RESULTS AND DISCUSSION

The general observations of main markers and time scale of embryonic development stages in G. mellonella was characterized in Table 1. The life cycle of G. mellonella at 30°C was described in Fig. 1.

External characteristics: Galleria mellonella eggs vary slightly in shape from spheroidal to ellipsoid, ovoid or obovoid, with a maximum×minimum diameter (mm) of 0.70±0.05×0.55±0.03 (n = 25). Early embryos (deposited during the first 3 h) have a larger surface and size in comparison with late stages. Aeropyles are sparsely distributed over the entire surface of the egg but there are fewer of them located on the anterior surface than elsewhere. Each egg bears 150-200 aeropyles with each opening measuring 1.6±0.6 μm (n = 25) (Fig. 1) and surrounded by a narrow to rather broad collar. The chorion is soft, fragile and thin (3.5 μm) immediately post-ovipostion (Fig. 1). The anterior end of the egg shows the petal of the micropylar rosette (Fig. 1). Superficial features of G. mellonella eggs were found to be identical to the description of Barbier and Chauvin³⁴.

Internal structures: The internal structure of newly laid eggs of G. mellonella has been compared with similarity to that described previously in D. saccharalis (Crambidae). Freshly deposited eggs contain a homogeneous granular yolk mass and were protected externally by a mucous substance secreted by the female. Different zones within the egg were visualized by Rhodamine B dye. One of these is the periplasm (PR), which is a layer of cytoplasm immediately under the chorion and vitelline membrane. Other zones include the yolk transition zone (TZ) comprising the region between periplasm and yolk and the yolk (YK). Only the central ooplasm (CO) was not clear with Rhodamine B staining. CO is the egg cytoplasm that contains the yolk, which is a nutritional reserve (Fig. 1). G mellonella eggs post-oviposition have posterior and anterior poles (Fig. 1).

Early embryogenesis: The characteristic features of early embryonic stages were summarized in Fig. 2 and 3. Previous studies that supported visualized markers of the early morphogenesis process in lepidopteran embryonic development were identified as follows.

Table 1:	Chronology of embryonic development of Galleria mellonella at 30±0.5°C, 85±2% RH
h: PO: Hours post-oviposition

Fig. 1:	Life cycle of Galleria mellonella. Embryogenesis stages covered

Fig. 2(a-b):	(a) Morphological micrographs of the eggs of G. mellonella., scanning electron micrographs of external surface, A: Aeropyles (arrows), B: Aeropyle openings, with measurements, C: Chorion section showing the thickness and D: Anterior pole of the egg, showing micropyle (arrows)
	(b) DIC micrographs of internal structure of the eggs after deposition, E: The egg showing the different zones of the ooplasm (PR: Periplasm, TZ: Transition zone, YK: Deutoplasm, CO: Central ooplasm) and F: Living egg just after oviposition (pp: Posterior egg pole, ap: Anterior egg pole)
	A: 50 μm, B: 10 μm, C: 5 μm, D: 20 μm, E and F: 100 μm

Fig. 3:	Overview of the development orientation in the embryos of G. mellonella. Scale bars of all micrographs: 100 μm

Maturation, fertilization and cleavage (0-4.5 h PO): The whole mount method is insufficient to visualize the nucleus of the female egg prior to entry of sperm that activate the female meiotic divisions. The formation phases of the cytoplasmic island (CI) in the primary post-oviposition eggs was shown in Fig. 4. A male pronucleus (MP) was clearly penetrated at completion time of CI formation. The MP was a black round spot in the yolk and its path behind it was clearly shown in the yolk.

Fig. 4(a-f):	DIC micrograph of the eggs of Galleria mellonella at 1st developmental stage. The development of the cytoplasmic island (CI) and its sequences is presented, (a) Egg at the beginning of CI formation, (b) The CI increases its emergence from AP side, (c) CI completion as a small ball independent from the yolk, (d) Bigger size of CI after completion shows differentiation during female mitosis divisions, (e) Kinetics of male pronucleus "MP" visualized inside the yolk and female pronucleus "FP" starts to appear as a black spot under CI and (f) FP migrates towards the MP before fertilization. Its path shows the events and the target place later
	Scale bars of all micrographs: 100 μm. AP: Anterior pole, PP: Posterior pole

