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The staining procedure is critical for investigating intra- and extra-cellular ultrastructure of microorganisms by transmission electron microscopy (TEM). Here, we propose a new ultra-low lead staining (ULLS) technique for preparing the ultrathin sections for TEM analysis. Sections of Enterobacter sp. (bacteria), Aspergillus niger (filamentous fungi), Rhodotorula mucilaginosa (fungi), and Chlamydomonas reinhardtii (microalgae) were tested. Compared with the sections prepared by the typical double-staining technique, ULLS-based sections showed evident advantages: (i) the staining process only required the addition of Pb(NO3)2; (ii) the Pb level during incubation was set as low as 1 mg/L, which had negligible toxicity to most microbial cells; (iii) the Pb cations were added during microbial culture, which avoided complicated sample preparation as in typical double staining. Taking C. reinhardtii as an example, the ULLS technique allowed fine investigation of microbial ultrastructure, e.g., starch granule, mitochondrion, Golgi apparatus, vacuole, and vesicle. Meanwhile, the physiological processes of the cells such as cell lysis and exocytosis were successfully captured, with relatively high contrast. This study hence shows a bright future on preparation of the high-quality ultrathin sections of microbial cells by the ULLS technique.
This study was conducted to monitor the cellular and molecular changes of buffalo cumulus–oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB− and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB− group. BCB− oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB− under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB−) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB− oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.
A survey on Anisakis simplex (sensu stricto (s.s.)) from blue whiting, Micromesistius poutassou, in the north-eastern Atlantic Ocean revealed the occurrence of high infection levels of third larval stages in visceral organs and flesh. Larvae were genetically identified with a multilocus approach as A. simplex (s.s.). Histochemical, immunohistochemical and ultrastructural observations were conducted on 30 M. poutassou specimens. Gonads, pyloric caeca and flesh harboured encapsulated larvae of A. simplex (s.s.) but no intense host reaction was encountered around the parasite in the above organs. In the liver, the most infected organ, the larvae co-occurred with the coccidian Goussia sp. Within the granuloma around the A. simplex (s.s.) larvae, two concentric layers were recognized, an inner mostly comprising electron-dense epithelioid cells and an outer layer made of less electron-dense epithelioid cells. Macrophages and macrophage aggregates (MAs) were abundant out of the granulomas, scattered in parenchyma, and inside the MAs, the presence of engulfed Goussia sp. was frequent. In liver tissue co-infected with Goussia sp. and A. simplex (s.s.), hepatocytes showed cytoplasmic rarefaction and acute cell swelling. Results suggest that the host-induced encapsulation of A. simplex (s.s.) larvae is a strategic compromise to minimize collateral tissue damage around the larval infection sites, to facilitate the survival of both parasite and host.
Interstitial cells of Cajal (ICC) play an essential role in the motility of the gastrointestinal tract, and they have been identified in many laboratory animals and in humans. However, the information of ICC in lower animals is still very limited. In the present study, ICC were identified in the gastric muscularis mucosae of an amphibian—the Chinese giant salamander, by c-Kit immunohistochemistry and transmission electron microscopy. ICC showed c-Kit immunoreactivity and had spindle-shaped cell bodies and 1–2 long processes. ICC were located between smooth muscle cells (SMC) in gastric muscularis mucosae. Ultrastructurally, ICC appeared as polygon-, spindle-, and awl-shaped with long cytoplasmic prolongations between SMC. ICC had distinctive characteristics, such as nuclei with peripheral electron-dense heterochromatin, caveolae, and abundant intracytoplasmatic vacuoles, mitochondria, and rough endoplasmic reticula. Moreover, lamellar bodies and two types of condensed granules were observed in the cytoplasm of ICC. Notably, ICC establish close contacts with each other. Moreover, ICC establish gap junctions with SMC. In addition, ICC were frequently observed close to nerve fibers. In summary, the present study demonstrated the presence of ICC in the gastric muscularis mucosae of the Chinese giant salamander.
This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa – Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.
