Characteristics of the caudal region of males of
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
However, helminths are the most common invertebrate parasites of amphibians. One well known example among trematodes is the monogenean genus
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. 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 Nollen (1975) who provided details of the tegument of the trematodes
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 microscope (Fig. 2). 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.
The analized amphibian species were:
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 gold-palladium 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-palladium.
In this study we basically followed the aforementioned procedure, with some modifications. Helminths were observed
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 formaldehyde-glutaraldehyde 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. 3. 5-9).
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 microscopy (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?
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.
There are several types of gross cuticular markings, namely,
Striations can be distributed uniformly along the whole body of the parasite (
In some parasitic nematodes, the cervical alae is modified as a
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 (
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.
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
In the lung nematodes of genus Rhabdias, which have an inflated cuticle, the cephalic structures are in most cases very difficult to observe (Fig. 9. 33). In oxyurid nematodes, eg.
On the other hand, in nematode parasites found in larval stage, the cephalic structures of adult forms are generally not present. For example,
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
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.
These structures are arranged in two longitudinal rows on the ventral surface of the males; the number of pairs of plectanes varies among species (
Table 1 shows characteristics of the posterior end of males for species of
|Cosmocerca spp.||Plectanes + rosette papillae||Adanal|
|Puncatations of rosette papillae||References|
|C. ornata||5 pairs||Not stablished||Not stablished||Not stablished||Navarro et al. (1988)|
|C. ornata||5 pairs||Not stablished||Not stablished||6-7 pad-like protuberances around the posterior border only||Grabda-Kazubska and Tenora (1991)|
|C. commutata||7 pairs||Not stablished||Several pairs||Interior and exterior rosette: 15||Grabda-Kazubska and Tenora (1991)|
|C. parva||5-7 pairs + 1 unpaired||1 + 2-4||3 pairs||Interior and exterior rosette: 12-16||Mordeglia and Digiani (1998)|
|C. podicipinus||5 pairs||3 pairs||Not stablished||Interior rosette: 11-12; exterior rosette: 12-15||González and Hamann (2010b)|
|C. parva||5-7 pairs||1 + 3 pairs||Not stablished||Interior rosette: 10-11; exterior rosette: 12-14||González and Hamann (2010b)|
|C. parva||4-5 pairs||1 + 3 pairs||Not stablished||Interior|
rosette: 12-15; exterior
|González and Hamann (2008)|
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) (Fig. 10.; Fig. 11. 44). In the genus Cosmocercoides, the caudal rosette papillae are not raised above the cuticular surface.
Phasmids: these are paired glandular sensory organs situated laterally in the caudal region and opening to the surface by a slit or pore (Fig. 14. 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. 14. 59).
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) (Fig. 15. 60, 61), an operculum (
The area of the body inmediately anterior and posterior to the vulvar opening is called vulvar region. In most females of nematode parasites of amphibians, this may be simply an opening transversal to the longitudinal body axis without any special striation, as in the genus
Nematodes usually have two spicules; each one is essentially a tube covered by a sclerotized cuticle and containing a central protoplasmic core. In terms of the taxonomy of nematode parasites of amphibians, the importance of the spicules lies in their morphology and size, and not in the presence of ornamentation on the cuticle of these structures. In this case the spicules can be studied with SEM only when they are outside the individual, i.e., when protruding from the cloaca (Figs. 17. 65-67).
The tegument of trematodes is syncytial and consists of a tegumental outer membrane (trilaminate), a matrix (with discoid bodies, membranous bodies and usually mitochondria) and a basal tegumental membrane. The tegument is variously interrupted by cytoplasmatic projections of gland cells and by openings of excretory pores. The tegumental surface often contains ornamentations such as spines between the outer and basal membranes; these are 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 papillae (Bušta and Našincová, 1987).
3.2.1. Tegument of suckers
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). Figure 17 (68-70) show some characteristics of the tegument of the oral and ventral suckers of digenean trematodes found in Argentinean amphibians.
3.2.2. Tegument of the ventral surface
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). Figure 19. (71-72) show the shape and distribution of spines found in the tegument of some digenean parasites of Argentinean amphibian.
The body surface of acanthocephalans has 5 layers. The outermost layer is the epicuticle, followed by the cuticle which is composed mainly of lipoproteins; the third layer has a homogeneous nature; the fourth layer possesses fibrous bands besides mitochondria, bladders and lacunar channels, and the fifth layer contains scarce fibres but larger and more abundant lacunar channels than in the previous layer (Olsen, 1974).
Regarding this group of helminth parasites, most SEM studies are focused on the hooks that they possess in the proboscis, as well as their body spines. Likewise, they present sensory structures with diverse ornamentations in the posterior part of the bursa.
