Dynamic Morphological Staging of Drosophila Pupal Development

Dynamic Morphological Staging of Drosophila Pupal Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dynamic Morphological Staging of Drosophila Pupal Development Xu Liwen, Chen Mei, Zhang Yumeng, Pang Xuan, Tang Jiaxu, Zhang Chenchen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6337002/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study provides a comprehensive analysis of the morphological-developmental characteristics of the pupal stage in Drosophila , a model organism for metamorphosis research. We have delineated the critical stages of pupal development, from the late third-instar larvae to the adult emergence, through continuous photography and time-lapse imaging. Our findings include the identification of the head-movement phase and head-static period as distinct transitional states preceding the pupal stage. We describe the dynamic changes in the pupal shell, including color transitions and the development of spiracles, which serve as reliable markers for staging. Additionally, we detail the morphological transformations of internal tissues and organs, such as the degeneration of larval tracheae, the retraction and degeneration of mouthparts, the formation of compound eyes and wings, and the development of appendages and bristles. Our research also highlights the significant morphological changes in pupal tissues, including the movement and displacement of bubbles, the appearance and migration of Malpighian tubules and corpora allata. In conclusion, we have identified several critical periods: P10 serves as a definitive marker for the onset of segmentation during the pupal stage, P40-P50 represents an optimal period for observing the initiation of changes in eye and wing coloration, and P70 marks the beginning of the investigation into the morphological changes of the adult within the Drosophila pupal case. We also found that compared with wild-type Drosophila , mutant fruit flies showed some differences in development time and pupal tissue organization. These observations offer a detailed developmental map of the Drosophila pupal stage, which is essential for accurate experimental staging and sampling. This study's findings not only contribute to the fundamental understanding of insect metamorphosis but also provide a valuable resource for researchers utilizing Drosophila as a model system to study gene expression, development, and behavior. Drosophila Metamorphosis Pupal Development Morphological Staging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1 Introduction Metamorphosis [ 1 ] encompasses drastic morphological and physiological transformations across an organism’s life cycle. This process encompasses different growth stages, accompanied by drastic changes in the organism's appearance and internal structure, in order to adapt to the demands of a variable ecological environment and specific living habits. Metamorphosis is not uncommon in the biological world; it occurs in insects [ 2 ], amphibians [ 3 ], certain fish [ 4 ], and some marine invertebrates [ 5 ]. Among them, the existence of metamorphosis in insects is the most widespread [ 6 ]. It is estimated that over 98% of insect species undergo varying degrees of metamorphosis during their developmental cycle [ 7 ]. Currently, insects are generally classified into three main categories based on their metamorphic development: ametabolous, hemimetabolous, and holometabolous [ 8 ]. Ametabolous insects refer to a group that undergoes only changes in body size during development, without any change in shape, such as Entognatha and Apterygota , which continue to molt throughout their lives [ 9 ]. Hemimetabolous development includes three main stages: egg, nymph and adult. In hemimetabolous development, the nymph gradually approaches the adult form, but there are no significant morphological changes. At this time, the living habits and diets of nymphs and adults remain highly consistent [ 10 ]. Grasshoppers [ 11 ] and cockroaches [ 12 ] are commonly used animal models for studying hemimetabolous development. In contrast, the metamorphosis of bees [ 13 , 14 ], dragonflies [ 15 ] and butterflies [ 16 ] begins with a fertilized egg and goes through four stages: egg, larva, pupa and adult. The larval stage focuses mainly on feeding and nutrient accumulation. The pupal stage involves extensive and intense changes in cellular organization, as well as the remodeling of body shape and organs, ultimately transforming into the adult form. After developing into an adult, it will complete the functions of reproduction and gene transmission, maintaining survival through activities such as flying and foraging. Metamorphosis that shows significant differences in morphology and behavior at each developmental stage is called complete metamorphosis [ 17 ]. Among all the metamorphic development methods of insects, holometabolous development accounts for the largest proportion [ 18 ]. Holometabolous insects are considered to be the most evolved group within the class Insecta, and they are also the most diverse in terms of species [ 10 ]. Therefore, most research is primarily focused on complete metamorphic insects [ 19 ]. Within this group, Drosophila has emerged as a key model organism due to its experimental tractability [ 20 , 21 ]. Currently, research on Drosophila mainly focuses on the larval and adult stages [ 22 ]. In contrast, the pupal stage, which occupies nearly 100 hours in the Drosophila 's life cycle, has relatively little research due to the unclear details of its overall development process. However, the pupal stage is a very important phase; during this time, the nutrients acquired during the larval stage gradually take effect within the pupal body, and both the imaginal discs and nerve cells develop [ 23 ]. The process of imaginal disc development into adult organs can be observed through a stereo microscope, but the central nervous system cannot be directly observed. If we can grasp the development of the adult organs corresponding to the central nervous system development during the proposed research period, it will greatly enhance the accuracy of experimental sampling time. As a classic research subject among holometabolous insects, Drosophila exhibits significant differences at various developmental stages, similar to other holometabolous insects. Therefore, it is essential to conduct detailed staging when performing in-depth mechanistic studies [ 24 ]. Currently, mainstream experimental protocols define the absolute time after pupation by hours after pupal formation (APF) to represent the staging of pupal development [ 25 , 26 ]. However, considering the differences in developmental speed, genetics and microenvironment among individual pupae, it is challenging to ensure that all pupae are at the same developmental stage under absolute time conditions. As development progresses towards the later stages of pupation, the synchronization among individuals decreases [ 27 ]. Sole reliance on temporal staging compromises accuracy and reproducibility, highlighting the need for a continuous morphological map of pupal development. Currently, many visualization imaging techniques have been applied to the study of insect metamorphosis, some of which have also been used in Drosophila [ 28 ]. Considering the advantages and disadvantages of various imaging techniques, due to the semi-transparent nature of the Drosophila 's cuticle, it is relatively convenient to observe the overall morphology of organ development during the pupal stage using optical microscopy after moistening the specimen. Correspondingly, there is indeed a substantial amount of research that applies morphological changes during the pupal stage to the study of metamorphic development mechanisms [ 29 , 30 ]. Compared to merely using a temporal scale to categorize the Drosophila life cycle, employing developmental staging definitions based on morphological characteristics can yield better comparative results when comparing multiple individuals [ 28 ]. However, most experiments observing the pupal stage of Drosophila involve removing the experimental animals from their pupal cases for filming and recording [ 31 , 32 ], during which the documentation of the characteristics of the pupal cases may be lost. Currently, the most detailed morphological staging of the Drosophila pupal stage was established in 1981, classifying 24 developmental stages from 