Nonalcoholic fatty liver disease (NAFLD) represents a hepatic manifestation of metabolic syndrome. The aim of this study was to examine the effect of betaine on ultrastructural changes in the mouse liver with methionine- and choline-deficient (MCD) diet-induced NAFLD. Male C57BL/6 mice were divided into groups: Control—fed with standard chow, BET—standard chow supplemented with betaine (1.5% w/v drinking water), MCD—fed with MCD diet, and MCD + BET—MCD diet with betaine supplementation for 6 weeks. Liver samples were taken for pathohistology and transmission electron microscopy. The MCD diet-induced steatosis, inflammation, and balloon-altered hepatocytes were alleviated by betaine. MCD diet induced an increase in mitochondrial size versus the control group (p < 0.01), which was decreased in the betaine-treated group. In the MCD diet-fed group, the total mitochondrial count decreased versus the control group (p < 0.01), while it increased in the MCD + BET group versus MCD (p < 0.01). Electron microscopy showed an increase in the number of autophagosomes in the MCD and MCD + BET group versus control, and a significant difference in autophagosomes number was detected in the MCD + BET group by comparison with the MCD diet-treated group (p < 0.05). Betaine decreases the number of enlarged mitochondria, alleviates steatosis, and increases the number of autophagosomes in the liver of mice with NAFLD.
We have previously presented a stereological analysis of organelle distribution in human prophase I oocytes. In the present study, using a similar stereological approach, we quantified the distribution of organelles in human metaphase I (MI) oocytes also retrieved after ovarian stimulation. Five MI oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. Kruskal–Wallis and Mann–Whitney U-tests with Bonferroni correction were used to compare the means of relative volumes (Vv) occupied by organelles. In all oocyte regions, the most abundant organelles were mitochondria and smooth endoplasmic reticulum (SER) elements. No significant differences were observed in Vv of mitochondria, dictyosomes, lysosomes, or SER small and medium vesicles, tubular aggregates and tubules. Significant differences were observed in other organelle distributions: cortical vesicles presented a higher Vv (P = 0.004) in the cortex than in the subcortex (0.96% vs 0.1%) or inner cytoplasm (0.96% vs 0.1%), vesicles with dense granular contents had a higher Vv (P = 0.005) in the cortex than in the subcortex (0.1% vs 0%), and SER large vesicles exhibited a higher Vv (P = 0.011) in the inner cytoplasm than in the subcortex (0.2% vs 0%). Future stereological analysis of metaphase II oocytes and a combined quantitative data of mature and immature oocytes, will enable a better understanding of oocyte organelle distribution during in vivo maturation. Combined with molecular approaches, this may help improve stimulation protocols and in vitro maturation methods.
Kapsulotaenia tidswelli is a proteocephalidean cestode that utilizes varanid lizards as definitive hosts. Fresh specimens of this cestode were observed with endogenous red pigmentation in the neck region that disappeared rapidly if specimens were not preserved in glutaraldehyde. The ultrastructural characteristics of the red pigment, which are described, suggest it is a carotenoid. Phylogenetic analysis confirmed a close relationship between K. tidswelli and other species of Kapsulotaenia for which sequence information is available. There is thus no reason to consider that the red pigmentation is because K. tidswelli is atypical, and it is proposed the carotenoids are likely to be associated with the diet of its varanid host.
Cement glands are one of the most conspicuous and distinctive elements of taxonomic interest in male Acanthocephala. Cement glands vary in shape, number and arrangement in different classes of the taxon. The glands and their products have a fundamental role in the reproductive process. Light and electron microscopy were used to investigate the ultrastructure of the cement apparatus, which includes both cement glands and the cement reservoir, in mature males of Centrorhynchus globocaudatus (Zeder, 1800). Centrorhynchus globocaudatus is an enteric parasite of birds of prey, including Falco tinnunculus (Linnaeus, 1758) and Buteo buteo (Linnaeus, 1758) from the province of Ferrara (northern Italy). The four elongated cement glands of C. globocaudatus are situated posterior to the testes. Sections through the cement glands show each gland is surrounded by a fibrous envelope with an approximate thickness of 0.6 μm. Beneath this envelope is an outer cytoplasmic layer thickness ranging from 22 to 26 μm, which contains a number of nuclei with diameters variable from 20 to 22 μm. The cytoplasmic layer is filled with prominent free ribosomes and many mitochondria with lamellar cristae. Secretory granules, measuring from 1 to 1.3 μm in diameter, are formed within the cytoplasmic layer. The cytoplasmic layer surrounds the luminal area for storage of the cement material in each gland. Cement gland ducts arise from the gland and extend towards a common cement reservoir in close contact with the seminal vesicle and Saefftigen's pouch. Microtubules, large secretory granules and rest of undefined organelles were also observed within the cement reservoir.
