José F. Maya V. Biologist, Los Andes University (ULA). Animal Physiology Group, School of Sciences, ULA. e-mail: mayajf@ula.ve

Ernesto Valiente. Biologist, University of Bucarest, Rumania. Structural and Chemical Analysis of Material Laboratory, ULA. Address: Facultad de Ciencias, ULA. Mérida 5101. Venezuela. e-mail: valienteer@latinmail.com

James O’Callaghan. Biologist, ULA. Ph.Sc. Venezuelan Institute for Scientific Research. Animal Physiology Group, School of Sciences, ULA. e-mail: jocall2002@hotmail.com

Using a modification of the standard protocol of sample processing for Scanning Electron Microscopy (SEM), we report ultrastructural characteristics of the ootheca of Periplaneta americana, an oval and semi-cylindrical structure, which protects each batch of eggs that is laid in order to ensure the final stage of organogenesis of these insects. It can be characterized geometrically as an oval case made up of two valves with convex outer surfaces and concave inner surfaces, a suture line that surrounds the valves, a zipper-like toothed rail structure (crest), adjacent segmentations that are perpendicular to the crest, teeth consisting of pre- and post-dental elements, transverse crevices, channel-like inner structures, an internal cavity and internal loci. Detailed observations lead to conclude that the crest is a dynamic zipper-like structure that allows exchange with the outside environment through internal longitudinal apertures. An updated model of the inner transversal section of the ootheca is proposed.

Con una modificación en el protocolo estándar de preparación de muestras para Microscopía Electrónica de Barrido (SEM) se reportan características ultraestructurales de la ooteca de Periplaneta americana, estructura semicilíndrica y ovalada que protege cada lote de huevos ovipuesto, para asegurar la fase final de organogénesis de estos insectos. Ésta puede ser caracterizada geométricamente como una cápsula ovalada constituida por dos valvas con superficies convexas externas y superficies internas cóncavas, una línea de sutura que rodea las valvas, una estructura dentada tipo cremallera (cresta), segmentaciones contiguas perpendiculares a la cresta, dientes constituidos por elementos pre- y post-dentales, ranuras transversales, estructuras internas tipo canal, una cavidad interna y loci internos. Observaciones detalladas permiten concluir que la cresta es una estructura dinámica, en forma de cremallera, que permite el intercambio con el ambiente externo a través de aperturas longitudinales internas. Se propone un modelo actualizado de la sección transversal interna de la ooteca.

Com uma modificação no protocolo patrão de preparação de amostras para Microscópio Eletrônico de Varrido (SEM) foram reportadas características ultra-estruturais da ooteca de Periplaneta americana, estrutura semicilíndrica e ovalada que protege cada lote de ovos oviposto, para assegurar a fase final de organogênese destes insetos. Esta pode ser caracterizada geometricamente como uma cápsula ovalada constituída por duas valvas com superfícies convexas externas e superfícies internas côncavas, uma linha de sutura que rodeia as valvas, uma estrutura dentada tipo cremalheira (cresta), segmentações contíguas perpendiculares à cresta, dentes constituídos por elementos pré- e pós-dentais, ranhuras transversais, estruturas internas tipo canal, uma cavidade interna e loci internos. As observações detalhadas permitiram concluir que a cresta é uma estrutura dinâmica, em forma de cremalheira, que permite o intercâmbio com o ambiente externo através de aberturas longitudinais internas. Propõe-se um modelo atualizado da seção transversal interna da ooteca.

Received: 11/19/2001. Modified: 03/07/2002. Accepted: 03/19/2002

The structure and development of Periplaneta americana has long been a subject of extensive study. This species has been used as a tool to improve knowledge about the morphophysiology of insects. However, limited research has been reported using scanning electron microscopy (SEM) techniques (Maya et al., 2000). It could be helpful to correlate existing data with information obtained with this technique.

