Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

O'Callaghan, James Study of ultrastructural details of the ootheca of periplaneta americana (dictyoptera: blattidae) using scanning electron microscopy Interciencia, vol. 27, núm. 5, mayo, 2002, pp. 243-246 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative STUDY OF ULTRASTRUCTURAL DETAILS OF THE OOTHECA OF Periplaneta americana (DICTYOPTERA: BLATTIDAE) USING SCANNING ELECTRON MICROSCOPY

José F. Maya V., Ernesto Valiente and James O’Callaghan

SUMMARY

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

RESUMEN

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

Introduction The ootheca of P. americana formation of the ootheca and of food by either simply drop- is an oval and semi-cylindrical its contents. The formation of ping it or gluing it to a sur- The structure and develop- structure formed by the sexual the ootheca and the structure face with a secretion from its ment of Periplaneta ameri- accessory glands (colleterial and function of the left mouth. The case is brown cana has long been a subject glands), which are made up of collaterial gland has been de- when deposited and turns of extensive study. This spe- right- and left-hand compo- scribed by Brunet (1952). Fe- black in a day or two. A typi- cies has been used as a tool nents. It has been reported that males of the American cock- cal egg case contains about 14 to improve knowledge about each gland consists of a mass roach lay their eggs one week to 16 eggs (Bell and Adiyodi, the morphophysiology of in- of branched tubules lying after mating and at the peak 1981). sects. However, limited re- freely in the haemocoel and of her reproductive period they During animal development search has been reported us- each has its own opening into are able to form about two the egg, once fertilized, fol- ing scanning electron micros- the genital vestibulum, an in- oothecae per week (Bell and lows common development copy (SEM) techniques (Ma- vagination of the posterior end Adiyodi, 1981). The females, phases that can be summa- ya et al., 2000). It could be of the abdomen concerned produce, on average, one egg rized as segmentation, gastru- helpful to correlate existing with the bringing together of case about once a month for lation and organogenesis data with information ob- eggs, stored sperms and other ten months. The female depos- (Smith and Wood, 1997). tained with this technique. materials used to complete the its the ootheca near a source Other authors use the terms

KEYWORDS / Morphophysiology / Ootheca / Periplaneta americana / Scanning Electron Microscopy / Received: 11/19/2001. Modified: 03/07/2002. Accepted: 03/19/2002

José F. Maya V. Biologist, Los Ernesto Valiente. Biologist, Uni- de Ciencias, ULA. Mérida James O’Callaghan. Biologist, Andes University (ULA). Ani- versity of Bucarest, Rumania. 5101. Venezuela. ULA. Ph.Sc. Venezuelan Insti- mal Physiology Group, School Structural and Chemical e-mail: [email protected] tute for Scientific Research. of Sciences, ULA. Analysis of Material Labora- Animal Physiology Group, e-mail: [email protected] tory, ULA. Address: Facultad School of Sciences, ULA. e-mail: [email protected]

