Ephemeroptera egg chorion characters: a test of their importance in assessing phylogenetic relationships

The egg chorion ultrastructures of the Hermanella-Traverella (Insecta: Ephemeroptera) species complex were studied from a comparative point of view and used for the first time in a cladistic analysis. Egg characters, along with other nymphal and adult morphological characters, were used to assess the phylogenetic relationships of the species complex. In order to test the value of egg characters, analyses were performed on three matrices: 1) egg characters alone, 2) adult and nymphal characters, and 3) adult, nymphal, and egg characters. The computer program Pee-Wee was used to carry out the analysis. The cladistic analysis confirmed the value and potential of egg chorionic characters in assessing the phylogenetic relationships among ephemeropteran species. Egg characters, when added to the nymphal and adult character matrix, provided extra support to the monophyletic nature of the Hermanella-Traverella complex. Previously weakly defined clades were also resolved based on the new evidence. In the species studied the egg chorionic structures as well as their shape did not change after oviposition or water immersion, remaining constant through the different maturation stages of each species (mature nymph, subimago, and imago). For this reason, the eggs are a valuable source of information to unambiguously identify and associate a nymph to its correspondent adult stage when rearing is not possible.     

Feeding behavior and related functional morphology of the mayflyEphemerella needhami (Ephemeroptera: Ephemerellidae)

The feeding behavior and functional morphology associated with feeding in Ephemerella needhamiMcDunnough larvae were studied using videomacroscopic techniques, gut content analysis, and scanning electron microscopy. Two stereotypic feeding cycles were employed by the larvae. In the maxillary brushing cycle, the maxillae are the primary food-gathering organs, with the main food being detritus deposited on the filamentous alga Cladophora.In the mandibular biting cycle, the mandibles are the primary food-gathering organs used to bite Cladophora filaments.Epiphytic diatoms on Cladophorawere another important part of the diet. Behavioral similarities are apparent in the choreography and synchronization of mouthpart movements among mayflies from several families. Functional morphological comparisons are drawn with hypognathous E. needhami, Cloeon dipterum, Baetis rhodani,and Siphlonurus aestivalis, aswell as the prognathous Heptageniidae. Differences in mouthpart usage and structure are related to the relative development of setal fields and combs and the feeding microhabitat.     

Egg surface structure of three clingfish species, using scanning electron microscopy

The three eastern Atlantic gobiesocid species Lepadogaster lepadogaster, Apletodon dentatus and Diplecogaster bimaculata share an attachment apparatus consisting of numerous bifurcating filaments that originate at the animal egg pole and are embedded in a mucous layer. The thickness of the mucous layer and zona radiata, and the structure and size of the micropyle vary among the three species. Because of lack of information about egg structure in paracanthopterygians and percomorphs with adhesive demersal eggs, the phylogenetic significance of this character complex remains unclear.     

Observations on the seasonal breeding biology and fine structure of the egg surface in the white piranha Serrasalmus brandtii from the São Francisco River basin,Brazil

In the Juramento Reservoir, south‐eastern Brazil, the white piranha Serrasalmus brandtii showed a prolonged reproductive season, with evidence for multiple spawning and a reproductive peak associated with seasonal rains. The egg surface exhibited a honeycomb‐like pore canal arrangement and an adhesive apparatus surrounding the micropyle. Electron microscopic analysis suggests a role for the micropylar cell and neighbouring follicular cells in secretion of substances for egg attachment.     