The ootheca of P. americana is an oval and semi-cylindrical structure formed by the sexual accessory glands (colleterial glands), which are made up of right- and left-hand components. It has been reported that each gland consists of a mass of branched tubules lying freely in the haemocoel and each has its own opening into the genital vestibulum, an invagination of the posterior end of the abdomen concerned with the bringing together of eggs, stored sperms and other materials used to complete the formation of the ootheca and its contents. The formation of the ootheca and the structure and function of the left collaterial gland has been described by Brunet (1952). Females of the American cockroach lay their eggs one week after mating and at the peak of her reproductive period they are able to form about two oothecae per week (Bell and Adiyodi, 1981). The females, produce, on average, one egg case about once a month for ten months. The female deposits the ootheca near a source of food by either simply dropping it or gluing it to a surface with a secretion from its mouth. The egg case is brown when deposited and turns black in a day or two. A typical egg case contains about 14 to 16 eggs (Bell and Adiyodi, 1981).

During animal development the egg, once fertilized, follows common development phases that can be summarized as segmentation, gastrulation and organogenesis (Smith and Wood, 1997). Other authors use the terms growth, differentiation and morphogenesis for these same processes (Curtis and Barnes, 1994). The term embryogenesis can be used to generalize all three phases stated above, but it is important to point out that the development process of a new being is not unique. There are different modalities according to each animal group (Watson et al., 1987; Curtis and Barnes, 1994). Previous SEM work on embryogenesis of vertebrates and invertebrates refers to phases between egg fertilization and the first segmentations, much before the gastrulation process occurs (Calarco and Epstein, 1973; Beam and Kessel, 1976; Turner and Mahowald, 1976).

This paper studies an aspect of morphogenesis of P. Americana during its final stage, and reports SEM ultrastructural details of the ootheca, the structure in which this final stage of organogenesis occurs.

P. americana specimens were cultured at 28°C. Pairs (male/female) of insects were selected and placed in a glass chamber under optimum conditions to guarantee mating in order to obtain oothecae. Sex selection was carried out according to Snodgrass (1935).

Oothecae were processed using a modification of the standard protocol of sample preparation for SEM observation (see Protocols for Scanning Electron Microscopy). The modification consisted in the use of Formaldehyde and Chloral Hydrate, as follows: fixation was achieved by immersion in a 3% Formaldehyde solution prepared in phosphate buffer pH 6.3 for 1h at 4°C. After fixation samples were washed three times at 5 min intervals in the same phosphate buffer. Post-fixation was carried out by immersion in a 3% Chloral Hydrate solution prepared in the same buffer for 0.5h at 4°C. Thereafter, the samples were washed 3 times in the phosphate buffer and dehydrated with increasingly concentrated ethanol, for 5 min at each stage. Drying was performed by placing the samples in a vacuum chamber for approximately 24h. The dried samples (oothecae) were then Silver covered using ionic deposition at 18mA and 0.3 millibars for 90 sec. Observations were made with a Hitachi S-2500 SEM.

Figure 2. Another view of the structures in Figure 1. A: adjacent segmentations; B: convex external surfaces; C: crest; E: remains of cellulosic material. Scale: 1.50mm.

The ootheca can be geometrically described as an oval and semi-cylindrical structure made up of two valves of semi-rough convex external surfaces with squashed endings. There is a suture line in the contact zone of the valves (Figures 1 and 2). In some micrographs, remains of cellulose material can be seen. Dental elements formed by bilobulated endings are present throughout the above mentioned suture line, separated by a zone called neck. We refer to these bilobulated endings, as pre- and post-dental elements. The whole dental structures, laying one behind the other throughout the suture line, form a zipper-like toothed rail structure known as crest, which has open and closed areas (Figures 3 and 4; Maya et al., 2000). The necks of the dental elements located in the open area have an average length of 220µm, while in the closed area it is of 260µm. There are adjacent segmentations with an average width of 730µm, which are perpendicular to the crest (Figures 1 and 2).

Figure 3. Zipper-like toothed rail structure of the crest (C), showing an open area (R) and a closed area (T). E: remains of cellulosic material. Scale: 400µm. Reproduced from Maya et al. (2000).