51 observable morphological sequences In recent years, many studies have continued to use the staging method from this literature [ 33 ], but this method also studies experimental animals after the removal of the pupal shell. Additionally, due to the earlier time period, imaging equipment was relatively outdated, leading to a rough classification of the early Drosophila pupal stage. In fact, during the prepupal stage, the central nervous system synapses of the Drosophila have already begun to degenerate [ 34 ], but earlier literature did not provide a relevant classification for this period. Therefore, based on existing research progress, we utilized a high-resolution stereomicroscope to observe and record the complete developmental changes of the wild-type Drosophila melanogaster pupal stage through continuous photography and time-lapse imaging, aiming to provide a basis for accurately defining the developmental stages of the pupa and for precise sampling. At the same time, in the central nervous system of the Drosophila , transmembrane proteins encoded by the Neurexin and Neuroligins genes are located on the presynaptic and postsynaptic membranes respectively. Their interaction is significant for forming synaptic connections and maintaining the normal structure and function of synapses [ 35 ]. Current research has clearly shown that the nervous systems of dnrx and dnlgs are inevitably affected during their development [ 36 ]. However, we are still unclear whether there are differences in the morphological development during the pupal stage between mutant Drosophila and wild-type Drosophila , and at which specific stage of the pupal phase certain homozygous lethal mutant strains are unable to continue normal development. Therefore, we used the method of observing the morphological development of wild-type Drosophila pupae to simultaneously observe the morphological development of the mutant pupae, recording the characteristic morphological features of dnrx and dnlgs during their development, and further linking these features to the poor development of their nervous systems. 2 Materials and Methods 2.1 Fly stocks and rearing conditions The flies used in this experiment include CS , dnrx 273 , dnlg2 KO70 , dnlg3 KO127 and dnlg4 KO10 . Pour 30 flies (half female and half male) into a large glass bottle or fly vial containing standard culture medium. Rear the flies at 25°C and expose them to a 12h:12h light-dark cycle. The rearing room was equipped with air conditioning and a humidifier to maintain constant conditions (25.0 ± 0.5°C, 60% relative humidity). 2.2 Sampling and Photography Methods during the Pupal Stage We began to record the characteristics of the late third instar larvae. The late third instar larvae tend to search for suitable pupation sites on the tube wall, but the shooting range of the camera is limited. They often actively avoid the light source of the camera, thus hindering the observation of the characteristic changes of the late third instar larvae entering the pupation stage. Based on this situation, we designed the following experiment utilizing the characteristic of late third-instar larvae stopping feeding and crawling up the tube walls: We initiated the experiment by inserting six to eight cleaned and dried pathological slides into the culture medium of each large glass bottle, followed by incubation under standard conditions for two to three days to facilitate pupation of the larvae on both sides of the slides. Alternatively, for sample collection, Drosophila tubes containing standard culture medium could be employed. In this scenario, a transparent polypropylene film was cut into rectangles measuring 5cm × 10cm, inserted into the culture medium, and incubated under standard conditions until the larvae migrated onto the polypropylene film. When third-instar larvae were observed to be motionless on the slides or polypropylene film, mechanical stimulation was applied using a soft-bristled brush. If the larvae resumed crawling, they were permitted to continue growing. However, if they remained motionless, they were deemed to have entered the Head-static period (HSP). Subsequently, the slides or polypropylene film were removed using tweezers, and larvae, pupae, and food residues on one side were wiped off with a wet tissue. Any excess food residues on the observation side were also cleaned off. The slide was then positioned under a high-resolution stereomicroscope (OLYMPUS SZX7), where the bottom and side light sources were adjusted, a magnification of 1.6× was selected, and the complete process of the respiratory openings extending from the late third-instar larvae to the P0 stage was captured. Based on the established morphological characteristics of the P0 stage, we collected ten typical P0 specimens with distinct morphological features and arranged them neatly on a new clean glass slide with the ventral, dorsal, and lateral sides facing up. A small amount of glycerin was applied to the surface of the samples using a brush to enhance surface contrast and reduce moisture evaporation from the pupae. The slide containing the samples was then placed on the imaging device, a magnification of 1.6× was selected, and continuous or intermittent video recording of the samples was conducted to observe the developmental status of the fly pupae from different observation angles. Concurrently, changes in the complete morphological characteristics of the pupal stage and their appearance times were recorded, obtaining time-marked characteristic organs such as tracheae, mouthparts, compound eyes, wings, appendages, bristles, and the overall shape of the pupa, as well as bubbles, Malpighian tubules, and the corpora allata . For the assembly of the shooting device, a piece of filter paper was placed on the bottom light source to prevent interference from the background color. A petri dish (90mm in diameter) was placed on a hollow support, the hollow bottom of which increases air flow and thereby reduces heating of the petri dish. Inside the petri dish, a glass slide was placed on top of a 3cm high support, and the polypropylene film containing the sample was positioned on the glass slide. The petri dish was then sealed with transparent cling film, and a 1cm × 1cm breathable hole was cut directly above the sample to prevent water mist interference during shooting and to maintain humidity. Finally, an appropriate amount of deionized water was injected into the petri dish; the water level did not cover the glass slide but maintained humidity throughout the shooting process. 2.3 Continuous video recording and time-point sampling observation during the pupal stage Due to the potential impact of transferring the P0 phase onto the slide with a brush on the development of the pupae, we validated the characteristics of the pupal development of fruit flies in the fly culture tubes based on the preliminary data obtained from the aforementioned experiments. We began the experiment by introducing more than 15 adult flies, with an equal ratio of males to females, that had eclosed for 3–7 days into culture tubes, with approximately 20–30 tubes used in total. These tubes were cultured under standard conditions for 2 days, after which the adult flies were extracted, and the tubes were cultured for an additional 3–4 days. During this period, we observed the presence of stationary white pupae, brown pupae, and larvae on the walls of the tubes. To systematically track the development, we used a marker to number each culture tube (A1, B1, C1, etc.) and marked the white pupae on the walls of each numbered tube, recording the initial marking time as T1. The marked tubes were then placed back into the incubator. After 1 hour, the tubes were removed, and new white pupae that had appeared on the tube walls were marked with a marker. The marking time was recorded as T2 (T2 = T1 + 1h), along with the corresponding number of newly marked pupae. This process was repeated every hour, with each marking session kept within a 10-minute window, until the required quantity of samples for the experiment was reached. It is important to note that the white pupae (P0) collected at T1 were not used for the experiments. Instead, the culture tubes containing different batches of P0 collected after T1 were placed in an incubator for continued cultivation. Pupae that had been cultivated for 2 hours were marked as P2, and similarly, those cultivated for 4 hours were marked as P4. The entire pupal period was divided into 24 stages: P0, P2, P4, P6, P8, P10, P12, P14, P16, P20, P24, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, and P90. Based on the time records, we collected samples from the tube walls using a brush at the corresponding time points and arranged them sequentially on a slide. By examining the consistent developmental characteristics observed in more than half of the flies at the same time point, we made judgments and corrections on the recorded time-marked morphological features. Finally, we compared the appearance time and characteristics of various morphological features, including tracheae, mouthparts, compound eyes, wings, appendages, bristles, and the overall shape of the pupa, as well as bubbles, Malpighian tubules, and the corpora allata . 