Seedling morphology of three mangrove species and its taxonomic implications

Seedling establishment is an important part of the life cycle of mangroves. Very early in the juvenile stage, they face adverse situations such as a variable and unstable substrate as well as a high-saline environment Therefore, the propagules should have some ability to float in saline water. Seedlings differ morphologically from the adult plants. Here, I examined the seedling growth stages in three species of mangrove:Aegialitis rotundifolia Roxb.,Aegiceras corniculatum (L) Blanco, andAvicennia marina (Forsk.) Vierh. that grow in the Sundarbans swamps of West Bengal. In all cases, fruits were the propagating units; seeds were inseparable from those fruits. Germination was rapid and required no dormancy period. The hypocotyi pierced the seed coat rather than emerging from the pericarp — a variation on the traditional viviparous type of germination. Seedlings then became exposed prior to abscission from parent plants. Based on germination patterns and differences in seedling morphology observed here, these species should be classified in a more relevant taxonomic group than was done previously.     

Formation and structure of egg envelopes in Russian sturgeon Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The covering of the eggs in Russian sturgeon Acipenser gueldenstaedtii consists of three envelopes (the vitelline envelope, chorion and extrachorion) and is equipped with multiple micropyles. The most proximal to the oocyte is the vitelline envelope that consists of four layers of filamentous and trabecular material. The structural components of this envelope are synthesized by the oocyte (primary envelope). The chorion encloses the vitelline envelope. The extrachorion covers the external surface of the egg. Examination of the arrangement of layers that comprise the egg envelopes together with the ultrastructure of follicular cells revealed that the chorion and extrachorion are secondary envelopes. They are secreted by follicular cells and are built of homogeneous material. During formation of egg envelopes, the follicular cells gradually diversify into three morphologically different populations: 1) cells covering the animal oocyte region (cuboid), (2) main body cells (cylindrical) and (3) micropylar cells. The apical surfaces of follicular cells from the first two populations form processes that remain connected with the oocyte plasma membrane by means of gap junctions. Micropylar cells are located at the animal region of the oocyte. Their apical parts bear projections that form a barrier to the deposition of materials for egg envelopes, resulting in the formation of the micropylar canal.     

Buoyancy variations in eggs of Atlantic cod (Gadus morhua L.) in relation to chorion thickness and egg size: theory and observations

Egg specific gravity is a function of egg volume, chorion volume, perivitelline space (PVS) and the specific gravity of chorion and ovoplasm. Experimental studies on cod ( Gadus morhua L.), a marine multiple batch spawner, showed that approx. forty per cent of the chorion volume is incorporated between batches and that the chorionic material has a mean specific gravity of 1·20 (range: 1·14–1·35). The chorion volume and specific gravity are low at the end of the spawning period in extremely fecund fish. Specific gravity of ovoplasm was observed to be 1·017 in marine eggs and calculated to be about 1·008 in brackish water eggs. Inherent variation in PVS is found to have little influence on egg specific gravity. With the other variables constant, egg specific gravity is inversely related to egg volume supported by studies on single batches of eggs. Measurements on every batch of eggs reveal that the production of large eggs (>1·3 mm) gives low density eggs initially and high density eggs towards the end of spawning, while the production of small eggs (<1·3mm) gives moderate specific gravity. These observations are discussed in relation to vertical distribution of the eggs and reproductive success of recruit and repeat spawners. There are indications that repeat spawners produce larger eggs.     

Conformational analysis of peptide analogues of silkmoth chorion protein segments using CD, NMR and molecular modelling.

Silkmoth proteins secreted from the follicular cells that surround the oocyte form a large extracellular assembly which is important for protecting and sustaining the structure of the oocyte and the developing embryo. These proteins have been classified into two major families (A and B). Sequence analysis showed conservation of a central domain containing long stretches of six amino acid residue repeats in both families, which have been suggested to be organized in beta-sheet structures. In this work NMR and CD spectra, as well as molecular calculations, have been used to investigate the conformational properties of two synthetic peptides (A and B), analogues of parts of the central domain of silkmoth chorion proteins of the A and B families, respectively. These peptides consist of three tandem repeats of the six-residue basic motif. Analysis of CD spectra of the two peptides in aqueous solutions and mixtures with organic solvents revealed beta-sheet and turn structural elements with a percentage higher than 40%. NOESY spectra at low temperatures (263-273 K) show sequential nOe connectivities (i, i + 1), indicative of a relative flexibility. The presence of HNi-HNi+1 cross-peaks and medium Halphai-HNi+1 connectivities, chemical shift deviations and temperature coefficient data provide, for the first time, experimental evidence that local folded structures around Gly residues occur in peptide segments of chorion proteins in solution. Simulated annealing calculations were used to examine the conformational space of the peptides and to probe the initial steps of amyloid fibril formation in the case of chorion proteins.     