Figure 4. Closed area of the crest (T) showing pre-dental element (1), neck (2), post-dental element (3) and thin transverse crevices (4). E: remains of cellulosic material. Scale: 200µm.

Figure 5. Detail showing pre-dental element (1), neck (2) and post-dental elements (3), as well as the thin transverse crevice (4) located between the two elements. E: remains of cellulosic material. Scale: 75µm.

Figure 6. Different view of the details in Figure 5. Scale: 120µm.

A very thin transverse crevice is present in the contact region of dental structures which are located on the closed area of the crest ( Figures 4, 5 and 6) and in the open area there is a longitudinal aperture between these dental elements with an average width of 10µm (Figure 7).

Concave (inner) surfaces of valves

These surfaces are made up of adjacent loci with an average width of 685µm. Each one is delimited by remains of external corion which are easily removed from the eggs (Figure 8). There is a great diversity of geometric structures in the external corion surface. Among these hexagonal and pentagonal forms are the most frequently observed (Figure 9).

Figure 8. The concave inner surface of valves (P), made up of adjacent loci (M) delimited by remains of external corion (N). C: crest. Scale: 1.50mm.

Figure 9. Diversity of geometric structures in the external corion surface, including pentagonal (h), hexagonal (g), and heptagonal (i) shapes. Scale: 50µm.

These concave surfaces show the inner side of the crest in which there are channel-like structures and an inner cavity with an average length of 100µm, both within a distance of 500µm between these loci and the crest (Figure 10).

Figure 10. Concave surface at the inner side of the crest (C), showing channel like structures (Z) and an inner cavity (X) between the adjacent loci (M) and the crest. Scale: 400µm.

Among the new ultrastructural details of the ootheca of P. americana reported herein are the average width of adjacent segmentations on outer surfaces (730µm), the average length of the neck in each dental element located on the open (220µm) and closed areas (260µm), the average width of the longitudinal aperture between dental structures (10µm), the separation distance between the crest and loci (500µm), the average width of adjacent segmentations on inner surfaces (685µm), the average length of the inner cavity (100µm) and the presence of channel-like structures on the inner side of the crest, laying within the separation distance between loci and the crest.

There is a relation of approximately 1:1 between the average width of adjacent segmentations on the outer surfaces and the average width of loci located on the inner surfaces, indicating that each segmentation corresponds to a specific loci on the inner surface, which are occupied by nymphs during the final stage of morphogenesis and are delimited by an external corion (Figure 8). The diversity of geometric structures observed on the external corion surface (Figure 9) could be related to its flexibility, which would allow a mass increase of nymphs that are completing their morphogenesis stage, as it has been reported by Valiente et al. (2001). Such reasoning could explain a dynamic mechanism by which the external corion surrounds the eggs in the inner side of the ootheca, such as mentioned for Aedeomya squamipennis by Petersen and Linley (1995).

Channel-like structures observed on inner surfaces of the valves might reflect the origin of longitudinal apertures on the open area of the crest. These apertures extend to the inner cavity, with an average width of 100µm between the loci and the crest, and the cavity is in immediate contact with the space occupied by nymphs (Figure 10). Therefore, the exchange between the inside and the outside of the ootheca could occur by a diffusion process through the external corion surrounding the eggs, in agreement with Wiglessworth and Beament (1960).

An updated model of the inner transversal section of the ootheca of P. americana is proposed, including new reports on this subject (Figure 11), which added to previous observations might contribute to have a better understanding of the morphophysiology of the ootheca of this species.

Figure 11. Inner transversal section of the ootheca.

Finally, micrographs show that the modification in experimental conditions used in the present work does not turn vulnerable the biological material employed. The presence of cellulosic material in some micrographs was a consequence of the oviposition process taking place on a paper surface.

The authors thank Jorge Fernández for his assistance in SEM techniques, Sócrates Pérez for the processing of the micrographs, Ramón Díaz for the design of the diagram, and Ian Bratt for help in translation.