In a previous research work aimed at discovering natural helminthicides as alternatives to conventional synthetic drugs, Piper retrofractum fruit hexane extract (PHE) has been shown to possess promising nematocidal activity against the third-stage infective larvae of Strongyloides stercoralis. Thus, this study was designed to evaluate the chemical composition and the impact of PHE on symptom and structural alterations of S. stercoralis. Chemical analysis of PHE by gas chromatography–mass spectrometry demonstrated 26 different compounds, constituting 100% of the total composition. The main components were 4-acetylphenyl (4-benzoylphenoxy) acetate (14.86%) and octyl methoxycinnamate (12.72%). Nematocidal bioassays revealed promising potential of PHE against S. stercoralis larvae, with an LC50 value of 0.059 mg/ml, while the reference drug ivermectin exerted higher efficacy, with an LC50 value of 0.020 µg/ml. Behavioural observations under light microscopy revealed that PHE-treated S. stercoralis larvae moved slowly, became paralysed and eventually died during 24 h of incubation. The dead larvae appeared under light microscope as straight worms with unknown vacuoles of different sizes inside their internal bodies. Morphological alterations of the PHE-treated S. stercoralis larvae, such as straight bodies with swollen cuticle, faded transverse annulations and faded longitudinal striations, as well as shallow and smooth lateral longitudinal grooves, were seen clearly under scanning electron microscopy. Ultrastructural changes in the treated larvae, such as protruded lateral longitudinal grooves, loose muscle with vacuolation, dissociation between the hypodermis and cuticle and marked intracellular disorganization with vacuolation, were detected under transmission electron microscopy. The results of this study provide evidence that PHE is toxic against S. stercoralis and also a potential new alternative for anti-Strongyloides chemotherapy.
The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is an important vector for the xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner), which is associated with olive quick decline syndrome in southern Italy. The mouthparts of Hemiptera have important roles in host plant selection, feeding behavior and for vectoring pathogens that cause plant diseases. In this study, the functional morphology of the sensory structures located on the labium tip and precibarium of P. spumarius was investigated using scanning and transmission electron microscopy. The labium tip is composed of two symmetrical sensory complexes, each with five different types of sensilla: aporous sensilla trichodea type 1 and 2; uniporous sensilla chaetica type 1 and 2; and multiporous sensilla basiconica. The precibarium of P. spumarius has two kinds of sensory structures: bulbous sensilla and papillae sensilla. In particular, two groups of sensilla are located on the epipharynx: a distal group that consists of ten papillae sensilla and a proximal group composed of six papillae sensilla and two bulbous sensilla, while the hypopharynx has only two papillae sensilla. The involvement of these sensory structures in the context of feeding behavior and pathogen transmission is discussed.
An array of nano-scale protrusions, called the nipple array, is found on the body surface of various invertebrates, and this structure is believed to decrease light reflectance and water wettability on the surface in the terrestrial environment. However, its potential functions have not been well studied in aquatic environments. Clavelina spp. are colonial ascidians that have the nipple array on their integumentary matrix (i.e. tunic). We examined the physical properties on the surface of the tunic of C. cyclus and C. obesa, such as hardness, wettability and refractive indices, to evaluate the functional importance of this structure. The tunic cuticle of both species was covered with the nipple array, and the cuticle of C. cyclus was bent into folds forming parallel plications. The Clavelina tunic was very soft and had high bubble- and oil-repellency. The refractive-index deviation between the tunic and seawater was 0.07–0.095 for 589-nm light (D-line). Rigorous coupled wave analysis (RCWA) showed that the nipple array slightly reduced reflectance on the surface and the parallel plications reduced the reflectance still more. The nanoimprinted plates imitating the parallel plications have higher bubble repellency and lower reflectance than the flat plates. These findings support the functional importance of the plications as well as the nipple array.