The CI have variable shapes and are observed near the anterior pole in the beginning of this stage. The male pronucleus migrated towards the center of the egg (fusion position) where it should meet with the female pronucleus (Fig. 3a, Fig. 4f). Female pronucleus meiosis in the CI was not visually discernible here but it is reported to occur before this migration according to observations of the same phase in other lepidopterans³⁵. In three out of ten observed cases, multiple (two or three) sperm cells were visualized penetrating the egg as polyspermy (Fig. 3b). One of the male pronuclei usually fuses with the female pronucleus during syngamy, while the other pronuclei degenerate in the yolk as the CI decomposes to disappear later. Polyspermy was likewise observed in B. mori eggs³⁶ but not in other lepidopteran eggs. The progeny of the second maturation division (one female pronucleus and two polar bodies) was difficult to observe inside the CI during this stage but the division that appeared in cytoplasmic islands may be related to that mitosis (Fig. 4d). Fertilization was estimated to occur at 1.5-2 h. PO, in accordance with the second cleavage of two nuclei observed at 3 h. PO, which usually took place immediately after fertilization (Fig. 5a).

Many cleavage nuclei (energids) produced by each cleavage within a cytoplasm surrounded by a cellular membrane and the interval time (3 h) between each sample observation made it difficult to determine the exact number of energids or divisions from fertilization to the final blastoderm. Most cleavage nuclei migrated firstly toward the anterior half of the periplasm (Fig. 5b), then to the posterior half of the embryo, to cover all the periplasm (Fig. 3d). At that migration time, several cleavage nuclei remained in the yolk, where they came to resemble primary yolk nuclei or vitellophages (the nuclei arriving in the periplasm made a layer that prevented capturing the vitellophages in micrographs). The cleavage mode observed in G. mellonella eggs is a superficial, thus like that seen in other lepidopteran eggs. The beginning cleavage center in the anterior part of the egg was detected also in the eggs of B. mori ³⁷, whereas, cleavage in the center of the egg was observed in two lepidopteran species, M. sexta and D. saccharalis³⁸. Maturation, fertilization and cleavage in G. mellonella were generally similar to markers observed in eggs of lepidopterans such as Endoclyta signifier (Walker) and E. excrescens (Butler)³⁹ B. mori ⁴⁰ and Mnesarchaea fusilella (Walker). The observation of early embryonic mitosis in G. mellonella indicates that it is analogous to that of B. mori.

Blastoderm formation (5-11 h PO): The cleavage nuclei, once they arrived in the periplasm, were evenly and sparsely distributed in the cytoplasm and were equidistantly separated. Gaps appeared between the future blastoderm cell borders on the anterior, posterior and dorsal surfaces of the egg (Fig. 5c). Blastoderm formation was complete at about 9 h. PO and its cells started to be found in different size and appearance according to the position inside the egg (Fig. 3d,e). In the posterior and posteroventral regions the cells were small, round and compactly arranged and each cell contained a single nucleus as well as small yolk granules. This region is the future embryonic area. In the anterior and anterodorsal regions, the blastodermal cells are large, these cells typically contained one or two nuclei and many basal yolk granules that appeared to be spatially separated from each other.

Fig. 5(a-h):	Schematic representation of DIC micrographs of the sequence of early embryonic development in G. mellonella, (a) Second mitotic cleavage of two nuclei after fertilization, (b) The cleavage nuclei (CN) at the time of first arriving at the anterior pole, (c) The gaps (arrows) among CN that have arrived at the periplasm, (d) Blastoderm differentiated to the embryonic area and extra-embryonic area, (e) Germ disk before complete separation from serosa cells, stained with TB, (f) Germ disk (germ rudimentary GR) separated from Serosa and starting to form Amnion membrane under Serosa covering the germ rudiment, (g) GR sinks into yolk while connected to serosa membrane by the Amnion membrane and (h) GR during its movement into the yolk to transform to germ band
	Scale bars of all micrographs: 100 μm

This region is extra-embryonic and later will become the serosa (Fig. 5d). Blastoderm formation in G. mellonella eggs is different from that occurring in B. mori due to the lack of a uniform syncytial blastoderm⁴⁰. The cleavage nuclei in the blastoderm become larger than the yolk nuclei as similar to C. suppressalis eggs. Blastulation of G. mellonella embryos is identical to that characterized in B. mori, with the same invasion of energids into the periplasm, starting anteriorly and subsequently occurring elsewhere. Conversely, in the eggs of two other lepidopteran species, M. sexta and D. saccharalis, the nucleus invasion of periplasm was synchronous due to the cleavage beginning at the egg center and all cleavage energids being equidistant from the periplasm. The blastodermal cells differ in structure between embryonic and extra-embryonic regions (Fig. 5d), this character was detected also in other lepidopterans, including B. mori M. sexta and Epiphyas postvittana (Walker)⁴¹. Shape and relative size of the Germ Disk (GD) is identical to P. rapae and C. suppressalis (Ditrysia).