3 Results 3.1 Definition of the Head-movement phase, Head-static period, and White Pupa Stage According to our aforementioned method, individuals at different developmental stages can be observed within the same field of view (Fig. 2 A-E, Video S1). In addition to the wandering third instar larvae (which we refer to as L3w) (Fig. 1 A) and the stationary brown pupae (Fig. 2 E), three other types of developmental individuals can also be seen. Two of these exhibit a shortened larval form, which we tentatively name the head-moving stage (Fig. 2 B) and the head-stationary stage (Fig. 2 C) based on their external morphology and behavioral characteristics. The third type resembles the brown pupa but has a white exterior, which we call the white pupa (Fig. 2 D). Head-movement phase In the L3w stage before the head movement stage, feeding has stopped, and individuals crawl around on the slide and the walls of the tube, aiming to find a suitable position to pupate. During the head movement stage, the body of the individual shortens, and the cuticle on the surface of the abdomen hardens and adheres to the slide. The mouthparts of the thorax extend and retract, causing the individual to sway back and forth in place (Fig. 2 B), with the mouthparts not fully retracted into the body. When illuminated with a light source or moved with a brush, individuals in the head movement stage will crawl again on the slide, relocating to other positions to pupate. Head-static period During this period, the entire epidermal stratum corneum of the individual hardens and shrinks, completely adhering to the slide, with both the head and tail unable to move (Fig. 2 C), and the mouthparts fully retracted into the body. When subjected to slight brush stimulation or weak light stimulation, the head and tail still do not move. If water is dripped and the stimulation time or intensity is extended, the head will first exhibit forward and backward movements, while the tail remains adhered to the slide. Intense stimulation can cause some individuals to crawl a short distance. White pupal stage (P0) The epidermis has completely hardened into a milky white pupal shell; the mouthparts retract into the pupal shell's rostrum, and the spiracles extend from the body and protrude on both sides of the rostrum (Fig. 2 D). There is no extension or crawling response to light or brush stimuli. Since the duration of the white pupal stage lasts for about 1 hour (according to literature), this paper considers the white pupal stage as the beginning of the pupal stage, marked as P0 (Fig. 3 A-C). 3.2 Morphological changes of pupal shell The hallmark characteristic changes of pupal stage included changes in pupal shell morphology and pupal tissue, among which the pupal shell morphology mainly included changes in spiracle and pupal shell color. 3.2.1 Spiracles change The spiracles were branched, hidden in the body during the head movement and head static stages (Fig. 2 B-C), and protruded on both sides of the snout during the white pupal stage (P0), with 4–7 branches of about 0.05 mm in length on each side. Thereafter, no significant morphological changes were observed in the spiracles until the pupal stage (Fig. 2 D-E). The changes in the location characteristics of the spiracle can provide a reference for distinguishing the white pupal stage from the head static stage and head movement stage. 3.2.2 Pupal shell color change In the white pupal stage, the pupal shell color can change significantly within 2 hours. At the white pupal stage, the pupal shell was milky white (Fig. 3 A). As development progressed, the pupal shell color gradually deepened from the posterior spiracle of the tail. As observed by serial photography, the pupal shell color changed to light brown about 1 hour after the fly entered the white pupal stage (Fig. 3 C). After 2 hours, the pupal shell color deepens to brown (Fig. 3 E) and enters the late pupal stage. With the appearance and maturation of bristles on the pupal surface inside the shell (Fig. 3 J-K), the pupal shell surface gradually deepens from brown to tan (Fig. 3 K). Once the pupal body emerges as an adult, the remaining pupal shell loses the foil of pupal body and bristles, and appears brown again under the stereoscope (Fig. 3 L). Pupal shell color changes rapidly in the early stage of pupal development and is an important basis for judging pupal P0 stage. 3.3 Morphological changes of pupal tissues In the pupal stage, the shape of pupal shell changes less, but the pupal tissue morphology in pupal shell changes significantly with the development process. During this period, most tissues and organs of larva will gradually disintegrate and fade in the pupal stage, and tissues and organs of adult will gradually develop or rebuild. The pupal tissues and organs were divided into static and dynamic morphological changes according to the criterion of whether they underwent obvious positional migration during metamorphosis. The former refers to the tissues and organs that changed morphological characteristics at the same position in the pupal body during the whole pupal stage, including tracheae, mouthparts, compound eyes, wings, appendages and bristles. The latter were the tissues and organs that showed characteristic changes during the whole pupal stage and dynamic position migration, including pupal shape, bubbles, Malpighian tubules and corpora allata . 3.3.1 Degeneration of the dorsal tracheae of the larva The tracheae is a structure for gas exchange between Drosophila and the outside world, and the dorsal tracheae of Drosophila larvae will show obvious morphological changes during the pupal stage. In the larval and white pupal stages (P0), the tracheae is curved in vivo (Fig. 4 A-B '), but the curved tracheae will straighten out after the P10 stage (Fig. 4 B-C,B '), and then complete the extinction process in two ways. In the first way, after the tracheae became straight, the tracheae were curved again, and the bending position was random, which might be the upper end, middle end or lower end (the bending position of the sample was the lower end again). The degree of bending was different in different individuals, and this re-bending could be observed more than one hour after entering P0 stage. Maximal bending was observed after approximately 2 hours of development (Fig. 4 D). The time it takes to straighten again varies from individual to individual (Fig. 4 E), and after P10 the tracheae become very blurred (Fig. 4 F). These blurred tracheae are disturbed by the movement of bubbles within the pupa shell within 10 to 20 minutes, and most of them disappear within a short time. Only a small segment of the upper and lower end of the tracheae of the original larvae were preserved (Fig. 4 G-H). The preserved upper and lower end tracheae are connected to the spiracle and posterior spiracle exposed outside the pupal shell, respectively, giving a relatively uniform appearance of the pupal shell throughout the pupal stage. In the second way, after the tracheae become straight, it will remain straight until P10, until the blur disappears (Fig. 4 C'-H'). The first type of tracheal degeneration accounted for only 20%, but most pupae showed the second type of tracheal degeneration (the number of samples observed was 10). The time of tracheal disappearance can be used as a reference for P10. 