Bunocotyle progenetica is a hemiuroid digenean whose sexual adults become fully developed and lay their eggs inside the rediae in the molluscan host. In this study, the fine structure of the germinal mass, brood cavity and birth canal in the B. progenetica rediae was examined using transmission electron and confocal microscopy. The large germinal mass attached to the body wall has a cellular composition typical for this organ. The characteristic traits of this germinal mass are weakly developed supporting tissue and the presence of deep lacunae opening into the brood cavity. These lacunae presumably participate in feeding the deeply lying embryos and facilitate their release into the brood cavity. The germinal mass is also characterized by intensive degeneration of cellular elements, which may represent a mechanism controlling the offspring number, limited in this species by the size of the redial brood cavity. The brood-cavity lining consists of flattened cells bearing lamellar projections and is connected anteriorly with the epithelium of the birth canal. The brood-cavity musculature, which is well developed in other hemiuroid digeneans, is significantly reduced in B. progenetica, most likely because their cystophorous cercariae remain inside the rediae, removing the need for muscle contractions pushing them through the brood cavity. The birth canal comprises three regions distinguished by the structure of the lining and muscle arrangement. The comparison of rediae of B. progenetica with parthenitae of other digeneans has shown that the organization of the redial reproductive apparatus in this species may have been influenced by life-cycle modification.
The aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.
The process of autophagy has been detected in the midgut epithelium of four millipede species: Julus scandinavius, Polyxenus lagurus, Archispirostreptus gigas, and Telodeinopus aoutii. It has been examined using transmission electron microscopy (TEM), which enabled differentiation of cells in the midgut epithelium, and some histochemical methods (light microscope and fluorescence microscope). While autophagy appeared in the cytoplasm of digestive, secretory, and regenerative cells in J. scandinavius and A. gigas, in the two other species, T. aoutii and P. lagurus, it was only detected in the digestive cells. Both types of macroautophagy, the selective and nonselective processes, are described using TEM. Phagophore formation appeared as the first step of autophagy. After its blind ends fusion, the autophagosomes were formed. The autophagosomes fused with lysosomes and were transformed into autolysosomes. As the final step of autophagy, the residual bodies were detected. Autophagic structures can be removed from the midgut epithelium via, e.g., atypical exocytosis. Additionally, in P. lagurus and J. scandinavius, it was observed as the neutralization of pathogens such as Rickettsia-like microorganisms. Autophagy and apoptosis ca be analyzed using TEM, while specific histochemical methods may confirm it.
Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis in humans, the most prevalent non-viral sexually transmitted disease (STD). Imidazole compounds are used for the treatment of trichomoniasis, and metronidazole is the most commonly prescribed. However, these compounds can lead to parasite resistance and unwanted side effects. Therefore, there is a need for an alternative treatment for this disease. Here, we explored the potential of clotrimazole (CTZ) and zinc compounds, as well as CTZ complexed with zinc salts ( acetate [Zn(CTZ)2(Ac)2] and  a chloride [Zn(CTZ)2Cl2] complexes) against T. vaginalis. We synthesized the zinc complexed CTZ compounds and determined their concentration values that inhibited parasite growth by 50% (IC50). We used scanning and transmission electron microscopy to visualize the ultrastructural alterations induced by CTZ and their zinc complexes. The incubation of the parasites with [Zn(CTZ)2(Ac)2] complex inhibited their growth, yielding an IC50 of 4.9 µm. Moreover, there were changes in the shape of treated parasites, including the formation of surface projections that subsequently detached from the cell, in addition to changes in the hydrogenosomes, endoplasmic reticulum and Golgi complex. We found [Zn(CTZ)2(Ac)2] to be a highly effective compound against T. vaginalis in vitro, suggesting its potential utility as an alternative chemotherapy for trichomoniasis.
