- Email: [email protected]
Ooxtaxonomy of eight Tettigonoidea species (Insecta: Orthoptera), description and comparison of the egg morphology
Accepted Manuscript Title: Ooxtaxonomy of eight Tettigonoidea species (Insecta: Orthoptera), description and comparison of the egg morphology Authors: Marta Franch Sas, Josep M. Olmo-Vidal, Marcos Roca-Cusachs, Juli Pujade-Villar PII: DOI: Reference:
S0968-4328(17)30046-X http://dx.doi.org/doi:10.1016/j.micron.2017.05.003 JMIC 2435
To appear in:
Micron
Received date: Revised date: Accepted date:
15-2-2017 11-5-2017 12-5-2017
Please cite this article as: Sas, Marta Franch, Olmo-Vidal, Josep M., RocaCusachs, Marcos, Pujade-Villar, Juli, Ooxtaxonomy of eight Tettigonoidea species (Insecta: Orthoptera), description and comparison of the egg morphology.Micron http://dx.doi.org/10.1016/j.micron.2017.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
OOXTAXONOMY OF EIGHT TETTIGONOIDEA SPECIES (INSECTA: ORTHOPTERA), DESCRIPTION AND COMPARISON OF THE EGG MORPHOLOGY Marta Franch Sas(a), Josep Mª Olmo-Vidal(b), Marcos Roca-Cusachs(a) & Juli Pujade-Villar(a,c) (a)
Department of Evolutionary Biology, Ecology and Environmental Sciences. Faculty of Biology, University of Barcelona. Avda. Diagoinal, 645. 08028-Barcelona, Catalunya. A/e: [email protected]; [email protected]; [email protected] (b) Servei de fauna y flora, Departament de Política Territorial i Sostenibilitat, Generalitat de Catalunya, Dr, Roux, 80, 08017 Barcelona, Catalunya. A/e: [email protected] (c) Corresponding author. [email protected]
Highlights
Make a morphological description of the eggs of the studies species and find the micropillar area. Use ootaxonomy as a tool to distinguish and group taxa.
ABSTRACT In the present study, eggs of eight species of the Tettigonoidea superfamily have been examined with the aim to find characteristic traits of each of the studied taxa. In addition, we aim to distinguish them through their eggshell morphology, a technique that nowadays is known as ootaxonomy. All the eggs analysed belong to four subfamilies of the Tettigoniidae family (Bradyporinae: Ephippiger diurnus cunii, Parasteropleurus perezii, Lluciapomaresius panteli, L. ortegai, Tettigoniinae: Decticus verrucivorus, Antaxius hispanicus, and Meconematinae: Cyrtaspis scutata) and from family Phaneropteridae: Phaneroptera nana. Observations and comparisons were made after optical and Scanning Electron Microscope (SEM) photographs. In all katydids evaluated, we observed that hexagonal cells usually compose the chorion, nevertheless, in some cases egg surfaces appearance is follicular or smooth. Micropylar areas are different among the species examined. Ootaxonomy allowed us to differentiate between the genera studied and the two more related species: Lluciapomaresius panteli and L. ortegai. Key words: Tettigonoidea, ootaxonomy, micropylar area. 1. INTRODUCTION Chorionic morphology of insect eggs provides in many taxa important information for their systematics and identification (Weber et al., 2003), even helping to discriminate between cryptic species (Vera Sánchez, 2014). It has also been used to help
with the placement of the species in their correct subfamily (Rentz et al., 2007) and in phylogenetic studies (Saenger & Helfert, 1994). In this particular case, chorion structures and micropylar area are useful for drawing systematic schemes (Mazzini, 1976, 1980) and the external egg morphology gives specific characteristics for each species (Çakici & Ergen, 2012). According to Tuck (1939) several authors (Uvarov, 1928; Bushland, 1934) have suggested that orthopteran eggs can be and are differentiated by chorionic sculptures. Orthopteran eggs are structured in three layers, from the most inner layer to the most external one, the layers are: vitelline membrane, endochorion and exochorion, all of them secreted by follicular cells through successive cycles of secretion (Gwynne, 2001). Follicular cells shape and draw more or less complex sketches on the surface of the eggs (Mazzini, 1987). Thus, the surface of tettigonid eggs can show a great variety of external morphologies, which range from simple surfaces without any drawing to surfaces with complex structures (Vera Sánchez, 2014). Furthermore, eggs can have divergent shapes: from extended, thin and cylindrical to short, thick and discoidal (Hartley, 1964). Generally, their surfaces display a blurred polygonal pattern and on the centre of each polygon there is a hole that communicates with the spongy layer under the chorion (Mazzini, 1976, 1987). However, this hole is not the aerophyl, which in Tettigonioidea superfamily is found on specialized zones on both egg poles (Mazzini, 1987). Egg and ovipositor shape normally reflect the oviposition habits (Gwynne, 2001). Tettigoniidae females lay their eggs in different substrates: on the ground, in plant stems, in bark fissures or in mosses. Eggs are placed, one by one, without shaping an ootheca (Gwynne, 2001; Tuck, 1939). The egg coloration ranges in a gradient from beige-brown to yellow. The aim of this study is to study for the first time by electron microscopy, the exochorion morphology of eight species of the Tettigonoidea superfamily [Antaxius hispanicus (Bolívar, 1887), Cyrtaspis scutata (Charpentier, 1825), Decticus verrucivorus (Linnaeus, 1758), Ephippiger ephippiger (Fiebig 1784), Lluciapomaresius panteli (Navás, 1899), L. ortegai (Pantel, 1896), Parasteropleurus perezii (Bolívar, 1877) and Phaneroptera nana (Fieber, 1853)] and to discuss genus and species differences (in Lluciapomaresius), and to provide new data about the Tettigonoidea’s ootaxonomy. 2. MATERIAL AND METHODS Most of the examined eggs were obtained from ovipositions of individuals in captivity. Individuals were collected as adults and kept in terrariums. The terrariums were constructed trying to simulate their environmental conditions, filling them with soil and plant species from the locality where the specimens were collected. In the particular case of Lluciapomaresius panteli, apart from taking the eggs laid in the terrarium, eggs were also taken “in situ” after observations of the oviposition in their natural environment and inspecting the moss from the area where the specimens had laid the eggs. The eggs obtained were fixed in 96% ethanol and processed following three different methodologies before taking photographs by an electron microscopy: (i) one egg of each species was cleaned via ultrasound during 30-60 seconds in alcoholic solution and then placed horizontally on a stub, (ii) one egg of the species
