Eggs
Macroscopically, eggs of Aedes aegypti are whitish in color at the time of oviposition, which quickly becomes black. Some characteristics were identified, such as shiny appearance, tapered extremities, bilateral symmetry, in addition to a flattened surface opposite to a convex surface (Fig. 1A). Morphometrically we observed that the linear dimension of the population of eggs referring to the length was 581.45 ± 39.73 µm and the width of 175.36 ± 11.59. The egg index (length / width ratio) was 3.32 ± 0.26 µm. Concerning the measurement of the micropyle (DM) disk diameter of the evaluated eggs, it remained at 18.75 ± 1.92 µm (Table 1). The results were also compared with those from the studies by Suman et al., (13), Linley (22) and Faull (Fig. 1B).
Table 1
Morphometric parameters related to the linear size of the population of Aedes aegypti eggs coming from the municipality of São Paulo-SP in relation to measures of central tendency and dispersion. [This Table 1 should be attached between the 236 and 237 lines]
|
Atributes Morphometric
|
Central tendency and dispersion measures
|
|
|
Mean
|
IC95%
|
Maximum Value
|
Minimum Value
|
Eggs
collected
in
Sao Paulo
|
Length
|
581.45 ± 39.73
|
569.65-593.25
|
655.20
|
521.40
|
Width
|
175.36 ± 11.59
|
171.92–178.80
|
199.60
|
156.50
|
Length/width
|
3.32 ± 0.26
|
3.24–3.40
|
4.13
|
2.78
|
Diameter
|
18.75 ± 1.92
|
18.18–19.32
|
22.18
|
14.27
|
Table 2. Comparative analysis of the morphometric findings of Aedes aegypti eggs identified in this study and the results of Suman et al, (2011), Linley (1989), Faull and Williams (2016).
|
A. aegyptia *
|
A. aegyptib**
|
A. aegyptic***
|
A. aegyptid ****
|
A. aegyptie****
|
Length
|
581.45±39.73
|
625.65±19.91
|
670.2±7.2
|
554.41±36.56
|
562.62 ± 30.85
|
Width
|
175.36±11.59
|
183.30±11.04
|
186.3±2.2
|
167.65±7.05
|
160.15 ± 9.73
|
Length/Width
|
3.32±0.26
|
--------
|
3.61± 0.05
|
3.31±0.18
|
3.52 ± 0.27
|
Diameter of micropilar disc
|
18.75±1.92
|
--------
|
--------
|
33.49 ± 3.9
|
34.19 ± 5.4
|
a.Mundim-Pombo et al b.Suman et al, 2011 c. Linley,1989 d. Faull and Wiliams.,2016 e. Faull and Williams, 2016
* Population of São Paulo eggs, **Population of India eggs ***Population of Florida USA eggs **** Population of Cairns-Australia eggs ****Population of Charters Towers eggs - Australia
Ultrastructural SEM showed that the extremities are characterized by poles, the anterior pole, where the entire micropylar apparatus (disc/crown of the micropyle, sectors of the disc of the micropyle and micropyle) is located and is slightly prominent when compared to the opposite pole. The microcapillary device maintains a prominent and continuous circle shape (Figs. 1C and 1D), while the posterior pole is more tapered in relation to the opposite side (Figs. 1C and 1E). In the external coating of the eggs, regularity was identified about the aspect of distribution and shape of their cells, most of them maintaining a hexagonal shape (Figs. 1C and 1E).
In the central region of the chorionic cells, larger diameter tubers are identified. The tubers are symmetrically arranged, there are small peripheral tubers arranged in an organized manner around larger central tubers (Fig. 1F, 1G and 1H). Both appear prominently in the exochorion, thus giving the appearance of high relief or texture.
Transmission microscopy was used in a complementary manner to analyze the chorion (Fig. 1I-L), where it was possible to identify the ornamentations of the exochorion and confirm that the impermeable characteristic of this structure prevents the entry of fixers and resin inside the cell in the sample processing, causing the compromise of these cells. Another finding in relation to the exochorion is that it was more electron dense when compared to its adjacent layer, the endocorion.
The impermeability characteristic of chorion drew attention, which made the analyzes related to it, as being extremely difficult and generating several repetitions in the different stages of this study.