3.3.2 Pupal stage mouthparts retraction and degeneration The black mouthparts of Drosophila larvae begin to retract after entering the head quiescent stage, and will completely retract and degenerate within 12 hours after entering the pupal stage. The process can be observed from the dorsal and ventral sides, which can be divided into four stages: slowing down of the mouth parts, shrinking of the mouth parts, immobile of the mouth parts, and abscission of the mouth parts. At L3w, the black mouthpiece at the top of the larva's head expands and expands to feed (Fig. 2 A; Fig. 5 A,A'). After entering the head movement stage, the mouthparts were still above the snout, continuously stretching back and forth and driving the chest movement, but the expansion of the mouthparts slowed down significantly during this period (Fig. 2 B; Fig. 5 B,B '). Here, we carried out a supplementary experiment, taking 10 third instar larvae (non-head movement stage, head static stage) and head movement stage, and counting the average number of mouth retraction per minute in each stage. According to the statistics, the third instar larvae (non-head movement phase, head-static phase) on average expand 67 times per minute and head movement stage larvae on average expand 33 times per minute. (Statistics are provided in the Supplementary Data) During the cephalostat phase, the mouthparts were retracted into the worm body (Fig. 2 C; Fig. 5 C,C '). Then, during the development from the ab static stage to the white pupal stage (P0) and 30 minutes into P0 stage, the mouthparts in the pupal body would continuously recluse, and the distance from the pupal shell to the rostral end gradually increased (Fig. 5 C-E,C '-E'). The mouthparts stopped constricting 30 min after entering the pupal stage and were quiescence after a period of in situ fibrillation (Fig. 5 E-F,E '-F'). After stage P10, the black mouth parts separated from the newly formed pupae to complete abscission, and thereafter remained in the same position in the pupal shell until emergence (Fig. 5 G-L, G '-L'). The changes of mouthpiece retraction during the pupal stage provided the basis for distinguishing the head movement stage from the head static stage, and was also a major feature of P10 in the pupal stage under standard culture conditions. 3.3.3 Compound eye formation and eye color changes Drosophila melanogaster has compound eyes. After 10 hours of development from P0, with the formation of the head, thorax and abdomen of the pupal body, the position of the compound eyes can be determined by the pupal shell, which is located on both sides of the head (Fig. 6 A,A '). At this time, the pupal shell was removed, and it was found that the pupal body was full of particles. When the pupal body was puncted with forceps, a large number of particles emerged, and adult tissues such as compound eyes did not form at this time (Fig. 6 B,B '). During the period from P35 to P40, the pupal shell of the sample from this period was removed, and the particles could not be observed. Adult tissues such as compound eyes formed, and a transparent film covered the tissues, which was called pupal cuticle. During this period, there was no pigment deposition in the compound eyes, and the eye color was consistent with the pupal shell (Fig. 6 C-D,C '-D'). Pigmentation only begins to appear around the contour of the compound eye (Fig. 6 A-E,A '-E'). During the next 10 hours, the pigment gradually deepened from the periphery to the middle of the compound eye until the entire compound eye became orange (Fig. 6 E-H,E '-H'). At this time, the pupa developed for about 55 hours. During the next 5 hours, the orange compound eye darkening to brownish brown (Fig. 6 H-J,H '-J'); After 10 hours of development to P70, the eye color changes from brown to red (Fig. 6 J-N,J '-N'); Then it enters the late pupal stage. Due to the gradual deepening of the pupal shell color, the color of the compound eye observed through the pupal shell also gradually deeps until it becomes red and black before emergence (Fig. 6 N-R,N '-R'). 3.3.4 Wing formation and color change The formation of wings was in the same period as the compound eye, about P35-P40. The pupal shell could not be observed through the pupal shell. When the pupal shell was removed, the wings were located under the pupal cuticle, close to the sides of the body, and the wings were transparent. Wing color change occurs during the late pupal stage and lasts about 10 hours. From P65-P70, the ends of the wings showed color and gradually deepened to gray, while the other parts of the wings showed no color change (Fig. 7 E-H). With the development, the wing color gradually deepened. At 75 hours of development, the end of the wing color deepened to black, and the root of the proximal end of the wing became gray (Fig. 7 H-K). After that, the color of the wings does not change again until emergence (Fig. 7 K-N). 3.3.5 Appendage formation and maturation The newly formed appendages were difficult to observe through the pupal shell. During P35-P45, when the pupal shell was removed, the three pairs of appendages were closely juxtaposed from top to bottom on the ventral side of the pupal body. The development of appendages was slow, and the landmark morphological changes of legs did not occur until after the P65 stage. Between P65 and P70, legs became brown in color and began to appear segmented (Fig. 8 A-B); At P75, the leg color was still brown, but the segments became prominent (Fig. 8 C). From P75 to P80, the legs grew black fine hair, and the leg color was black at this time (Fig. 8 D,D '). During this period, the sexual comb structure was observed on the first tarsus of the forefoot of the male individual (Fig. 8 D), which was black patches. After development to P80, the sex of Drosophila can be distinguished by sexual comb. At P90, the legs of males and females developed differently, and the legs of males did not show obvious changes (Fig. 8 D-F). However, at this time, the legs of females developed faster than those of males, and the fine hairs on the legs became hard (Fig. 8 D '-F'). If the pupae shell was removed at this time, the females could walk. 3.3.6 Formation and hardening of bristles Pupal bristles were classified into three categories based on the position of adult bristles: head bristles, chest bristles, and abdominal bristles. The morphological changes of the three types of bristles occurred in the late pupal stage, about P65-P85. Head bristles were first observed between P65 and P70 (Fig. 9 A-B); Chest bristles were observed from P70 to P75 (Fig. 9 B-C). Abdominal bristles were last observed between P75 and P80 (Fig. 9 C-D); All three types of bristles develop and mature within the next 5 hours. The bristles are black and hard (Fig. 9 E-F). 3.3.7 Pupal head, thorax and abdomen segments and their morphological displacement changes At P0 stage, the newly formed pupal body was not separated from the pupal shell and retained most of the larval structure (Fig. 10 A). After development to P10, the pupal body first moved to the snout as a whole under the action of bubbles at the lower end of the shell (Fig. 10 B). Within 10 minutes, the pupal body moved to the tail to complete the separation of the pupal body from the pupal shell and form the head, thorax and abdomen. At this time, the outline of each part was not obvious (Fig. 10 B-C). The whole pupal body gradually moved downward until it was about 1/4 from the upper end of the pupal shell, during which the outline of the head, thorax and abdomen gradually appeared (Fig. 10 C-G). One hour before emergence, the pupal abdominal segment gradually elongates, causing the head to move up to the front of the pupal shell. The pupal shell is poked and the pupal shell is left behind by the emerging adult (Fig. 10 H-N). The separation of the pupal body from the pupal shell to form the head, thorax and abdomen was one of the most significant temporal morphological changes in the pupal stage. Based on this morphological change, it could be quickly inferred whether the pupal development time was before or after P10. 