In our study we found larval stages belonging to the genus
4. Helminth parasites of Argentinean amphibian studied with SEM: Synthesis and new contributions. Research perspectives
Up to the present, studies performed with scanning electron microscopy techniques on helminth parasites of Argentinean amphibians have included three families of nematodes: Rhabdiasidae,
New contributions presented in this work include, for the Class Nematoda, the families Molineoidae (
Future research in this topic should focus on extending the geographical areas studied while at the same time, expanding the examination to other possible amphibian hosts.
Reports about helminth parasites of Argentinean amphibians studied under SEM refer mainly to specimens collected in host from the Northeast region, specifically Corrientes province, and the Northwest region, with only one record for Salta province so; thus, there is still a vast portion of the Argentinean territory that has not yet been studied (Fig. 21.).
Lavilla et al. (2000) reported a total of 271 amphibian species for Argentina (167 anurans and 4 gymnophions); of these, only 32 (11.8%) have been cited as hosts for helminth parasites (González and Hamann, 2004, 2005, 2006a, 2006b, 2007a, 2007b, 2008, 2009, 2010a, 2010b, 2011; Hamann and Pérez, 1999; Hamann and González, 2009; Hamann et al., 2006a, 2006b, 2009a, 2009b, 2010; Lajmanovich and Martinez de Ferrato, 1995; Lunaschi and Drago, 2007; Ramallo et al., 2007a, 2007b, 2008). Of all the anuran families, the most studied for helminth parasites are Hylidae, Bufonidae, Leiuperidae and Leptodactylidae. Nine species of hylids have been studied for helminth parasites, but only one of these studies included SEM: Scinax nasicus (González and Hamann, 2008; Hamann and González, 2009); similarly, seven species of bufonids have been studied for helminth parasites, but SEM was employed in only two cases:
The SEM study of the tegument of helminth species (e.g. morphology of spines) collected in different localities could detect possible intraspecific variation related to geographical location; this phenomenon has been highly documented in both trematode (Grabda-Kazubska and Combes, 1981; Kennedy 1980a) and nematode (Chitwood, 1957) parasites of amphibians. Similarly, variations related to occurrence in a wide range of phylogenetically unrelated hosts, i.e. the cases of generalist helminths could detect possible intraspecific variation related with the host age or diet, previous exposure to the parasite, presence of another parasite and number of specimens present (Chitwood, 1957; Haley, 1962; Kennedy, 1980b; Watertor, 1967).
5. Importance of the use of scanning electron microscope for the study of helminth parasites of amphibians
In helminth parasites, all morphological aspects must be studied under light microscope, because these structures are very important in the context of their systematic classification. Some examples of these traits include, in the case of parasitic nematodes, the type of esophagus (oxyuroid, rhabditoid, strongyloid), presence of ventriculus and its shape, and the caecum and its shape; in the females, the arrangement of ovaries (prodelphic, amphidelphic or opisthodelphic), number of uteri (monodelphic or didelphic), structure of the ovoyector and the vagina and, in the males, the structure and measurements of the gubernaculum. In the case of trematodes, the distribution of vitelline follicles, the position of the testes and ovary, size of the eggs, position of oral and ventral suckers, reproductive structures, among others, are characteristics of taxonomic importance. Finally, regarding the internal anatomy of acanthocephalans, some particularly relevant structures are the proboscis receptacle, lemnisci, retractor muscle, testis, seminal vesicle, cement gland, Saefftigen's pouch, etc. Thus, the scanning electron microscope represents an additional tool for the study of this group of organisms. The importance of SEM lies in its ability to provide three-dimensional images with high magnification that allow understanding the spatial relationships among surface structures. It could be used to separate species that appear morphologically identical when examined under light microscope, validate species and demonstrate differences between populations or races (Gibbons, 1986; Hirschmann, 1983).
We thank Secretaría General de Ciencia y Técnica of Universidad Nacional del Nordeste, Corrientes, Argentina, for supporting partially this work.
We are grateful Dr. Graciela T. Navone, Dr. Julia I. Díaz, Dr. María del Rosario Robles at the Centro de Estudios Parasitológicos y de Vectores, La Plata, Argentina, Licentiate Rodrigo Cajade at Centro de Ecología Aplicada del Litoral, Corrientes, Argentina, Dr. Lorena Sereno at Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Brazil, Dr. Viviane Gularte Tavares dos Santos at Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Brazil, for helping with literature search.
We are grateful Dr. Marta I. Duré and Dr. Eduardo F. Schaefer at Centro de Ecología Aplicada del Litoral for the photographs of the host and for helping with the edition of the map.
We thank to Graphic Designer Cecilia Rios Encina for help in photograph edition.
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