MAY 2002, VOL. 27 Nº 5 0378-1844/02/05/243-04 $ 3.00/0 243 RESUMO

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

the use of Formaldehyde and ionic deposition at 18mA and Chloral Hydrate, as follows: 0.3 millibars for 90 sec. Ob- fixation was achieved by im- servations were made with a mersion in a 3% Formalde- Hitachi S-2500 SEM. hyde solution prepared in phosphate buffer pH 6.3 for Results 1h at 4°C. After fixation samples were washed three Convex (outer) surfaces times at 5 min intervals in the of valves Figure 1. Mosaic assembly of the ootheca of Periplaneta Americana. same phosphate buffer. Post- A: adjacent segmentations; B: convex external surfaces; C: crest; D: fixation was carried out by The ootheca can be geo- suture line; E: remains of cellulosic material. Scale: 1.5mm. immersion in a 3% Chloral metrically described as an Hydrate solution prepared in oval and semi-cylindrical growth, differentiation and chamber under optimum con- the same buffer for 0.5h at structure made up of two morphogenesis for these same ditions to guarantee mating in 4°C. Thereafter, the samples valves of semi-rough convex processes (Curtis and Barnes, order to obtain oothecae. Sex were washed 3 times in the external surfaces with 1994). The term embryogen- selection was carried out ac- phosphate buffer and dehy- squashed endings. There is a esis can be used to generalize cording to Snodgrass (1935). drated with increasingly con- suture line in the contact zone all three phases stated above, Oothecae were processed centrated ethanol, for 5 min of the valves (Figures 1 and but it is important to point using a modification of the at each stage. Drying was 2). In some micrographs, re- out that the development pro- standard protocol of sample performed by placing the mains of cellulose material cess of a new being is not preparation for SEM observa- samples in a vacuum chamber can be seen. Dental elements unique. There are different tion (see Protocols for Scan- for approximately 24h. The formed by bilobulated endings modalities according to each ning Electron Microscopy). dried samples (oothecae) were are present throughout the animal group (Watson et al., The modification consisted in then Silver covered using above mentioned suture line, 1987; Curtis and Barnes, 1994). Previous SEM work on embryogenesis of verte- brates and invertebrates refers to phases between egg fertili- zation and the first segmenta- tions, much before the gastru- lation process occurs (Calarco and Epstein, 1973; Beam and Kessel, 1976; Turner and Mahowald, 1976). This paper studies an aspect of morphogenesis of P. Ameri- cana during its final stage, and reports SEM ultrastructural de- tails of the ootheca, the struc- ture in which this final stage of organogenesis occurs.

Materials and Methods

P. americana specimens Figure 2. Another view of the structures in Fig- Figure 3. Zipper-like toothed rail structure of the were cultured at 28°C. Pairs ure 1. A: adjacent segmentations; B: convex ex- crest (C), showing an open area (R) and a closed (male/female) of insects were ternal surfaces; C: crest; E: remains of cellulosic area (T). E: remains of cellulosic material. Scale: selected and placed in a glass material. Scale: 1.50mm. 400µm. Reproduced from Maya et al. (2000).

244 MAY 2002, VOL. 27 Nº 5 Figure 4. Closed area of the crest (T) show- Figure 5. Detail showing pre-dental element Figure 6. Different view of the details in Fig- ing pre-dental element (1), neck (2), post- (1), neck (2) and post-dental elements (3), as ure 5. Scale: 120µm. dental element (3) and thin transverse crev- well as the thin transverse crevice (4) located ices (4). E: remains of cellulosic material. between the two elements. E: remains of cel- Scale: 200µm. lulosic material. Scale: 75µm. separated by a zone called the open area have an average (Figures 4, 5 and 6) and in one is delimited by remains neck. We refer to these length of 220µm, while in the the open area there is a lon- of external corion which are bilobulated endings, as pre- closed area it is of 260µm. gitudinal aperture between easily removed from the eggs and post-dental elements. The There are adjacent segmenta- these dental elements with an (Figure 8). There is a great whole dental structures, lay- tions with an average width average width of 10µm (Fig- diversity of geometric struc- ing one behind the other of 730µm, which are perpen- ure 7). tures in the external corion throughout the suture line, dicular to the crest (Figures 1 surface. Among these hexago- form a zipper-like toothed rail and 2). Concave (inner) surfaces nal and pentagonal forms are structure known as crest, A very thin transverse of valves the most frequently observed which has open and closed crevice is present in the con- (Figure 9). areas (Figures 3 and 4; Maya tact region of dental struc- These surfaces are made up These concave surfaces et al., 2000). The necks of tures which are located on of adjacent loci with an aver- show the inner side of the the dental elements located in the closed area of the crest age width of 685µm. Each crest in which there are chan-

Figure 7. Open area of the crest (R) showing Figure 8. The concave inner surface of valves Figure 9. Diversity of geometric structures in a longitudinal aperture (5) between pairs of (P), made up of adjacent loci (M) delimited the external corion surface, including pen- dental structures (6). E: remains of cellulosic by remains of external corion (N). C: crest. tagonal (h), hexagonal (g), and heptagonal (i) material. Scale: 150µm. Reproduced from Scale: 1.50mm. shapes. Scale: 50µm. Maya et al. (2000).