3.3.8 Appearance and displacement of bubbles in pupae Bubbles were observed both in the pupal stage and in the pupal stage, and both of them had obvious characteristics of expansion and migration. Bubbles in the pupal body appeared earlier and were divided into lateral and central pupal bubbles according to the location of their appearance. Pupal side bubbles appear below the abdominal unilateral (or left or right) tracheae shortly after the pupal shell color darkens to brown and after P2 development, at which point the bubble diameter is smaller than the spacing of the two tracheae (Fig. 11 A). In the subsequent development, the bubbles will gradually expand, and the diameter of P8 bubbles will be close to the middle distance between the two sides of the tracheae (Fig. 11 B-D). Over the next 2 hours, the bubble moves toward the middle and reaches its maximum diameter, which is larger than the middle distance between the tracheae on each side (Fig. 11 D-F). At P10-P12, the bubbles disappeared (Fig. 11 G). When the pupal central bubble developed to P2, it appeared in the central position of the pupal body. Because the initial bubble had a small diameter and would be blocked by the filth in the pupal body, only when the pupal shell was punctured, the small bubbles would escape from the pupal body and float on the dissection fluid. Therefore, the central bubbles in the pupa are observed through the pupal shell more slowly than the ventral bubbles appear. There were differences in the time when the central bubbles were observed among different individuals, but at P8, due to the large size of the central bubbles (Fig. 11 D '), large central bubbles could be observed through the pupal shell in all the samples observed. The bubbles in the central and side of the pupal body disappeared about 11 hours after development (Fig. 11 G '). 3.3.9 Outer pupal bubble formation and displacement The outer pupal bubble refers to the air bubble between the pupal shell and the pupal body, which formed later. The bubble diameter reached the maximum in the body, and appeared on the day before the pupal body separated from the pupal shell, at which time the development was about 10–12 hours. The specific process is as follows (Video S2): after the air bubbles inside the pupa expand to the maximum, the outer air bubbles appear in the pupa shell and the pupa tail and expand and move rapidly, pushing the pupa body to the head as a whole, so that the pupa tail and the pupa shell are separated (Fig. 12 A). Then the outer pupal bubble moves forward and continues to expand from the side (or left or right) of the pupal body, moves upward and breaks off at the pupal chest to form two bubbles. One bubble continues to move toward the head and disappears at the snout, and the other bubble moves downward back to the tail of the pupal shell (Fig. 12 C-E), continues to expand forward and move, and divides into two bubbles at the chest again. The two bubbles again flow toward the rostral and caudal portion of the pupal shell, respectively (Fig. 12 E-F). The repeated migration and rupture of bubbles outside the pupal body, and the repeated flow through the head, thorax and abdomen of the pupal body are the necessary conditions for the separation of the pupal shell from the pupal body to form the head, thorax and abdomen. 3.3.10 Appearance and migration of Malpighian tubules Malpighian tubules are excreting organs in Drosophila , which contain a pair of tubular structures. During metamorphosis, a pair of Malpighian tubules will show obvious morphological changes and positional migration. Malpighian tubules appear at the thoracoabdominal junction within 10–20 minutes after the pupal body separates from the pupal shell to form the head, thorax and abdomen, and a pair of Malpighian tubules are elongated in morphology (Fig. 13 A-B). As development progresses, the elongated Malpighian tubules gradually blur at the thoracoabdominal junction and develop to P13, when the Malpighian tubules are not visible through the pupa shell. According to the literature, the Malpighian tubules are obscured by internal tissues at this time and enter the white Malpighian tubule stage (Fig. 13 B-D), which lasts for 7 to 8 hours (Fig. 13 D-F). In some of the individuals observed during this period, the occlusion of the Malpighian tubule was incomplete and still indistinct. After P20, the occluded Malpighian tubules gradually appeared from the abdomen (Fig. 13 G). Over the next 5–10 hours, a pair of Malpighian tubule will move either symmetrically down or asymmetrically toward the tail in different individuals. Symmetrically down means that in some individuals a pair of Malpighian tubule will move down a certain distance at the same time and stop, while asymmetrically down means that in some individuals a pair of Malpighian tubule will move down first and then the other pair will move down. The two Malpighian tubules are placed one up and the other down on the abdomen, but the two Malpighian tubules eventually show symmetry (Fig. 13 M) in the late stage of development. Malpighian tubules appeared again after P20, becoming shorter and thicker in morphology with positional shift, and gradually becoming darker and more obvious in color (Fig. 13 H-M). Malpighian tubules disappeared from the abdomen about ten hours before emergence (Fig. 13 N). 3.3.11 Appearance and migration of corpora allata In the observed samples (N = 10), the appearance time of the corpora allata structure varied greatly among different individuals. The corpora allata structure was first observed in the individuals during the period of P24 to P30, located above A pair of Malpighian tubules, at the thoracoabdominal junction, and was punctate (Fig. 14 A,A '). It is observed in individuals between P45 and P50 at the latest, at the middle and upper end of a pair of Malpighian tubules, ventrally, and punctate (Fig. 14 B,B '). After P50 stage, the color of the corpora allata in all observed samples deepened, gradually became obvious, and moved down from the middle and upper end of the Malpighian tubule to the median position (Fig. 14 B-C,B '-C'). The time required for different individuals to move to the median position also varied greatly. A strip-like shape (Fig. 14 C,C '); According to the results of continuous photography, the emerging corpora allata will move up and down in the middle of the Malpighian tubule with a small amplitude (Fig. 14 C-F,C '-F'), and become blurred with Malpighian tubule within ten hours before emergence and finally disappear from the abdomen (Fig. 14 F-G,F '-G'). The appearance and migration of corpora allata vary greatly in time among individuals, and generally it is not used alone to judge and distinguish pupal development, but usually plays an auxiliary role in combination with other features such as Malpighian tubules. 3.4 Differences in morphological changes between mutant and wild-type fruit flies during the pupal stage We selected four mutant fruit flies and the wild-type Drosophila CS, all at the P0 stage, and observed their pupal development under a stereomicroscope. We found differences in developmental time and pupal tissue morphology between the mutants and the wild type. 3.4.1 Differences between dnrx 273 and CS during pupal development: The dnrx 273 (The genotype is homozygous.) exhibited a short-body phenotype and only had larval and pupal stages, with no adult form. When we synchronously observed the development of the dnrx 273 (The genotype is homozygous.) and the CS , both exhibited similar pupal tissue changes such as mouthpart retraction and tracheal bending. At the P8 stage, the internal bubbles in both expanded to their maximum size, then gradually moved to the center, and external bubbles began to form. In the wild-type Drosophila , external bubbles flowed between the rostrum and the pupal case tail, rapidly completing the separation of the pupal case from the pupal body and the formation of the head, thorax, and abdomen. However, in the dnrx 273 (The genotype is homozygous.), the head, thorax, and abdomen failed to form, and the external bubbles expanded from the rostrum to the tail, with the pupal contents continuously leaking out (Video S3). As the CS continued to develop, the dnrx 273 (The genotype is homozygous.) pupae showed no further changes. Therefore, we concluded that the dnrx 273 (The genotype is homozygous.) is lethal during the pupal stage, with death occurring approximately between P8 and P12. 3.4.2 Differences between dnlg2 KO70 and CS during pupal development: Compared to the CS , the dnlg2 KO70 exhibited similar timing in head, thorax, and abdomen segmentation, and in some cases, the time from P0 to segmentation was even shorter. However, the developmental timing of various tissues within the pupa was inconsistent. The appearance of Malpighian tubules in dnlg2 KO70 was delayed, making them more difficult to observe from outside the pupal case, and the corpora allata within the tubules was also hard to detect. Additionally, the timing of compound eye and eye color changes was prolonged in dnlg2 KO70 . As the three types of bristles appeared sequentially, the eye color gradually deepened to a vermilion hue, whereas the wild-type Drosophila exhibited a deep red color (Video S4). 