and Nollen, 1975). Figures 71-72 show the shape and distribution of spines found in the

Fig. 71-72. Tegument of the ventral surface of Macroderoididae. 71. Spines of the anterior

papillae (Bušta and Našincová, 1987).

**3.2.1 Tegument of suckers**

in Argentinean amphibians.

ventral sucker of Paramphistomatidae.

**3.2.2 Tegument of the ventral surface**

tegument of some digenean parasites of Argentinean amphibian.

third of the body. 72. Spines of the posterior third of body.

## **3.1.5 Vulva**