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes

The unfertilized egg envelope of medaka ( Oryzias latipes ) consists of two major groups of subunits, ZI-1,2 (74–76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N -termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N -terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57–65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca²⁺ and Zn²⁺ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.     

The dual roles of Armigeres subalbatus prophenoloxidase V in parasite melanization and egg chorion melanization in the mosquito Ar. subalbatus

Phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning, cuticle formation and melanotic encapsulaction of pathogens. Previously, we identified five POs, designated As-pro-PO I–V, from the mosquito Armigeres subalbatus and demonstrated that the functions of As-pro-PO I, II and III, were associated with filarial parasite melanization, blood feeding and cuticle formation, respectively. In the present study, we delineate the dual functions of As-pro-PO V. We found that the level of As-pro-PO V mRNA in mosquitoes was significantly increased after microfilaria challenge or blood feeding, and decreased to normal level after oviposition. Knockdown of As-pro-PO V by dsRNA resulted in significant decreases in the degree of microfilaria melanization, egg chronic melanization rates and egg hatching rates in Ar. subalbatus. Further transfection and electrophoretic mobility-shift assays verified the As-pro-PO V gene might regulated by both AP-1, a putative immune-related regulatory element and CdxA, a developmental regulatory element. The binding of AP-1 and CdxA motif with mosquito nuclear extracts was significantly enhanced after microfilaria challenge and blood-feeding in Ar. subalbatus, respectively. These results indicate that As-pro-PO V is a critical enzyme that is required for both an effective melanization immune response and egg chorion melanization in this mosquito.     

Egg-laying behavior and morphological and chemical characterization of egg surface and egg attachment glue of the digger wasp Ampulex compressa (Hymenoptera,Ampulicidae)

For providing their offspring females of the digger wasp species Ampulex compressa hunt cockroaches, paralyze them and attach as a rule one egg to the coxa of one of the mid legs of their prey. We observed the egg-laying behavior and examined with light- and scanning microscopy (i) nearly mature eggs from ovaries of freshly dissected females and (ii) eggs immediately after their deposition on the coxae of their prey. The length of the white bean-shaped eggs varied between 2.2 and 3.0 mm, their diameter between 0.66 and 0.72 mm, and their weight between 345 and 832 μg. The surface of fresh, untreated eggs shows even at higher magnifications (>20.000×) a smooth appearance. However, after conventional fixation, dehydration with ethyl-alcohol and critical-point drying the egg-surface exhibited a little bit texture. The eggs are at two-third of their underside glued to the coxa of the prey. With the naked eye the glue appears as a compact mass. The eggs may be mechanically removed from the substrate (their attachment site); however, in doing so the viscous attachment glue appears in a more fibrous consistence. The polypeptide composition washed off the egg surface and the glue revealed no similarities, whereas the molecular mass of two polypeptides were similar between glue and the Dufour's gland contents.     