Lluciapomaresius panteli, Lluciapomaresius ortegai and Parasteropleurus perezii were immersed in hexamethyldisilazane (HMDS) during five minutes before placing the eggs horizontally on the stubs, and (iii) the same as the previous procedure, but then they were cleaned with ultrasound during 30-60 seconds in alcoholic solution and were placed vertically on the stubs. All samples were covered with a 30-40 nm layer of gold using a Sputter Jeol JFC-1100. SEM images were taken with a Quanta 200 (Fei, Co) microscope of 10-20 kV voltage. The measurements given refer normally to the arithmetic mean of the length and width of the eggs. In samples n > 1 the value given is the exact measured value. Three cells where measured in all eggs in order to obtain the mean cell diameter. 3. RESULTS 3.1. Ephippiger diurnus cunii (Bolívar, 1877) One individual captured at Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona). n = 1: Maximum length and width of egg: 5.0 mm x 1.4 mm. Mean cell’s diameter: 28.36±1.49 µm. Long and fusiform. Egg poles are different: one of the ends is more pointed than the other (Fig. 1). Hexagonal and pentagonal follicular cells form a uniform pattern throughout the area of the egg (Fig. 2-3). The surface pattern is not uniform at the poles and in the micropylar area (Figs. 4-6). The micropylar area is made up of two rose-like shaped subsets of pores (Figs. 4-6). 3.2. Parasteropleurus perezii (Bolívar, 1877) Captured individuals from Parc Natural de la Serra de Montsant (Priorat, Tarragona), from Parc Natural dels Ports (Terres de l’Ebre, Tarragona) and from Coll d'Estenalles (Sant Llorenç del Munt, Mura, Barcelona). Size for n = 3: Mean length and width of egg: 5.13±0.07 mm x 1.6±0.20 mm. Mean cell’s diameter: 34.78±4.35 µm. Long and symmetric (not symmetric sensu Hartley). The egg poles are different: one is more rounded than the other (Fig. 7). Hexagonal follicular cells throughout the area (Fig. 9). Depending on the condition of the egg, their follicular cells can be more protrusive (Fig. 8). The surface pattern is not uniform at the poles and in the micropylar area (Fig. 10-12). The micropylar area is formed by 10 small protrusive tubes. 3.3. Lluciapomaresius panteli (Navás, 1899) Captured individuals from Parc Natural de la Serra de Montsant (Priorat, Tarragona). Size for n = 3: Mean length and width of egg: 4.91±0.17 mm x 1.24±0.14 mm. Mean cell’s diameter: 34.33±3.95 µm. Long egg. The egg poles are different: one pole is more pointed than the other (Fig. 13). Follicular cells form hexagons that cover all the surface of the egg (Figs. 1415). Big intracellular spaces. The pattern is only broken in the poles and in the micropylar area (Fig. 16). The micropylar area is formed by 8-10 rose-like shaped subsets with a centered micropyl (Figs. 16-18).
3.4. Lluciapomaresius ortegai (Pantel, 1896) Captured individuals from Parc Natural dels Ports (Terres de l’Ebre, Tarragona). Size for n = 3: Mean length and width of egg: 4.51±0.52 mm x 1.4 2±0.04 mm. Mean cell’s diameter: 33.33±3.41 µm. Asymmetric egg. The egg poles are different: one is oval while the other is pointed (Fig. 19). Follicular cells are hexagonal across the whole egg surface (Figs. 2021) but not in contact with one another Intracellular spaces. The pattern is not uniform in the poles and in the micropylar area. The micropylar area is formed by an ensemble of groups of pores very close together and two roses more separated from this grouping (Figs. 22-24). 3.5. Phaneroptera nana (Fieber, 1853) Captured individuals from Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona). n = 1: Maximum length and width of egg: 3.47 mm x 1.47 mm. Cell are absent. Oval-shaped egg (Fig. 25). Smooth surface. Follicular cells do not form any pattern (Figs. 26-27). Poles and micropylar area are not smooth. The micropylar area is located at the side and enters the egg and it enters the egg surface (Fig. 28). Micropylar area made up by six short tubes that arise from the surface (Figs. 29-30). 3.6. Decticus verrucivorus (Linnaeus, 1758) Captured individuals from Pirineu (Catalunya). n = 1: Maximum length and width of egg: 5.13 mm x 1.47 mm. Mean diameter of cells: 25.81±5.59 µm. Long and symmetric egg (Fig. 31). Follicular cells at the surface are superficial and have rounded walls and margins (Figs. 32-33). Pattern not uniform in the poles. Micropylar area found exclusively in one of the poles (Fig. 34). Micropylar area formed by three groups of three circles each group. Each group has at least one micropyl inside one of the circles (Figs. 35-36). 3.7. Antaxius hispanicus (Bolívar, 1887) Captured individuals from Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona). n = 1: Maximum length and width of egg: 4.33 mm x 1.27 mm. Mean cell’s diameter: 20.97±1.61 µm. Symmetric and long egg (Fig. 37). The egg poles are different; one is more rounded than the other. Micropyles are found in a wart-like protrusion looking near one of the poles. Surface of follicular cells polyhedral (rhomboids, pentagonal and hexagonal) with sides not clearly defined across the complete surface (Figs. 38-40). This pattern is broken in the egg poles. 3.8. Cyrtaspis scutata (Charpentier, 1825) Captured individuals from Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona).