3.4.3 Differences between dnlg3 KO127 and CS during pupal development: In the early pupal stage, the slender Malpighian tubules were difficult to observe in dnlg3 KO127 . However, after P20, the tubules and corpora allata became clearly visible. Along the Z-axis, the corpora allata in dnlg3 KO127 was positioned closer to the dorsal side compared to the Malpighian tubules. As development progressed, the corpora allata moved from the side of the tubules near the rostrum to the side near the tail, with vigorous shaking (Video S5). 3.4.4 Differences between dnlg4 KO10 and CS during pupal development: Compared to the CS , the dnlg4 KO10 developed more slowly, with a longer developmental time. Its compound eyes exhibited an orange-red color. In the CS , eye color changes were observed before the Malpighian tubules became clearly visible. In contrast, in dnlg4 KO10 , eye color deepened only after the appearance of head bristles, eventually turning orange-red (Video S6). 4 Discussion 4.1 Definition of head-movement phase and head static phase It is known that the insect pupal stage will undergo drastic morphological degeneration, and thus this transitional period may also have a corresponding continuity of morphological degeneration. The pupal stage of insects can be divided into prepupal stage, cryptocephalous pupa stage, and cephalous pupa stage, such as Eristalinus aeneus and Eristalis tenax [ 37 ]. The prepupal stage refers to the stage when the final instar larvae of whole insects shorten their body, stop feeding, search for a suitable place, and enter the stage of imactivity before pupation. In the cryptocephalic pupa, there are bubbles in the pupa to maintain a constant body volume, and the hidden head can be observed through the transparent cuticle. When the pupa emerges, the head of the worm has broken through the shell, and thereafter, other physical features gradually emerge. However, in Drosophila , some researchers divided the pupal stage into the prepupal stage and the head pupal stage according to the morphology [ 27 ]. In the prepupal stage, it was further divided into four stages according to the pupal shell color and the bubbles in the pupal shell. The first stage was the white pupal stage, when the flies had completely stopped peristalsis, and the pupal shell was white in color. From the white pupal stage to the bubble moving to the head, the head everting, and all the tracheal connection with the pupa outside were disconnected, the stage was considered as the prepupal stage. In fact, there is still a transition period of about 20 hours from larval to pupal stage, during which there are still obvious morphological changes in Drosophila . Therefore, according to the characteristics of the head mouthparts, we divided the transition period into the head movement period and the head-static period, which had obvious morphological characteristics. The characteristics and differences of the two are briefly described as follows: Head-movement phase: the body of the individual is partially shortened, the cuticle on the surface of its ventral segment stiffens and adheres to the slide, and the mouthparts are still protruding outside the head. The individual swayed and wriggled back and forth in place under the telescopic drive of the thoracic articulator. Moving individuals with a light source or a brush, they will swim and crawl again on the slide and move to other locations to pupate. Head static phase: the cuticle of the body surface of the individual is hardened and shriveled, completely adheres to the slide, and neither head nor tail can move. When slight brush stimulation or weak light stimulation was given, the head and tail still did not move. If the stimulation time or intensity was prolonged, the head would first expand back and forth along the direction of the body, and the tail would still adhere to the slide. If the activity was too intense, the whole individual might move forward for a small distance. However, the time from the head-movement phase to the head static phase is uncertain, and both brushing and light can stimulate the head quiescence phase to resume crawling. According to our observation and experimental verification, under normal circumstances, the duration from the head-movement phase to P0 phase is generally 1–2 hours. If the brush is used continuously to stimulate, the transition time of the two will be prolonged, and there will be no head static phase in the normal growth state, and the behavior of mouth organ retraction and stomatal extension will occur simultaneously. Therefore, we inferred that the third instar larvae would successively experience the head movement stage, head static stage, and white pupal stage (P0) after entering the late stage. The head movement stage and head quiescence stage were regarded as the transition states of the third instar larva and before pupation. 4.2 Definition and collection of P0 stage (high synchronization, 1 hour duration). The pupal shell was milky white and the epidermis was completely indurated; the spiracles extended from the body surface and protruded on both sides of the snout; there was no stretching and peristalsis of the body when stimulated (such as light stimulation and brush stimulation). In this article, we also define white pupae as pupa 0 hours, which is P0. The characteristics of P0 that we observed are consistent with, and supplemented by, multiple literatures that have identified the white pupae with eversion of stomata and shortening of the body [ 38 , 39 ]. The white pupa is generally considered to be the earliest stage of pupal development [ 40 ]. At this time, it has already formed a hardened cuticle, but the internal structure of pupa is similar to that of larva and has not yet begun to undergo drastic changes. Therefore, we also determined P0 as the starting point of pupation, and the determination of the starting point of pupation is crucial for the fine division of pupal stage. The experiment was divided into continuous video recording and time-point sampling observation. The experimental methods used in each observation mode are innovative. For continuous video recording, we took advantage of the characteristic that the third instar larvae will climb up the tube wall in the late stage to find a suitable position for pupuium formation. A pathological slide or polypropylene film was inserted into the medium, so that the larvae could climb up the support by themselves, which was conducive to the subsequent removal of the support and the observation of the individual larvae distributed on the support through the bottom light source. This method has not been reported in the literature before. Compared with directly selecting white pupae from the tube wall [ 27 ], the intelligent use of support reduces the physical stimulation of the brush to the experimental animals and the possibility of contamination of the culture tube. At the same time, it is more beneficial to the observation of the late third instar larvae to the white pupal stage, which lays a foundation for us to more carefully distinguish the emergence and movement stage from the head static stage. For the observation of time-point sampling, considering that the operation performed during the transfer of P0 stage individuals may affect the ontogenesis of the pupal stage, we cultured the individuals in the pupal stage at the same time interval in the Drosophila observation tube, and the 2h of culture was marked as P2. Will start from P0, a total of 24 The patients were divided into two stages (P0, P2, P4, P6, P8, P10, P12, P14, P16, P20, P24, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, P90) and those observed on the above slide The appearance time and characteristics of the tracheae, mouthparts, compound eyes, wings, appendages, bristles and pupal body, bubbles, Malpighian tubules, and corpora allata were compared and verified. By selecting a large number of pupal flies at the same moment, the recorded morphological characteristics of pupal stage at a specific moment were verified, which increased the accuracy and scientificity of the pupal developmental appearance described. 