MAY 2002, VOL. 27 Nº 5 245 each segmentation corresponds to a specific loci on the inner surface, which are occu- pied by nymphs during the final stage of morpho- genesis and are delimited by an external corion (Figure 8). The diversity of geo- metric structures observed on the external corion surface (Figure 9) Figure 11. Inner transversal section of the ootheca. could be related to its flexibility, which would al- Figure 10. Concave surface at the inner side low a mass in- Finally, micrographs show Maya JF, Valiente E, O’Callaghan J (2000) Ultrastructural study of of the crest (C), showing channel like struc- crease of nymphs that the modification in ex- tures (Z) and an inner cavity (X) between the the crest in the ootheca of Pe- that are complet- perimental conditions used in riplaneta americana using a adjacent loci (M) and the crest. Scale: the present work does not scanning electron microscope. 400µm. ing their morpho- genesis stage, as turn vulnerable the biological Rev. Ecol. Lat. Am. 7: 11-16. material employed. The pres- it has been re- Petersen J, Linley J (1995) De- nel-like structures and an in- ported by Valiente et al. ence of cellulosic material in scription of the egg of Aedeo- ner cavity with an average (2001). Such reasoning could some micrographs was a con- myia squamipennis (Diptera: length of 100µm, both within explain a dynamic mechanism sequence of the oviposition Culicidae). J. Medical Ento- a distance of 500µm between by which the external corion process taking place on a pa- mol. 32: 888-894. per surface. these loci and the crest (Fig- surrounds the eggs in the in- Protocols For Scanning Electron ure 10). ner side of the ootheca, such Microscopy (SEM) (http:// as mentioned for Aedeomya ACKNOWLEDGMENTS imf.bio. swt.edu/cellbiology/ SEMstuff/SEMtechs.htm). pp. Discussion squamipennis by Petersen and 1-6. Linley (1995). The authors thank Jorge Among the new ultrastruc- Channel-like structures ob- Fernández for his assistance Smith C, Wood E (1997) Diferen- tural details of the ootheca of served on inner surfaces of in SEM techniques, Sócrates ciación y Desarrollo en P. americana reported herein the valves might reflect the Pérez for the processing of Biología Molecular. 1st Edi- the micrographs, Ramón Díaz tion. Addison-Wesley Iberoa- are the average width of adja- origin of longitudinal aper- mericana. Wilmington, Dela- cent segmentations on outer tures on the open area of the for the design of the diagram, ware. Chapter 12. pp. 324-325. surfaces (730µm), the average crest. These apertures extend and Ian Bratt for help in length of the neck in each to the inner cavity, with an translation. Snodgrass RE (1935) Principles of Morphology. McGraw- dental element located on the average width of 100µm be- Hill. USA. pp. 227-236. open (220µm) and closed ar- tween the loci and the crest, REFERENCES eas (260µm), the average and the cavity is in immedi- Turner F, Mahowald A (1976) width of the longitudinal ap- ate contact with the space Beam H, Kessel R (1976) Cytoken- Scanning Electron Microscopy esis: A Comparative Study of of Drosophila Embryogenesis. erture between dental struc- occupied by nymphs (Figure Cytoplasmic Divisions in Ani- Developmental Biol. 50: 95- tures (10µm), the separation 10). Therefore, the exchange mal Cells. American Scientist 108. distance between the crest between the inside and the 54: 279-290. and loci (500µm), the average outside of the ootheca could Valiente E, Morales D, O´Cal- Bell WJ, Adiyodi KG (1981) The laghan J, Albornoz E, Zamora width of adjacent segmenta- occur by a diffusion process American . 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Sci. 93: 47-69. within the separation distance the ootheca of P. americana Calarco P, Epstein, C (1973) Cell Watson JA, Hopkins N, Roberts J, between loci and the crest. is proposed, including new Surface Changes During Pre- Steitz J, Wiener A (1987) Mo- lecular Biology of Gene. 4th There is a relation of ap- reports on this subject (Fig- implatation Development in the Mouse. Developmental Biology Edition. The Benjamin/Cum- proximately 1:1 between the ure 11), which added to pre- 32: 208-213. mings Publishing Co. Califor- average width of adjacent vious observations might nia. pp. 785-789. segmentations on the outer contribute to have a better Curtis H, Barnes N (1994) Bio- logía. 5th Edition. Editorial Wiglessworth VB, Beament JW surfaces and the average understanding of the morpho- Médica Hispanoamericana. (1960) The Principles of Insect width of loci located on the physiology of the ootheca of Buenos Aires. Chapter 45. pp. Physiology. 7th Edition. 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