n = 1: Maximum length and width of egg: 2.80 mm x 0.93 mm. Mean cell’s diameter: 29.51±1.64 µm. Symmetric and long egg (Fig. 41). The egg poles are different: one is more pointed than the other. Follicular cells slightly marked (Fig. 43), with irregular rhomboid cells that cover the complete surface (Fig. 42). Poles have a different pattern. Micropylar area is found on the side of the egg and is composed by two cone-shaped tubes. 4. DISCUSSION Concerning the methodology used in the sample processing, we have observed that dirt (filaments of plant or animal origin dust and sand) on the egg surface is detached after the eggs are cleaned by ultrasound. This method helps to make structures better visible. Different placements of the eggs on the stab were used, and it was observed that vertical placement is the better way to inspect the eggs external morphology, as long as the micropylar area is not at the tip of the egg pole. The micropylar area is usually located near one of the poles but not at the tip. In case that the micropylar area is located at the bottom or top pole, eggs shall be placed laterally on the stab. Vertical placement allowed us to analyse all sides of the egg and top pole. Exploration of the external morphology using SEM has allowed us to identify similar patterns. In species of which several eggs have been studied, no visible interspecific morphological variation was observed; in our study, egg morphology appears to be stable within species. The taxonomy of the studied groups is shown below according to Zhou et al. (2017) and the main characteristics of the eggs species are summarized in Table 1. Superfamily Tettigonoidea Krauss, 1902 Family Phaner opteridae Burmeister, 1838 Tribe Phaneropterini Burmeister, 1838 Phaneroptera nana (Fieber, 1853) Family Tettigoniidae Krauss, 1902 Subfamily Bradyporinae Burmeister, 1838 Tribe Ephippigerini Brunner von Wattenwyl, 1878 Ephippiger diurnus cunii (Bolívar, 1877) Lluciapomaresius panteli (Navás, 1899) Lluciapomaresius ortegai (Pantel, 1896) Parasteropleurus perezzi (Bolívar, 1877) Subfamily Meconematinae Burmeister, 1838 Tribe Meconematini Burmeister, 1838 Cyrtaspis scutata (Charpentier, 1825) Subfamily Tettigoniinae Krauss, 1902 Tribe Decticini Herman, 1874 Decticus verrucivorus (Linnaeus, 1758) Tribe Platycleidini Brunner von Wattenwyl, 1893 Antaxius hispanicus (Bolívar, 1887) The observed traits allow us to highlight similarities and differences in the external egg morphology of the studied species. The tribe Ephippigerini has a clear tendency to have hexagonal cells on the egg surface. As observed, follicular cells of Parasteropleurus perezi, Lluciapomaresius
panteli and Lluciapomaresius ortegai (Figs. 9, 15, 21 respectively) have a similar external appearance and form the same pattern across their surface. Ephippiger diurnus cunii is the taxon with the most different egg morphology within this tribe. Ootaxonomy (Mazzini, 1987) has allowed us to detect similarities and distinguish between the eggs of the studied genera Parasteropleurus and Lluciapomaresius. In addition, this technique has allowed us to separate the species L. ortegai and L. panteli, providing additional distinctive traits for their morphological characterization. The micropylar area and the separation among chorion cells are diagnostic for the genera of the tribe Ephippigerini studied here: cells of Parasteropleurus are more close to each other (Fig. 9), whereas in Lluciapomaresius (Figs. 15, 21) the distance between the cells is larger. Furthermore, and taking in consideration the low amount of replicas, differences between the intercellular spaces among the species of Lluciapomaresius show that there is no intraspecific variation, while cells are smaller in L. ortegai than in L. panteli. Concerning the micropylar area, in Parasteropleurus it is made of six tubes while in Lluciapomaresius it is made by a with a more or less protrusive, variable rose-shaped micropyl with no tube structure. The species of the genus Lluciapomaresius can be differentiated by the micropylar area: in L. panteli it is composed by a group of 8-10 rose-like shaped subsets (Figs. 16-18) while in L. ortegai it is composed by an ensemble of a group of pores very close together and two roses more separated from this grouping (Figs. 22-24). The egg of Phaneroptera nana is the only egg that shows an oval and flattened shape with a completely smooth surface. The family Phaneropteridae, where Phaneroptera belongs to, is characterized by showing this ellipsoidal shape as Bey-Bienko (1954) and Mazzini (1976) pointed out. Another peculiarity observed is the carina at the border of the ventral and dorsal egg regions, which has also been found in the phanaeropterinae species Poecilimon cervus Karabag, 1950 (Yilmaz et al., 2012), and it surrounds the whole egg except the micropylar area at the posterior pole. If we follow Mazzini’s (1976) description, the micropylar area is located at a close distance from the anterior pole of the egg but if we follow Yilmaz et al. (2012), the micropylar area must be located at the posterior pole. These observations also match with Hartley (1964), who says that the micropylar area is located at the posterior pole. So, according to our study the posterior pole of the egg of Phaneroptera nana has a more pointed shape with some pointed protusions on the chorion. These protusions are more pronounced and packed than at the anterior pole, which has a rounded shape and the chorion has a more relaxed pattern. Decticus verrucivorus has a micropylar area composed by three groups of three circles each, with at least one micropyl in one of the circles. Morphologically, the micropylar area is different from the rest of the chorion surface and it matches the description made by Mazzini (1976), which says: it can be isolated or in groups of three, it is found at a 0.8 mm distance from the anterior pole. In this case, the position of the anterior and posterior poles respectively is known. Contrary to our results, Mazzini (1976) suggested that for each micopillar area, 4 to 8 micropyls can be found. Furthermore, we have found that the micropylar area in the species Antaxius hispanicus is found in a protrusion. The micropylar area has never been found at the terminal poles of the egg, instead, it has been found close to one of the egg poles, but never at the pole itself. This result matches with the representative scheme of the eggs of some orthopteran families made by Mazzini (1987). Some authors in previous studies
(Metz & Monroy, 1966; Mazzini, 1987; Çakici & Ergen, 2012) mention that the micropylar area is located either at the anterior pole or in the ventral region of the egg. However, in British Tettigoniidae (Hartley, 1964) the micropylar area was found at the ventral side near the posterior pole of the egg. Localizing the micropylar area in the eggs would permit to easily identify the position of the egg poles (Vera Sánchez, 2014; Hartley 1964), and therefore this should be checked carefully as their localization varies depending on each taxonomic group. The fact of not finding the micropylar areas in some studied eggs (mostly at the beginning of this study) is probably due to the egg mounting technique used. If the egg is placed horizontally, some parts may remain hidden. For future observations, it is better to place the eggs vertically in the stab, as an overview of all sides of the egg can be observed. In some cases in which the micropylar area is not found, this is not due to a mounting error, but due to the fact that sometimes there are no micropyls as Çakici & Ergen (2012) reported on an unfertilized egg of Gryllus bimaculatus (Orthoptera: Gryllidae). 5. ACKNOWLEDGEMENTS We would like to thank all the people that helped in this project (collectors of the individuals, egg treatment procedures, execution and analysis of the images and with the revisions of the article). We are also very thankful with Joan Barat for his final text commentaries and corrections that improved this manuscript as well as all his support provided during the project. Last but not least, we would like to give our special thanks to all the institutions: Parc del Montsant, Parc dels Ports, the Department of Territory and Sostenibility and to the UB Servei Cientifico-Tècnic for all the help provided. In addition the Authors would like to thank gratefully the anonymous reviewers for all the comments that improved greatly the final version of the manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. 6. REFERENCES Bey-Bienko, G.Ya. (1954) Orthoptera Vol. II. No. 2. Tettigonioidea Phaneropterinae, Fauna of the U.S.S.R. Zoological Institute Akademii Nauk SSSR, 381 pp. [English translation 1965 Jerusalem (Israel Program for Scientific Translations)] Bushland, R. C. 1934. A study of the sculpture of the chorion of the eggs of eighteen South Dakota grasshoppers (Acridiidae). Unpublished thesis. South Dakota State College of Agriculture and Mechanic Arts. 31 p. Çakici, Ö. & Ergen, G. 2012. External egg morphology of Melanogryllus desertus (Pallas, 1771) (Orthoptera: Gryllidae). Biharean Biol., 6: 122–125. Gwynne, D. T. 2001. Katydids and bush-crickets. Reproductive behaviour and evolution of the Tettigoniidae. Cornell University Press. Ithaca. New York. 319 pp. Hartley, J. C. 1964. The structure of the eggs of the British Tettigoniidae (Orthoptera). Proceedings of the Royal Entomological Society of London Series A General Entomology, 39: 111-117. Mazzini, M. 1976. Sula fine struttura del micropilo negli insetti. IV. Le sculture corionidae come mezzo di identificazione della uova degli ortotteri Tettigoniodei. Redia, 59: 109–134.