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori

Summary Three -type early chorion gene copies (6F76.1, 6F76.2, and 6F76.3) are dispersed in the late region of chorion locus Chl-2. Detailed analysis of the 5-flanking region and the intron of 6176.1 shows that they contain sequences that are homologous to Bombyx mori Bm l repeat elements. Interestingly, the Bm l -type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6176.1 5-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion -gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.Offprint requests to: G. Rodakis     

The comparative gastrointestinal morphology of five species of muroid rodents found in Saudi Arabia

Meriones rex (King jird), Meriones libycus (Libyan jird), Acomys dimidiatus (Eastern spiny mouse), Acomys cahirinus (Egyptian spiny mouse), and Dipodillus dasyurus (Wagner's dipodil) are five species of small rodents of the superfamily Muroidea with distributions in Eastern Africa, Egypt, and the desert regions of the Arabian Peninsula. Water is scarce in these regions and may result in relatively low‐digestible food. The aim of the present study is to describe and compare the gastrointestinal tract morphology and morphometry of these five species in order to elucidate whether morphology is influenced by phylogeny or dietary preference. Each segment of the gastrointestinal tract of each species was macroscopically examined and the length and basal surface area of each segment was measured. Standard histologic procedures were performed to determine a surface enlargement factor to determine the mucosal luminal surface area. A unilocular‐hemiglandular stomach was observed in all the species examined. The caeca of all the species were long and arranged into a loose spiral toward the caecal tip with the ileocaecal and caeco‐colic openings positioned close together. Two rows of oblique folds could be observed in the proximal colon of all species except in D. dasyurus which had longitudinal folds. Morphometric analysis showed the largest stomach in A. cahirinus and the largest caecum and colon in M. libycus. All the species can be grouped in the family Muridae in two subfamilies and similarities were observed including the hemiglandular stomach and relatively large caecum. It could be concluded that phylogeny plays an important role in determining gastrointestinal morphology while diet plays a subordinate role in the desert rodents in the present study. J. Morphol. 275:980–990, 2014. © 2014 Wiley Periodicals, Inc.     

Community structure of Ephemeroptera in Siberian streams

In recent decades, the relationships between environmental conditions and community structures of stream macroinvertebrates have been investigated in many parts of the world. However, knowledge about assemblages of mayflies and other stream macroinvertebrates in Siberia (northern Asia) and Asia is limited. In fact, the patterns in mayfly species richness and assemblage structure in relation to environmental parameters have not been previously examined in western Siberia. The aim of the present study was to examine the relationship between Ephemeroptera community structure and physical parameters along a river altitude/longitude gradient in Siberia. The results showed that maximum species richness was at relatively low altitudes, high water temperatures, slow current velocities, medium stream widths, medium‐small substrate particle size, and the presence of macrophytes. The mayfly assemblage was separated using TWINSPAN classification into eight distinct groups, which differed significantly with respect to at least one measured environmental factor. Multivariate ordination (detrended correspondence analysis) revealed that mayfly assemblages are structured by a single dominant gradient of altitude‐related environmental variables; altitude and water temperature were the best predictors. Ordination further revealed that mayfly assemblages are structured by altitude‐related environmental factors at high elevations, whereas in the lowlands these factors are less important.     

三种黄花石蒜植物的花粉形态比较

利用光学显微镜和扫描电镜对三种开黄花的石蒜属(Lycoris Herb.)植物的花粉形态进行观察和比较.结果表明:三种植物花粉均为两侧对称,赤道面观呈舟形或肾形,极面观为椭圆形或长椭圆形,萌发孔类型为远极单槽,槽长几达两端;三种植物花粉外壁具网状纹饰,且在网眼底壁及网脊多附有小乳突.通过花粉特征及其他性状的比较研究,对三种黄花石蒜植物的分类鉴定和系统演化关系进行了初步探讨.     