Embryo
The embryonic development of Aedes aegypti occurs inside eggs of up to 1 mm in length (Fig. 1A), dark in color and capable of adhering firmly to various substrates. In the period that comprises the initial third of the development of the Aedes aegypti embryo, it was possible to verify the scarce presence of bristles in the cephalic region and relatively homogeneous structures in terms of their general morphological aspects. Rupture of the chorion (Figs. 2A and 2B), which in turn will open the area outside the egg and release the larva. The identification of this region as being the cephalic region is possible because this region coincides with the micropillar apparatus.
In the histological analysis, elements characteristic of the ornamentation of the exochorion were identified, since surrounding this region are the central (larger) and peripheral tubercles (Fig. 2I and 2J). In the interior of the egg, embryonic cells are identified, and specifically in the initial third of development (5 hours), it was possible to observe in a semi-thin section, the presence of round cells with a peripheral nucleus and several cells presenting two nuclei, a process suggestive of cell division (Fig. 2K, 2M and 2N). Still in the initial stage of embryonic development, after 18 hours from the analysis of the semi-fine cut, folding of the embryo was found, around the egg internally in about 60% of it, a situation called the extension of the germ band and occurs at the moment of gastrulation when the ventral blastoderm undergoes extension (Fig. 2C, 2D and 2E), it is also suggested that the process of cellular activity is maintaining itself intensely after 18 hours of the onset of cell development, as a large number of cells were found in the process of division (Fig. 2G and 2H).
The intermediate phase (Fig. 3A-F) of development brings characteristics such as the increase in the presence of bristles, including palatal brushes, which in the future will allow the larva to perform movements promoting water flow to bring food to be consumed. There is also a separation of the cephalic and thoracic region and loss of homogeneity of the general morphological aspect. At the developing embryo stage, is evident the presence of a membrane surrounding the embryo and, therefore, located between the embryo and the chorion, this extraembryonic membrane is the serous layer (Fig. 3G).
Still at this stage, embryos were analyzed after 25 hours (Fig. 19C) and 30 hours of development (Fig. 3H to 3M). During this period, the process of formation of the abdominal and thoracic segments and structures such as the respiratory siphon and the last abdominal segment in the form of a caudal appendix (Fig. 3L), a spike that will promote the cleft to release the larva at the time of hatching, was observed of the egg, in addition to the presence of bristles in the cephalic region (Fig. 3M).
In the final third of development (Fig. 4A-D) the body of the future larva already has the divisions in the head, thorax and abdominal segments, but the indicator for completion of growth is mainly the presence of the chorion-breaking spike (Fig. 4C-H). This structure persists until the first larval stage (Figs. 4I and 4J), a factor that differentiates the first and second larval stages. In the period of completion of embryogenesis, prominence in the chorion was identified (Fig. 4E), specifically in the anterior region, which indicates that the larva is ready to gain the aquatic environment. The prominence identification site was the one that gave rise to the rupture of the transverse fissure in this region by the chorion-breaking spike (Fig. 4F-H), in the egg after completion of the embryonic development. This region will be the one where the larva will leave the egg. Histologically, the final stage of development (Fig. 4K, 4L, 4Q) shows that segmentation is better defined, as well as the primitive intestine (Fig. 4M), the discreet presence of bristles in the cephalic region is replaced by the abundant presence of these structures (Fig. 4P).
It is known that an important element of the cytoskeleton is actin, and this, in turn, was marked by the reaction with phalloidin, which then made it possible to understand its distribution by cells, while the nucleus was marked by propidium iodide. The images obtained by confocal microscopy made it possible to follow the sequence in embryonic development after 18 hours of development (initial phase) until the moment when the larva is ready to gain the environment (Fig. 5A to 5F). In this analysis it was possible to identify the embryo in a situation of extension of the germ band (Fig. 5B), after 30 hours of development the serous cell layer surrounding the embryo (Fig. 5B) was clear, and at the end of the development it was verified in 40 hours, the process of segmentation and dorsal closure (Fig. 5C) and within 50 hours an embryo was observed whose thoracic and abdominal segmentation was well defined (Fig. 5D), it is possible to observe the moment of the larvae hatching (Fig. 5E) and for end the embryo after 7 days diapause, with its development finished, ready to gain the external environment. In this case, the presence of visible segmentation was found and between the fifth and sixth abdominal segment, it was possible to show the intestine (Fig. 5F).