Mazzini, M. 1980. Chorion sculptures and amino acid composition of orthopteran eggs. In ‘16th International Congress of Entomology, Kyoto. p.186. Mazzini, M. 1987. An overview of egg structure in Orthopteroid insects. B. M. Baccetti (Ed). Ellis Horwood Ltd. Chichester, Englan.pdf. Evol. Biol. Orthopteroid Insects. Vol. II Vo: 358–372. Metz, C.B. & Monroy, A. 1966. Biology Of Fertilization V1: Model Systems And Oogenesis. METZ, C.B. and MONROY, A. (Eds.). 390 pp. Academic Press, New York. 99–116. Tuck, J. B. 1939. The identification of the eggs of grasshoppers by means of the chorionic sculpturing. MS thesis. Kansas State College. Rentz D.C.F., Su, Y.N. & Ueshima, N. 2007 Studies in Australian Tettigoniidae. A new genus of listroscelidine katydids from northern Australia orthoptera: Tettigoniidae; Listroscelidinae). Transactions of the American Entomological Society (Philadelphia) 133(3-4): 279-296. Saenger, K. & Helfert, B. 1994 Comparative studies on number and position of the micropyles and the shape of the eggs of Saga pedo, S. natoliae and S. ephippigera (Orthoptera: Tettigoniidae). Entomologia Generalis, 19(1-2): 4956. Uvarov, B. P. 1928. Locusts and grasshoppers. London. Imperial Bureau of Entomology. 273 p. Vera Sánchez, A. 2014. Estudio comparativo de la morfología de huevos en Tettigoniinae (Insecta: Orthoptera) de Chile, mediante una aproximación con SEM. Gayana (Concepc.), 78: 144–151 Webber, B L, Rentz, D C F, Ueshima, N, Woodrow, I E 2003 Leucopodoptera eumundii, a new genus and species of katydid from the tropical rainforests of north Queensland, Australia (Orthoptera: Tettigoniidae: Phaneropterinae: Holochlorini). Journal of Orthoptera Research, 12(1) 2003: 79-88. Yilmaz, I., Suludere, Z. & Candan, S. 2012. Structure of the egg of Poecilimon cervus Karabag (Orthoptera: Tettigoniidae) and ultrastructural features. Turkiye Entomoloji Dergisi, 36: 549-556. Zhou, Z., Zhao, L., Liu, N., Guo, H., Guan, B., Di, J. & Shi, F. 2017. Towards a higherlevel Ensifera phylogeny inferred from mitogenome sequences. Molecular Phylogenetics and Evolution, 108:22-33. .
LEGENDS
FIGURE 1-6 Eggs from Ephippiger diurnus cunii: 1 General view, 2 surface general view, 3 follicular cells, 4 micropylar area general view (AM), 5 detail of the first micropylar rose-shaped (RS), 6 detail of the second micropylar roseshaped (RS).
FIGURE 7-12 Eggs from Parasteropleurus perezii: 7 General view, 8 surface general view, 9 hexagonal follicular cells, 10 micropylar area general view (AM), 1112 detail of the miropyles (M).
FIGURE 13-18 Eggs from Lluciapomaresius panteli: 13 General view, 14 surface general view, 15 follicular cells, 16 micropylar area general view (AM), 17 micropylar area zoom view, 18 detail of two micropylar rose-shaped (RS).
FIGURE 19-24 Eggs from Lluciapomaresius ortegai: 19 General view, 20 surface general view, 21 follicular cells, 22 micropylar area general view (AM), 23 micropylar area zoom view, 24 detail of a piece of the micropylar area.
FIGURE 25-30 Eggs from Phaneroptera nana: 25 General view, 26 surface general view, 27 follicular cells, 28 micropylar area general view (AM), 29 micropylar area zoom view with the micropyl, 30 detail of the micropyl.
FIGURE 31-36 Eggs from Decticus verrucivorus: 31 General view, 32 surface general view, 33 follicular cells, 34 AM: micropylar area (AM), 35 micropylar area zoom view, 36 micropyl (M).
FIGURE 37-40 Eggs from Antaxius hispanicus: 37 General view, 38 surface general view, 39 follicular cells, 40 micropylar warts. FIGURE 41-46 Eggs from Cyrtaspis scutata: 41 General view, 42 surface general view, 43 follicular cells, 44 micropylar area (AM), 45 micropylar area general view, 46 micropyles (M).
TABLE 1. Studied species with some of the traits that characterize the surface of the eggs. Species
Micropillar area shape
Egg surface
Ephippiger ephippiger
Two rose-like shaped structures
Fused hexagonal and pentagonal cells
Parasteropleurus perezii
Tubes
Hexagonal cells with irregular walls
Lluciapomaresius panteli
8-10 rose-like shaped subsets (here only roselike pictured)
Separataed hexagonal cells
Lluciapomaresius ortegai
Big structure plus two roselike shaped subsets
Hexagonal cells
Decticus verrucivorus
3 groups of 3 circles each one
Shallow follicular cells
In a warts-like shape
Polyhedral follicular cells (rhomboid, pentagonal and hexagonal)
Antaxius hispanicus
Phaneroptera sp.