4.3 The guiding significance of easily observable physical features in development Different developmental characteristics of organs are helpful for the study of their own and even the study of the nervous system. We take the post-pupal development time as a certain reference to divide the morphological-developmental characteristics at different time points, and on the premise of basically referring to the post-pupal development time, we provide morphological evidence to support it, which can effectively improve the efficiency and accuracy of the experiment. The pupal shell of Drosophila serves as a protective and developmental environment during its life cycle. During the white pupal stage, the pupal shell gradually hardened and became milky white, and gradually deepened after 1 h until it became tan at the late pupal stage. The gas exchange between flies and the outside world is done by the respiratory system. During the development from the silent stage to the white pupa stage, branched spiracles developed on both sides of the snout, one end extending from the body surface, and the other end connected with the internal tracheae. As the development progressed, the tracheae began to become blurred at about P10. During the next 30 minutes to 1 hour, the connection between the tracheae and the breathing hole is gradually disconnected and the tracheae is eliminated. In the larval stage, fruit flies mainly feed on rotten fruits and other organic matter, and the mouthparts are mainly masticatory. After the head quiescence stage, the black mouthparts of the larva begin to retract. After experiencing four stages of retraction, retraction, immobile and abscission, the degeneration of the larval mouthparts is completed in P10. The three-segment outline of head, thorax and abdomen of Drosophila adult was obviously formed after P10. After that, many structures also underwent dramatic changes in the interior of Drosophila : Malpighian tubules for excretion appeared at the thoracoabdominal junction within 10–20 minutes after the adult outline was formed, underwent white Malpighian tubules and yellow Malpighian tubules, and disappeared from the abdomen about ten hours before eclosion. During this period, the corpora allata , which supplies nutrients to the pupae, can be observed above the Malpighian tubule, but its appearance and migration vary greatly among individuals. It will become blurred along with Malpighian tubule within ten hours before emergence and eventually disappear from the abdomen. From P35, adult tissues such as compound eyes were formed, and the pupal cuticle covered the tissue. The two wings were located under the pupal cuticle and were transparent in color. The three pairs of appendages were closely juxtaposed from top to bottom on the ventral side of the pupa. Both compound eyes and wings become progressively darker as development progresses. After P65, the legs turned brown and began to segment. After P80, the sex of flies could be distinguished by whether the sexual comb structure was observed on the first tarsus of the forefoot, and bristles began to form on the head, chest and abdomen. After P90, the female flies were able to walk after removing the pupal shell. Thanks to these organs and structures, Drosophila can adapt to various environments and complete various stages of its life cycle. Our demonstration of the pupal stage enables researchers to use this ideal model organism to explore biological questions such as gene expression, development and behavior. Based on the published literature, there are relatively many studies on the P8-P12 [ 41 , 42 ], P40-P50 [ 26 , 43 ] and P70 stages [ 25 ] in the pupal stage of Drosophila . In order to make the selection of stages more accurate in the future research, the typical developmental characteristics of these stages are briefly described as follows: P8-P12: pupal segmentation is the most significant change in this period, and the overall morphology of the larval stage is gradually divided into head, thorax and abdomen. Whether there is segmentation can be clearly seen can be used as a marker to determine the P10 stage. The bubbles in pupae developed gradually from P8 to P10 to the largest volume. At P8, the bubble diameter was slightly smaller than the distance between the two sides of the tracheae, and was between P6 and P10. At P10, the bubble moved to the middle and reached the largest diameter, which was larger than the middle distance between the two sides of the tracheae. The outer pupal air bubbles began to appear at P10 to make up for the reduction in pupal shell volume after segmentation. P40-p50: In this period, for non-white-eyed flies, eye color will be a good characteristic observation point. At P40, adult tissues such as compound eyes have been formed, the compound eyes are covered with a transparent film, and transparent wings have appeared. From P45, there is pigmentation around the compound eyes, and at this time, the color of compound eyes and wings is gradually deepened. P70: At this point, the form of the adult worm in the pupal shell of the Drosophila becomes more pronounced. The color of the compound eyes had deepened to red, and the ends of the bilateral wings were gray. The legs were brown in color and already segmented. Head bristles can already be observed and chest bristles are beginning to appear. During the concurrent observation of morphological development in mutant and wild-type Drosophila pupae, we made several notable discoveries. In 1996, researchers had already identified that dnrx 273 exhibited homozygous lethality, although the underlying cause of death was not reported[ 44 ]. In our study, we observed that the homozygous lethality of dnrx 273 occurred approximately between pupal stages P8 and P12. During this period, wild-type fruit flies exhibit characteristic morphological changes, including the gradual disappearance of internal bubbles, separation of the pupal case from the pupal body, and segmentation of the head, thorax, and abdomen. Based on these observations, we preliminarily hypothesize that the function of the nrx is most critical during this specific stage and may be closely related to cellular differentiation, tissue remodeling, or organ formation occurring at this time. Additionally, these findings provide a more precise basis for developmental stage classification in subsequent studies and facilitate targeted genetic modifications during specific stages. Furthermore, we found that dnlgs generally exhibited prolonged overall development time, most notably characterized by delayed color changes in the compound eyes. Although the protein encoded by the Neuroligin gene is primarily associated with the development and function of the nervous system, abnormalities in the nervous system may indirectly affect the synchrony and coordination of morphological changes through various mechanisms. This is particularly significant during the pupal stage, a critical phase of metamorphosis, where precise intercellular signaling is required for the formation of specific structures. 5 Conclusion According to the above characteristic changes of the time signature of the pupal stage under the constant temperature condition and the observation results of the gap sampling of the pupal stage (Figure S1 -S5, Video S7), the time differential characteristics of the 24 stages of the pupal stage were summarized in the following table(Table 1 ), so as to provide a judgment basis for the subsequent sampling of the pupal stage. Declarations Conflict of interest No potential conflict of interest was reported by the authors. Funding This work was supported by the National Natural Science Foundation of China Grant 32070811(G,G) and 32271013(G,J). Author Contribution Gan guangming, Chen Mei, Xu Liwen contributed to the conception of the study;Chen Mei, Xu Liwen, Pang Xuan, Zhang Yumeng performed the experiment;Xu Liwen, Chen Mei, Tang Jiaxu performed the data analyses and wrote the manuscript;Zhang Chenchen, Geng junhua, Xie Wei, Gan Guangming helped perform the analysis with constructive discussions. Data availability statement All datasets presented in the study are included in the article/supplementary material. References Nielsen C. The origin of metamorphosis. Evol Dev. 2000;2(3):127–9. Truman JW, Riddiford LM. The origins of insect metamorphosis. Nature. 1999;401(6752):447–52. Brown DD, Cai L. Amphibian metamorphosis Dev Biol. 2007;306(1):20–33. Cheng Y, et al. Expression and Regulation of pde6h by Thyroid Hormone During Metamorphosis in Paralichthys olivaceus. Front Physiol. 