Germ disk, serosa formation and beginning of amnion (12-22.5 h PO): Soon after blastoderm formation, cells of the embryonic area actively proliferate to form a GD in the posterior region at about 12 h. PO (Fig. 5e, 3g). The GD is delimited from the extraembryonic area as commonly found in P. rapae embryos. During the formation of the GD, the amnio-serosal fold is formed along the margin of the GD (Fig. 5f, 6A, 3i). Then it shifts its position and the presumptive serosal cells flatten and fuse with another side above the separated GD to form the final serosal membrane which covers the entire egg surface (Fig. 5e, f).

Fig. 6:	DIC micrograph of the embryos of Galleria mellonella, A: Amnion formation starts during GD separation from serosa membrane, A’: GD sinks into yolk and assumes a contrasting shape from the two pole sides during anatrepsis movement, B: Embryos similar to cross section and crescent shape show differentiation of median plate (MP), C, C’: Completed amnion formation behind the extended germ band, D, D’: Completed germ band surrounded the yolk showing the connection between amnion and serosa at the end of the last stage, E: The final germ band formation
	Scale bars. A, E: 50 μm, B, C, D, D' and E: 100 μm

It begins to migrate across the GD at 15 h PO. In the G. mellonella embryos, immediately after splitting of the GD from the serosa cells, the presumptive serosa closes above the GD and is known also as the primary serosa cuticle. This synthesizes of serosa cuticle is similar to that which occurs in M. sexta⁴². Because the serosal cuticle is secreted from extra-embryonic serosal cells, it is not considered an embryonic cuticle as found in the mosquito Aedes aegypti (Linnaeus). The GD now is large and broad and occupies more than half the surface area of the egg (Fig. 5e). The GD differentiates into a germ rudiment and the amnion and the extra-embryonic area becomes a serosal membrane. The first sign of folding is usually seen laterally along either half of the GD (Fig. 5f). The rudimentary amnion of G. mellonella is initially represented by a few cells around the margin of the GD, like that detected in M. sexta. In G. mellonella, the formation of two embryonic membranes (amnion and serosa) follows the fault type (F-type) which was described in monotrysian and ditrysian embryos. This type is highly specialized and rare among insects, having been observed only in lepidopterans. In that type, GD condenses toward the ventral midline, with its margin delimited from the extra-embryonic area (Fig. 5g, h). The only specific difference in the serosa formation of G. mellonella eggs is that the margins of the developing serosa fuse before the start of anatrepsis movement and the amnion starts before the germ disk sinks into the yolk. Similar features were also found in the eggs of other basal moths except M. sexta, in which the amnion starts to cover the ventral face of the GD before anatrepsis occurs⁴³. It can confirm that serosa formation was completed at around 18 h PO and that the beginning of the amnion followed directly after this event.

Immersion of germ disk into the yolk, formation of the germ band during anatrepsis movement (22-31 h PO): After immersion into the yolk, the GD starts to differentiate into an amnion and germ rudiment before forming the germ band during anatrepsis movement. This movement starts post-completion of the serosa formation and sinking of the GD slightly into the yolk. The beginning of anatrepsis facilitates the amnion formation prior to the germ disk shortening in the process to form the germ band. Free margins of the GD bend toward the egg surface to grow into the narrow space between the serosa and GD, which fuse to initiate the process of forming the amnion (Fig. 5f, g, 6A). The long time needed for amnion formation may be related to the change in shape of the GD toward forming the germ band and its free movement in the yolk (Fig. 7d). The amnion continues to extend and is completed at about 32 h. PO (Fig. 6D,D’). These changes due to elongation of the germ band are distinct from anatrepsis, in that the GD initially extends in length and then shortens (Fig. 6B,E)⁴⁴, before it returns to increase in length again to become the final germ band (Fig. 6D). Connection between the serosa and the future cranial part of the embryo was observed during elongation of the germ band post-differentiation in the next stage (Fig. 6E’). Immersion of the GD into the yolk as anatrepsis movement was the first half of blastokinesis movement (Fig. 7). The extra-embryonic membrane formation in lepidopteran species differs and the difference is linked to anatrepsis movement.