Cyrtaspis sp.
Six tubes
Smooth surface
3-4 Tubes
Follicular cells with rhomboid shape and irregular walls
S0968-4328(17)30046-X http://dx.doi.org/doi:10.1016/j.micron.2017.05.003 JMIC 2435
To appear in:
Micron
Received date: Revised date: Accepted date:
15-2-2017 11-5-2017 12-5-2017
Please cite this article as: Sas, Marta Franch, Olmo-Vidal, Josep M., RocaCusachs, Marcos, Pujade-Villar, Juli, Ooxtaxonomy of eight Tettigonoidea species (Insecta: Orthoptera), description and comparison of the egg morphology.Micron http://dx.doi.org/10.1016/j.micron.2017.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
OOXTAXONOMY OF EIGHT TETTIGONOIDEA SPECIES (INSECTA: ORTHOPTERA), DESCRIPTION AND COMPARISON OF THE EGG MORPHOLOGY Marta Franch Sas(a), Josep Mª Olmo-Vidal(b), Marcos Roca-Cusachs(a) & Juli Pujade-Villar(a,c) (a)
Department of Evolutionary Biology, Ecology and Environmental Sciences. Faculty of Biology, University of Barcelona. Avda. Diagoinal, 645. 08028-Barcelona, Catalunya. A/e: [email protected]; [email protected]; [email protected] (b) Servei de fauna y flora, Departament de Política Territorial i Sostenibilitat, Generalitat de Catalunya, Dr, Roux, 80, 08017 Barcelona, Catalunya. A/e: [email protected] (c) Corresponding author. [email protected]
Highlights
Make a morphological description of the eggs of the studies species and find the micropillar area. Use ootaxonomy as a tool to distinguish and group taxa.
ABSTRACT In the present study, eggs of eight species of the Tettigonoidea superfamily have been examined with the aim to find characteristic traits of each of the studied taxa. In addition, we aim to distinguish them through their eggshell morphology, a technique that nowadays is known as ootaxonomy. All the eggs analysed belong to four subfamilies of the Tettigoniidae family (Bradyporinae: Ephippiger diurnus cunii, Parasteropleurus perezii, Lluciapomaresius panteli, L. ortegai, Tettigoniinae: Decticus verrucivorus, Antaxius hispanicus, and Meconematinae: Cyrtaspis scutata) and from family Phaneropteridae: Phaneroptera nana. Observations and comparisons were made after optical and Scanning Electron Microscope (SEM) photographs. In all katydids evaluated, we observed that hexagonal cells usually compose the chorion, nevertheless, in some cases egg surfaces appearance is follicular or smooth. Micropylar areas are different among the species examined. Ootaxonomy allowed us to differentiate between the genera studied and the two more related species: Lluciapomaresius panteli and L. ortegai. Key words: Tettigonoidea, ootaxonomy, micropylar area. 1. INTRODUCTION Chorionic morphology of insect eggs provides in many taxa important information for their systematics and identification (Weber et al., 2003), even helping to discriminate between cryptic species (Vera Sánchez, 2014). It has also been used to help
with the placement of the species in their correct subfamily (Rentz et al., 2007) and in phylogenetic studies (Saenger & Helfert, 1994). In this particular case, chorion structures and micropylar area are useful for drawing systematic schemes (Mazzini, 1976, 1980) and the external egg morphology gives specific characteristics for each species (Çakici & Ergen, 2012). According to Tuck (1939) several authors (Uvarov, 1928; Bushland, 1934) have suggested that orthopteran eggs can be and are differentiated by chorionic sculptures. Orthopteran eggs are structured in three layers, from the most inner layer to the most external one, the layers are: vitelline membrane, endochorion and exochorion, all of them secreted by follicular cells through successive cycles of secretion (Gwynne, 2001). Follicular cells shape and draw more or less complex sketches on the surface of the eggs (Mazzini, 1987). Thus, the surface of tettigonid eggs can show a great variety of external morphologies, which range from simple surfaces without any drawing to surfaces with complex structures (Vera Sánchez, 2014). Furthermore, eggs can have divergent shapes: from extended, thin and cylindrical to short, thick and discoidal (Hartley, 1964). Generally, their surfaces display a blurred polygonal pattern and on the centre of each polygon there is a hole that communicates with the spongy layer under the chorion (Mazzini, 1976, 1987). However, this hole is not the aerophyl, which in Tettigonioidea superfamily is found on specialized zones on both egg poles (Mazzini, 1987). Egg and ovipositor shape normally reflect the oviposition habits (Gwynne, 2001). Tettigoniidae females lay their eggs in different substrates: on the ground, in plant stems, in bark fissures or in mosses. Eggs are placed, one by one, without shaping an ootheca (Gwynne, 2001; Tuck, 1939). The egg coloration ranges in a gradient from beige-brown to yellow. The aim of this study is to study for the first time by electron microscopy, the exochorion morphology of eight species of the Tettigonoidea superfamily [Antaxius hispanicus (Bolívar, 1887), Cyrtaspis scutata (Charpentier, 1825), Decticus verrucivorus (Linnaeus, 1758), Ephippiger ephippiger (Fiebig 1784), Lluciapomaresius panteli (Navás, 1899), L. ortegai (Pantel, 1896), Parasteropleurus perezii (Bolívar, 1877) and Phaneroptera nana (Fieber, 1853)] and to discuss genus and species differences (in Lluciapomaresius), and to provide new data about the Tettigonoidea’s ootaxonomy. 2. MATERIAL AND METHODS Most of the examined eggs were obtained from ovipositions of individuals in captivity. Individuals were collected as adults and kept in terrariums. The terrariums were constructed trying to simulate their environmental conditions, filling them with soil and plant species from the locality where the specimens were collected. In the particular case of Lluciapomaresius panteli, apart from taking the eggs laid in the terrarium, eggs were also taken “in situ” after observations of the oviposition in their natural environment and inspecting the moss from the area where the specimens had laid the eggs. The eggs obtained were fixed in 96% ethanol and processed following three different methodologies before taking photographs by an electron microscopy: (i) one egg of each species was cleaned via ultrasound during 30-60 seconds in alcoholic solution and then placed horizontally on a stub, (ii) one egg of the species