Sequence analysis of a small early chorion gene subfamily interspersed within the late gene locus in Bombyx mori

A comprehensive sequence analysis of three early chorion genes (6F6.1, 6F6.2, 6F6.3) which form a small subfamily is presented. Two main features characterize this subfamily: (1) the 6F6 gene copies are -branch genes and, unlike typical chorion genes which are organized in divergent gene pairs, they are unpaired, and (2) they are not clustered in genetic locus Ch3 but are dispersed in Ch1-2, which is about 3 to 4 centiMorgans away and contains middle and late chorion genes. Sequence comparisons show that members of this subfamily exhibit high identity values in their major coding region (94–96%) and that similarities also extend, but to a lesser degree, into their noncoding regions. The putative 6F6 promoter regions have no significant similarities with the corresponding regions of other early -genes but quite surprisingly share common elements with middle and late genes. The main difference among the 6F6 gene introns is the presence of inserted sequences: the insert into 6F6.2 (IR; 248 bp) is flanked by a 102–103-bp inverted repeat, while those into 6F6.1 (FIB; 184 bp) and 6F6.3 (HOPE; 951 bp) are carried by a partial Bm1 element. HOPE has features of a non-LTR retrotransposable element. Preliminary experiments indicate that the copy number of IR and HOPE in the Bombyx mori genome is about 5,000 and 20,000, respectively. The great similarity of 6F6 genes cannot be accounted for by selective pressure but rather appears to be the result of gene-conversion-like events, which are supposed to operate frequently in middle and late chorion genes but not in other known early -genes. Using the relative position and orientation of the 6F6 gene copies, it is possible to propose an evolutionary scheme for the formation of chorion locus Ch1-2. Correspondence to: G.C. Rodakis     

Comparative morphology of the egg of the castniid palm borer,Paysandisia archon (Burmeister, 1880) (Lepidoptera: Castniidae)

Paysandisia archon (Burmeister, 1880) is an attractive neotropical castniid moth whose presence in Europe was recently reported. Its larvae are endophagous, feeding inside the trunks and branches of several species of palm trees (Arecaceae). The present paper deals with the morphology and biometry of the egg of this moth, comparing them with those of other castniid species.

The egg is a typical castniid egg, fusiform, upright sensu Döring, light cream or creamy pink when freshly laid, 4.69 ± 0.37 mm long and 1.56 ± 0.11 mm wide. Larvae emerge by gently splitting the chorion along one of the longitudinal ridges, on the half closer to the micropyle. SEM, TEM and LSCM photographs showing ultrastructural details of the egg are shown for the first time. The micropylar rosette (c. 54 μm in diameter) has generally 14–17 cells; in its centre lies the micropylar pit (c. 6 μm in diameter) which bears 12–16 micropylar canal openings (= micropyles) around its periphery. The pathways followed by those canals through the chorion have been figured. Eggs sampled in the wild (so laid by several females) were found to have a slightly variable number of ridges: most bore seven ridges (68.87%), although a significant portion (30.46%) bore eight and 1 egg (0.67%) bore only six; this against the currently accepted rule of five‐ridged eggs for Castniini (i.e. Neotropical castniids) to which Paysandisia archon belongs. It has also been found that the same female specimen has the capability of producing six‐, seven‐ or eight‐ridged eggs. Five types of egg irregularities affecting the longitudinal ridges are also figured and described. Transverse striae on the egg of P. archon are about 122. Aeropyles (c. 4 μm in diameter) occur on the ridges, at the intersections between the latter and two contiguous (left and right of the ridge) transverse striae, amounting to c. 854 on a seven‐ridged egg and to c. 976 on an eight‐ridged egg. Occasionally minute aeropyles ('microaeropyles') (c. 1.96–3.13 μm in diameter) also occur on transverse striae located close to both egg poles.

The chorion of P. archon shows the typical ditrysian fine structure with very thin basal layer (C‐1), 0.3–0.2 μm thick, gas‐filled trabecular layer (C‐2), c. 0.9 μm thick, and lamellar layer (C‐3), its thickness varying between 18.5 and 13 μm due to the bumpy external surface of the chorion. Aeropylar canals, that penetrate layer C‐3, connect the air‐containing inner chorionic meshwork (the trabecular layer C‐2) with the surrounding air; their outer part forms a big bulbous cavity (which opens to the outside through the small opening seen in external SEM images) and, underneath, a narrow canal follows, leading into the trabecular layer (C‐2).