Myxozoans are widespread and common endoparasites of fish with complex life cycles, infecting vertebrate and invertebrate hosts. There are two classes: Myxosporea and Malacosporea. To date about 2500 myxosporean species have been described. By comparison, there are only five described malacosporean species. Malacosporean development in the invertebrate hosts (freshwater bryozoans) has been relatively well studied but is poorly known in fish hosts. Our aim was to investigate the presence and development of malacosporeans infecting a diversity of fish from Brazil, Europe and the USA. We examined kidney from 256 fish belonging variously to the Salmonidae, Cyprinidae, Nemacheilidae, Esocidae, Percidae, Polyodontidae, Serrasalmidae, Cichlidae and Pimelodidae. Malacosporean infections were detected and identified by polymerase chain reaction and small subunit ribosomal DNA sequencing, and the presence of sporogonic stages was evaluated by ultrastructural examination. We found five malacosporean infections in populations of seven European fish species (brown trout, rainbow trout, white fish, dace, roach, gudgeon and stone loach). Ultrastructural analyses revealed sporogonic stages in kidney tubules of three fish species (brown trout, roach and stone loach), providing evidence that fish belonging to at least three families are true hosts. These results expand the range of fish hosts exploited by malacosporeans to complete their life cycle.
Little is known of the olfactory mechanisms of host detection in the ovipositors of endoparasitoids and ectoparasitoids. An endoparasitoid Aprostocetus causalis La Salle & Wu (Hymenoptera: Eulophidae) and an ectoparasitoid Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae: Tetrastichinae) are the two parasitoids of the eucalyptus gall wasp Leptocybe spp. Structures and sense organs of ovipositors of A. causalis and Q. mendeli were studied using scanning and transmission electron microscopy, which provided essential information for exploring the mechanism of host detection by endoparasitoid and ectoparasitoid. The ovipositors of two parasitoids consisted of the first and second valvulae and ended in a pointed tip. There were three types of microtrichia, two types of sensilla chaetica, and one type of sensilla campaniformia on the ovipositors of A. causalis and Q. mendeli. However, Q. mendeli has the fourth type of microtrichia on the ovipositor. The morphology, types, distribution, length, and width of these sensilla and microtrichia were described, and their possible functions are discussed in conjunction with the stinging, oviposition, and the host selection process.
Mullets inhabit a wide range of habitats from tropical to temperate regions and play a critical role in their ecosystems. This commercially important fish group constitutes a significant source of food in several geographic regions, and the production of some species for consumption is an increasing trend. About 64 myxosporean species have been reported in mullets, some of which are cryptic, as is the case of Myxobolus exiguus, and M. muelleri. This paper provides, for the first time, a detailed and critical revision of the data available for myxobolids reported in mullets, determining the species that have bona fide mugiliform fish hosts, in accordance with the original species descriptions, the available molecular data and the currently accepted taxonomic and phylogenetic criteria. Phylogenetic analyses using Bayesian inference and maximum-likelihood methodologies suggest that the evolutionary history of myxobolids with bona fide mugiliform fish hosts reflects that of its vertebrate hosts, while reinforcing known evolutionary factors and old systematic issues of the clade of myxobolids. A comprehensive morphological, ultrastructural and molecular redescription is also provided for the cryptic species M. exiguus, from infections in the visceral peritoneum of the thinlip-grey mullet Chelon ramada in the River Minho, Portugal.
Brycon orbignyanus is an important large teleost that is currently on the list of endangered species, therefore studies on its reproductive biology and embryology are fundamental to help species conservation and recovery. The objective of this research was to characterize the events that occur during extrusion, fertilization and embryonic development of the species. The samples were collected at predetermined times, fixed and processed for light microscopy and scanning electron microscopy. The greenish oocytes were spherical, had translucent chorion and a mean diameter of 1.3±0.11 mm. The eggs had well defined animal and vegetative poles approximately 18 min post-fertilization. Stages from 2 to 128 blastomeres occurred between 20 min and 3 h post-fertilization (hPF), when the morula was characterized. The blastula stage was observed between 2 and 3 hPF, and the gastrula between 3 and 7 hPF, when the embryonic shield emerged and the cellular migration with the consequent formation of epiblast and hypoblast. At 8 hPF, the formation of the neural tube, above the notochord and the encephalic region, was observed, delimiting the forebrain, mesencephalon and rhombencephalon regions. From 11 hPF onward, the optic vesicle was formed close to the forebrain and the embryo tail was well developed. The optic vesicle was observed from 12 hPF onward, and the tail showed an intense movement that culminated with the rupture of the chorion and consequent hatching of the larva at 13 hPF and 27°C.