Lluciapomaresius panteli, Lluciapomaresius ortegai and Parasteropleurus perezii were immersed in hexamethyldisilazane (HMDS) during five minutes before placing the eggs horizontally on the stubs, and (iii) the same as the previous procedure, but then they were cleaned with ultrasound during 30-60 seconds in alcoholic solution and were placed vertically on the stubs. All samples were covered with a 30-40 nm layer of gold using a Sputter Jeol JFC-1100. SEM images were taken with a Quanta 200 (Fei, Co) microscope of 10-20 kV voltage. The measurements given refer normally to the arithmetic mean of the length and width of the eggs. In samples n > 1 the value given is the exact measured value. Three cells where measured in all eggs in order to obtain the mean cell diameter. 3. RESULTS 3.1. Ephippiger diurnus cunii (Bolívar, 1877) One individual captured at Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona). n = 1: Maximum length and width of egg: 5.0 mm x 1.4 mm. Mean cell’s diameter: 28.36±1.49 µm. Long and fusiform. Egg poles are different: one of the ends is more pointed than the other (Fig. 1). Hexagonal and pentagonal follicular cells form a uniform pattern throughout the area of the egg (Fig. 2-3). The surface pattern is not uniform at the poles and in the micropylar area (Figs. 4-6). The micropylar area is made up of two rose-like shaped subsets of pores (Figs. 4-6). 3.2. Parasteropleurus perezii (Bolívar, 1877) Captured individuals from Parc Natural de la Serra de Montsant (Priorat, Tarragona), from Parc Natural dels Ports (Terres de l’Ebre, Tarragona) and from Coll d'Estenalles (Sant Llorenç del Munt, Mura, Barcelona). Size for n = 3: Mean length and width of egg: 5.13±0.07 mm x 1.6±0.20 mm. Mean cell’s diameter: 34.78±4.35 µm. Long and symmetric (not symmetric sensu Hartley). The egg poles are different: one is more rounded than the other (Fig. 7). Hexagonal follicular cells throughout the area (Fig. 9). Depending on the condition of the egg, their follicular cells can be more protrusive (Fig. 8). The surface pattern is not uniform at the poles and in the micropylar area (Fig. 10-12). The micropylar area is formed by 10 small protrusive tubes. 3.3. Lluciapomaresius panteli (Navás, 1899) Captured individuals from Parc Natural de la Serra de Montsant (Priorat, Tarragona). Size for n = 3: Mean length and width of egg: 4.91±0.17 mm x 1.24±0.14 mm. Mean cell’s diameter: 34.33±3.95 µm. Long egg. The egg poles are different: one pole is more pointed than the other (Fig. 13). Follicular cells form hexagons that cover all the surface of the egg (Figs. 1415). Big intracellular spaces. The pattern is only broken in the poles and in the micropylar area (Fig. 16). The micropylar area is formed by 8-10 rose-like shaped subsets with a centered micropyl (Figs. 16-18).
3.4. Lluciapomaresius ortegai (Pantel, 1896) Captured individuals from Parc Natural dels Ports (Terres de l’Ebre, Tarragona). Size for n = 3: Mean length and width of egg: 4.51±0.52 mm x 1.4 2±0.04 mm. Mean cell’s diameter: 33.33±3.41 µm. Asymmetric egg. The egg poles are different: one is oval while the other is pointed (Fig. 19). Follicular cells are hexagonal across the whole egg surface (Figs. 2021) but not in contact with one another Intracellular spaces. The pattern is not uniform in the poles and in the micropylar area. The micropylar area is formed by an ensemble of groups of pores very close together and two roses more separated from this grouping (Figs. 22-24). 3.5. Phaneroptera nana (Fieber, 1853) Captured individuals from Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona). n = 1: Maximum length and width of egg: 3.47 mm x 1.47 mm. Cell are absent. Oval-shaped egg (Fig. 25). Smooth surface. Follicular cells do not form any pattern (Figs. 26-27). Poles and micropylar area are not smooth. The micropylar area is located at the side and enters the egg and it enters the egg surface (Fig. 28). Micropylar area made up by six short tubes that arise from the surface (Figs. 29-30). 3.6. Decticus verrucivorus (Linnaeus, 1758) Captured individuals from Pirineu (Catalunya). n = 1: Maximum length and width of egg: 5.13 mm x 1.47 mm. Mean diameter of cells: 25.81±5.59 µm. Long and symmetric egg (Fig. 31). Follicular cells at the surface are superficial and have rounded walls and margins (Figs. 32-33). Pattern not uniform in the poles. Micropylar area found exclusively in one of the poles (Fig. 34). Micropylar area formed by three groups of three circles each group. Each group has at least one micropyl inside one of the circles (Figs. 35-36). 3.7. Antaxius hispanicus (Bolívar, 1887) Captured individuals from Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona). n = 1: Maximum length and width of egg: 4.33 mm x 1.27 mm. Mean cell’s diameter: 20.97±1.61 µm. Symmetric and long egg (Fig. 37). The egg poles are different; one is more rounded than the other. Micropyles are found in a wart-like protrusion looking near one of the poles. Surface of follicular cells polyhedral (rhomboids, pentagonal and hexagonal) with sides not clearly defined across the complete surface (Figs. 38-40). This pattern is broken in the egg poles. 3.8. Cyrtaspis scutata (Charpentier, 1825) Captured individuals from Coll d’Estenalles (Sant Llorenç del Munt, Mura, Barcelona).