Fig. 7(a-f):	DIC micrograph of the anatrepsis movement of Galleria mellonella embryos at 4th developmental stage (22-31 h PO), showing some parts of anatrepsis movement for formatting the germ band (24-40. PO), embryos stained with TB and Rhodamine B, (a) GD starts to sink into the center of the yolk and becomes compact from its borders, (b) GD becomes more compacted from two sides, (c) GD undergoes increased thickening and constriction at both ends, (d) Constriction of the two sides with the opposite wrap to both sides shows the white amniotic gap in ventral side of GD (black arrows) and its connection with serosa cells, (e) In this stage, the GD resembling the letter C but with shorter size and (f) Elongation and continued compression of the germ band edges (26h. PO)
	Scale bars of all micrographs: 100 μm

The GD begins its midline shifts transitorily towards the dorsal or ventral side of the egg and begins to roll up dorsally into a flattened vesicle (Fig. 7b, c, d). The GD is passively pulled into the yolk at the posterior pole as a consequence of concentric, anteriorly directed contractions of the yolk systems. These contractions are usually associated with a cleavage of the yolk in the eggs of some species⁴⁵. Following the cellular blastoderm formation, the embryos of most insects undergo a process of cell contraction or condensation, except for the most derived holometabolous species⁴⁶. The contractions of the GD and its increased thickness during this process were the first step in changing its cells from cuboidal to columnar shape. The GD rotates around its anteroposterior axis, causing its ventral side to face the lateral side of the egg and to become laterally compressed (Fig. 7e). Upon reaching its maximum length, the germ band assumes a C-shape (Fig. 6E). In the final stage, two embryonic membranes, serosa and amnion, are completed. The primary serosa cuticle surrounds the yolk and germ band, while the amnion membrane covers the ventral face of the germ band and stays connected to the serosa. Anatrepsis movement and formation of the serosa and amnion membranes in G. mellonella were identified from resemblance to these same events in C. suppressalis embryos.

CONCLUSION

The process of development of the greater wax moth is similar in most parts to the type of development generally seen in Lepidoptera but with some of its own specifications. The whole mount method with short interval collection time could be an effortless way to study the embryogenesis of insect eggs that are difficult to use in sectioning preparation methods and it could reveal morphological markers that have a phylogenetic relation in same-family species. The extra-embryonic area and the formation of membranes are unknown in G. mellonella, it is important to understand the tissue specification, the regulation of cell shape changes and tissue interactions during morphogenesis and ascertain the functions of these membranes between morphogenetic events.

SIGNIFICANCE STATEMENT

This study is discovering the characteristics of morphogenesis of embryonic development stages in G. mellonella that would be beneficial for selection of proper embryonic stage that needed for cryopreservation to succeed. This study will help the researcher to uncover the critical embryonic stages and their characters to withstanding the effects damage to the low temperature that may many researchers were not able to explore during their design for cryopreservation in Lepidopteran embryos. Thus a new theory on the importance of the early development stages for resistance the cold stress may be arrived at.

ACKNOWLEDGMENTS

This study was partly supported by a grant for "Storage at low and ultra-low temperatures of entomopathogenic microorganisms, nematodes and arthropods important in agricultural defence strategies" from the Agriculture and Forestry Ministry of Italy and "Protection of the environment and agriculture against the risk of importation of exotic arthropods" from the National Research Program PREVENTO of Italy. I gratefully acknowledge the valuable support of Prof. Y. Kobayashi (Tokyo Metropolitano Univ., Japan). I would like to thank my research advisors Dr. Pio F. Roversi (CRA-ABP, Florence, Italy) and Prof. Donatella Battaglia (Basilicata University, Italy) for their great support and to make this research exist. I wish to acknowledge the help provided by Dr. Elena Cosi and Ms. Claudia Benvenuto (CRA-ABP, Florence, Italy) for their support in the preparation of samples and SEM images. I am particularly grateful for the assistance given by Dr. Michael Poteser (Medical University of Graz, Austria), Prof. Christian Sturmbauer (Karl Franzens University of Graz, Austria) for their careful revisions on this manuscript. I would like to express my great appreciation to Terry L. Harrison (University of Illinois, USA) for performing the valuable revision and editing.