n = 1: Maximum length and width of egg: 2.80 mm x 0.93 mm. Mean cell’s diameter: 29.51±1.64 µm. Symmetric and long egg (Fig. 41). The egg poles are different: one is more pointed than the other. Follicular cells slightly marked (Fig. 43), with irregular rhomboid cells that cover the complete surface (Fig. 42). Poles have a different pattern. Micropylar area is found on the side of the egg and is composed by two cone-shaped tubes. 4. DISCUSSION Concerning the methodology used in the sample processing, we have observed that dirt (filaments of plant or animal origin dust and sand) on the egg surface is detached after the eggs are cleaned by ultrasound. This method helps to make structures better visible. Different placements of the eggs on the stab were used, and it was observed that vertical placement is the better way to inspect the eggs external morphology, as long as the micropylar area is not at the tip of the egg pole. The micropylar area is usually located near one of the poles but not at the tip. In case that the micropylar area is located at the bottom or top pole, eggs shall be placed laterally on the stab. Vertical placement allowed us to analyse all sides of the egg and top pole. Exploration of the external morphology using SEM has allowed us to identify similar patterns. In species of which several eggs have been studied, no visible interspecific morphological variation was observed; in our study, egg morphology appears to be stable within species. The taxonomy of the studied groups is shown below according to Zhou et al. (2017) and the main characteristics of the eggs species are summarized in Table 1. Superfamily Tettigonoidea Krauss, 1902 Family Phaner opteridae Burmeister, 1838 Tribe Phaneropterini Burmeister, 1838 Phaneroptera nana (Fieber, 1853) Family Tettigoniidae Krauss, 1902 Subfamily Bradyporinae Burmeister, 1838 Tribe Ephippigerini Brunner von Wattenwyl, 1878 Ephippiger diurnus cunii (Bolívar, 1877) Lluciapomaresius panteli (Navás, 1899) Lluciapomaresius ortegai (Pantel, 1896) Parasteropleurus perezzi (Bolívar, 1877) Subfamily Meconematinae Burmeister, 1838 Tribe Meconematini Burmeister, 1838 Cyrtaspis scutata (Charpentier, 1825) Subfamily Tettigoniinae Krauss, 1902 Tribe Decticini Herman, 1874 Decticus verrucivorus (Linnaeus, 1758) Tribe Platycleidini Brunner von Wattenwyl, 1893 Antaxius hispanicus (Bolívar, 1887) The observed traits allow us to highlight similarities and differences in the external egg morphology of the studied species. The tribe Ephippigerini has a clear tendency to have hexagonal cells on the egg surface. As observed, follicular cells of Parasteropleurus perezi, Lluciapomaresius
panteli and Lluciapomaresius ortegai (Figs. 9, 15, 21 respectively) have a similar external appearance and form the same pattern across their surface. Ephippiger diurnus cunii is the taxon with the most different egg morphology within this tribe. Ootaxonomy (Mazzini, 1987) has allowed us to detect similarities and distinguish between the eggs of the studied genera Parasteropleurus and Lluciapomaresius. In addition, this technique has allowed us to separate the species L. ortegai and L. panteli, providing additional distinctive traits for their morphological characterization. The micropylar area and the separation among chorion cells are diagnostic for the genera of the tribe Ephippigerini studied here: cells of Parasteropleurus are more close to each other (Fig. 9), whereas in Lluciapomaresius (Figs. 15, 21) the distance between the cells is larger. Furthermore, and taking in consideration the low amount of replicas, differences between the intercellular spaces among the species of Lluciapomaresius show that there is no intraspecific variation, while cells are smaller in L. ortegai than in L. panteli. Concerning the micropylar area, in Parasteropleurus it is made of six tubes while in Lluciapomaresius it is made by a with a more or less protrusive, variable rose-shaped micropyl with no tube structure. The species of the genus Lluciapomaresius can be differentiated by the micropylar area: in L. panteli it is composed by a group of 8-10 rose-like shaped subsets (Figs. 16-18) while in L. ortegai it is composed by an ensemble of a group of pores very close together and two roses more separated from this grouping (Figs. 22-24). The egg of Phaneroptera nana is the only egg that shows an oval and flattened shape with a completely smooth surface. The family Phaneropteridae, where Phaneroptera belongs to, is characterized by showing this ellipsoidal shape as Bey-Bienko (1954) and Mazzini (1976) pointed out. Another peculiarity observed is the carina at the border of the ventral and dorsal egg regions, which has also been found in the phanaeropterinae species Poecilimon cervus Karabag, 1950 (Yilmaz et al., 2012), and it surrounds the whole egg except the micropylar area at the posterior pole. If we follow Mazzini’s (1976) description, the micropylar area is located at a close distance from the anterior pole of the egg but if we follow Yilmaz et al. (2012), the micropylar area must be located at the posterior pole. These observations also match with Hartley (1964), who says that the micropylar area is located at the posterior pole. So, according to our study the posterior pole of the egg of Phaneroptera nana has a more pointed shape with some pointed protusions on the chorion. These protusions are more pronounced and packed than at the anterior pole, which has a rounded shape and the chorion has a more relaxed pattern. Decticus verrucivorus has a micropylar area composed by three groups of three circles each, with at least one micropyl in one of the circles. Morphologically, the micropylar area is different from the rest of the chorion surface and it matches the description made by Mazzini (1976), which says: it can be isolated or in groups of three, it is found at a 0.8 mm distance from the anterior pole. In this case, the position of the anterior and posterior poles respectively is known. Contrary to our results, Mazzini (1976) suggested that for each micopillar area, 4 to 8 micropyls can be found. Furthermore, we have found that the micropylar area in the species Antaxius hispanicus is found in a protrusion. The micropylar area has never been found at the terminal poles of the egg, instead, it has been found close to one of the egg poles, but never at the pole itself. This result matches with the representative scheme of the eggs of some orthopteran families made by Mazzini (1987). Some authors in previous studies
(Metz & Monroy, 1966; Mazzini, 1987; Çakici & Ergen, 2012) mention that the micropylar area is located either at the anterior pole or in the ventral region of the egg. However, in British Tettigoniidae (Hartley, 1964) the micropylar area was found at the ventral side near the posterior pole of the egg. Localizing the micropylar area in the eggs would permit to easily identify the position of the egg poles (Vera Sánchez, 2014; Hartley 1964), and therefore this should be checked carefully as their localization varies depending on each taxonomic group. The fact of not finding the micropylar areas in some studied eggs (mostly at the beginning of this study) is probably due to the egg mounting technique used. If the egg is placed horizontally, some parts may remain hidden. For future observations, it is better to place the eggs vertically in the stab, as an overview of all sides of the egg can be observed. In some cases in which the micropylar area is not found, this is not due to a mounting error, but due to the fact that sometimes there are no micropyls as Çakici & Ergen (2012) reported on an unfertilized egg of Gryllus bimaculatus (Orthoptera: Gryllidae). 5. ACKNOWLEDGEMENTS We would like to thank all the people that helped in this project (collectors of the individuals, egg treatment procedures, execution and analysis of the images and with the revisions of the article). We are also very thankful with Joan Barat for his final text commentaries and corrections that improved this manuscript as well as all his support provided during the project. Last but not least, we would like to give our special thanks to all the institutions: Parc del Montsant, Parc dels Ports, the Department of Territory and Sostenibility and to the UB Servei Cientifico-Tècnic for all the help provided. In addition the Authors would like to thank gratefully the anonymous reviewers for all the comments that improved greatly the final version of the manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. 6. REFERENCES Bey-Bienko, G.Ya. (1954) Orthoptera Vol. II. No. 2. Tettigonioidea Phaneropterinae, Fauna of the U.S.S.R. Zoological Institute Akademii Nauk SSSR, 381 pp. [English translation 1965 Jerusalem (Israel Program for Scientific Translations)] Bushland, R. C. 1934. A study of the sculpture of the chorion of the eggs of eighteen South Dakota grasshoppers (Acridiidae). Unpublished thesis. South Dakota State College of Agriculture and Mechanic Arts. 31 p. Çakici, Ö. & Ergen, G. 2012. External egg morphology of Melanogryllus desertus (Pallas, 1771) (Orthoptera: Gryllidae). Biharean Biol., 6: 122–125. Gwynne, D. T. 2001. Katydids and bush-crickets. Reproductive behaviour and evolution of the Tettigoniidae. Cornell University Press. Ithaca. New York. 319 pp. Hartley, J. C. 1964. The structure of the eggs of the British Tettigoniidae (Orthoptera). Proceedings of the Royal Entomological Society of London Series A General Entomology, 39: 111-117. Mazzini, M. 1976. Sula fine struttura del micropilo negli insetti. IV. Le sculture corionidae come mezzo di identificazione della uova degli ortotteri Tettigoniodei. Redia, 59: 109–134.
Mazzini, M. 1980. Chorion sculptures and amino acid composition of orthopteran eggs. In ‘16th International Congress of Entomology, Kyoto. p.186. Mazzini, M. 1987. An overview of egg structure in Orthopteroid insects. B. M. Baccetti (Ed). Ellis Horwood Ltd. Chichester, Englan.pdf. Evol. Biol. Orthopteroid Insects. Vol. II Vo: 358–372. Metz, C.B. & Monroy, A. 1966. Biology Of Fertilization V1: Model Systems And Oogenesis. METZ, C.B. and MONROY, A. (Eds.). 390 pp. Academic Press, New York. 99–116. Tuck, J. B. 1939. The identification of the eggs of grasshoppers by means of the chorionic sculpturing. MS thesis. Kansas State College. Rentz D.C.F., Su, Y.N. & Ueshima, N. 2007 Studies in Australian Tettigoniidae. A new genus of listroscelidine katydids from northern Australia orthoptera: Tettigoniidae; Listroscelidinae). Transactions of the American Entomological Society (Philadelphia) 133(3-4): 279-296. Saenger, K. & Helfert, B. 1994 Comparative studies on number and position of the micropyles and the shape of the eggs of Saga pedo, S. natoliae and S. ephippigera (Orthoptera: Tettigoniidae). Entomologia Generalis, 19(1-2): 4956. Uvarov, B. P. 1928. Locusts and grasshoppers. London. Imperial Bureau of Entomology. 273 p. Vera Sánchez, A. 2014. Estudio comparativo de la morfología de huevos en Tettigoniinae (Insecta: Orthoptera) de Chile, mediante una aproximación con SEM. Gayana (Concepc.), 78: 144–151 Webber, B L, Rentz, D C F, Ueshima, N, Woodrow, I E 2003 Leucopodoptera eumundii, a new genus and species of katydid from the tropical rainforests of north Queensland, Australia (Orthoptera: Tettigoniidae: Phaneropterinae: Holochlorini). Journal of Orthoptera Research, 12(1) 2003: 79-88. Yilmaz, I., Suludere, Z. & Candan, S. 2012. Structure of the egg of Poecilimon cervus Karabag (Orthoptera: Tettigoniidae) and ultrastructural features. Turkiye Entomoloji Dergisi, 36: 549-556. Zhou, Z., Zhao, L., Liu, N., Guo, H., Guan, B., Di, J. & Shi, F. 2017. Towards a higherlevel Ensifera phylogeny inferred from mitogenome sequences. Molecular Phylogenetics and Evolution, 108:22-33. .
LEGENDS
FIGURE 1-6 Eggs from Ephippiger diurnus cunii: 1 General view, 2 surface general view, 3 follicular cells, 4 micropylar area general view (AM), 5 detail of the first micropylar rose-shaped (RS), 6 detail of the second micropylar roseshaped (RS).
FIGURE 7-12 Eggs from Parasteropleurus perezii: 7 General view, 8 surface general view, 9 hexagonal follicular cells, 10 micropylar area general view (AM), 1112 detail of the miropyles (M).
FIGURE 13-18 Eggs from Lluciapomaresius panteli: 13 General view, 14 surface general view, 15 follicular cells, 16 micropylar area general view (AM), 17 micropylar area zoom view, 18 detail of two micropylar rose-shaped (RS).
FIGURE 19-24 Eggs from Lluciapomaresius ortegai: 19 General view, 20 surface general view, 21 follicular cells, 22 micropylar area general view (AM), 23 micropylar area zoom view, 24 detail of a piece of the micropylar area.
FIGURE 25-30 Eggs from Phaneroptera nana: 25 General view, 26 surface general view, 27 follicular cells, 28 micropylar area general view (AM), 29 micropylar area zoom view with the micropyl, 30 detail of the micropyl.
FIGURE 31-36 Eggs from Decticus verrucivorus: 31 General view, 32 surface general view, 33 follicular cells, 34 AM: micropylar area (AM), 35 micropylar area zoom view, 36 micropyl (M).
FIGURE 37-40 Eggs from Antaxius hispanicus: 37 General view, 38 surface general view, 39 follicular cells, 40 micropylar warts. FIGURE 41-46 Eggs from Cyrtaspis scutata: 41 General view, 42 surface general view, 43 follicular cells, 44 micropylar area (AM), 45 micropylar area general view, 46 micropyles (M).
TABLE 1. Studied species with some of the traits that characterize the surface of the eggs. Species
Micropillar area shape
Egg surface
Ephippiger ephippiger
Two rose-like shaped structures
Fused hexagonal and pentagonal cells
Parasteropleurus perezii
Tubes
Hexagonal cells with irregular walls
Lluciapomaresius panteli
8-10 rose-like shaped subsets (here only roselike pictured)
Separataed hexagonal cells
Lluciapomaresius ortegai
Big structure plus two roselike shaped subsets
Hexagonal cells
Decticus verrucivorus
3 groups of 3 circles each one
Shallow follicular cells
In a warts-like shape
Polyhedral follicular cells (rhomboid, pentagonal and hexagonal)
Antaxius hispanicus
Phaneroptera sp.
Cyrtaspis sp.
Six tubes
Smooth surface
3-4 Tubes
Follicular cells with rhomboid shape and irregular walls