2020;11:244. Xu F, et al. Transcriptional regulation analysis reveals the complexity of metamorphosis in the Pacific oyster (Crassostrea gigas). 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Dynamics of endoreplication during Drosophila posterior scutellar macrochaete development. PLoS ONE. 2012;7(6):e38714. Hartenstein V, Posakony JW. Development of adult sensilla on the wing and notum of Drosophila melanogaster. Development. 1989;107(2):389–405. Taniguchi K, et al. Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells. BMC Dev Biol. 2014;14:46. Baumgartner S, et al. A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell. 1996;87(6):1059–68. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterial.pdf SupplymentalMaterialVideo.zip image15.png Table 1. Development of morphological characteristics at the pupal stage under constant temperature Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6337002","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449551532,"identity":"57fcac4b-d90f-438a-acd8-19c28875e572","order_by":0,"name":"Xu Liwen","email":"","orcid":"","institution":"School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Liwen","suffix":""},{"id":449551533,"identity":"78d3592d-fcce-4e89-beba-f1733c10d2c7","order_by":1,"name":"Chen Mei","email":"","orcid":"","institution":"School of Life Science and Technology, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Mei","suffix":""},{"id":449551534,"identity":"71a5f750-50b3-4b1f-8c61-6e30fe295a5e","order_by":2,"name":"Zhang Yumeng","email":"","orcid":"","institution":"School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Yumeng","suffix":""},{"id":449551535,"identity":"8bf6d78e-fd4b-4812-8c19-6a6cab5eb08c","order_by":3,"name":"Pang Xuan","email":"","orcid":"","institution":"School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Pang","middleName":"","lastName":"Xuan","suffix":""},{"id":449551536,"identity":"e83b5f73-e948-4e60-a6c5-292e55bf6162","order_by":4,"name":"Tang Jiaxu","email":"","orcid":"","institution":"School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Tang","middleName":"","lastName":"Jiaxu","suffix":""},{"id":449551537,"identity":"c2c45763-8e00-412e-85b7-3c054f8fbe94","order_by":5,"name":"Zhang Chenchen","email":"","orcid":"","institution":"School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Chenchen","suffix":""},{"id":449551538,"identity":"34f6bccf-124d-41c6-be13-10f4a665d1d3","order_by":6,"name":"Geng junhua","email":"","orcid":"","institution":"School of Life Science and Technology, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Geng","middleName":"","lastName":"junhua","suffix":""},{"id":449551556,"identity":"865fe8bf-dc3a-443f-8922-1a670dfb05ab","order_by":7,"name":"Xie Wei","email":"","orcid":"","institution":"School of Life Science and Technology, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":false,"prefix":"","firstName":"Xie","middleName":"","lastName":"Wei","suffix":""},{"id":449551557,"identity":"09e46247-015f-414b-ad97-fa4a9648eeef","order_by":8,"name":"Gan Guangming","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYFAD9gYwxdhAvBaeAyRrkUggUou8+9nDr3lq7titnfn84WMeBhvZDQeYnz3Ap8XwTF6aNc+xZ8nbbucYG/MwpBlvOMBmboBXS0OOmTEP2+Fks9s5bNI8DIcTNxzgYZPAq6X/DVDLP6CWm8ef/+Zh+E9Yi7xEjvFj3rbDdmY3GMyYeRgOENZiIPHGjHFu3+EEszM5xpJzDJKNZx5mM8NvS3+O8Yc33w7bmx0//vDDmwo72b7jzc/w23KAAeyMxAYIF4iZ8akH2dLAwPwBSNsTUDcKRsEoGAUjGQAAMXpLtP4ZAKUAAAAASUVORK5CYII=","orcid":"","institution":"School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China","correspondingAuthor":true,"prefix":"","firstName":"Gan","middleName":"","lastName":"Guangming","suffix":""}],"badges":[],"createdAt":"2025-03-30 06:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6337002/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6337002/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82141422,"identity":"cf72f309-88c1-435a-a7c5-aa7f8a20f8d9","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCollection Process of P0\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntroduce some flies (＞ 15 adults, half male and half female) that have been eclosed for 3-7 days into culture tubes(A). Egg laying for 2 days(A-B). Embryo development(B-C). Appearance of larval stage and pupal stage flies in the tube(C). Marked white pupa(P0) on the wall of each numbered culture tube, and record the marking time as T1(D-E). Marked new white pupae on the wall of the tube at 1-hour intervals, labeled time Tn [Tn = T1 + (n-1)h] and recorded the number of labeled pupae(D-H).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/60591e3f05b42cb455eef09e.png"},{"id":82145070,"identity":"bcdc9c4b-226e-4e41-a529-f83901d0f8c3","added_by":"auto","created_at":"2025-05-07 06:50:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":212471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the external morphology and spiracle characteristics of the head movement, head stasis, and white pupal stages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe third instar larva L3w crawled (A). During the head movement stage, the abdominal somite was rigid and adhered to the slide, and the mouth organs were located on the top of the body and stretched back and forth to drive the thoracic somite to swing in place (B). The whole body adhered to the slide and the mouthparts retracted to the inner surface of the body during the head static phase (C). At the white pupal stage, the cuticle is completely hardened into the pupal shell, the mouthparts are retracted to the pupal shell, and the spiracle is extended (D). Brown pupae with brown pupal shell. Black lines indicate rigid abdomen during head movement; Red arrow indicates mouthpiece position; The blue arrow indicates the position of the breathing hole. Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/871f9bf6ddba3993aff83e19.png"},{"id":82141425,"identity":"f68d8898-78a6-4b2c-bca5-26005e5d9201","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":461717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePupal shell color change in the pupal stage (back view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the pupal stage, the color of the pupal shell changed from milky white to brown with time (A-D). The brown pupal shell was maintained for a long time until the late pupae (D-I), which further deepened to black brown (J-K) because the pupal body was full of bristles. The remaining shell after pupa emergence loses its bristles and becomes brown (L). Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/f406f7aeb5f39b8b55d195db.png"},{"id":82143738,"identity":"fc1a286c-9db6-4523-9610-82a97fb8c1bb","added_by":"auto","created_at":"2025-05-07 06:42:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":568964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegeneration of the tracheae of the pupal stage larvae (back view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were two patterns of degeneration in the pupal stage: one was curved during pupation (A), followed by rapid bulge and straightening (A-C), then curved again, and straightened again after reaching the maximum curvature (D-E). The straightened tracheae developed into A blur after ten hours, leaving a small segment at each upper and lower end of the tracheae (F-H). The second pattern was A rapid bulge and straightening of the tracheae after pupation (A '-B'), which was not curved during subsequent development until it became blurred after 10 H, leaving a small segment at each end of the tracheae (C-H). Black arrows indicate larval tracheae on both sides of the pupal body. Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/bede472f0feb9414dd5008e8.png"},{"id":82143740,"identity":"72ee27a8-14b7-47c1-b11f-8ee57a602a01","added_by":"auto","created_at":"2025-05-07 06:42:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":445237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRetraction and degeneration of larval mouthparts in the pupal stage (dorsal and ventral views)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-F: ventral view of mouthparts degeneration; Black mouthparts, protruding and rostral, rapidly retract in late larval L3w stage (A,A '); During head movement, the mouthpart was still located at the shortened snout, and the expansion speed slowed down (B, B '). In the cephalostat stage, the mouthparts retract into the body and gradually retract into the worm, with an increase in rostral distance (C-E,C '-E'), until 30 minutes into the pupal stage, when the mouthparts cease to retract (E-F,E '-F').\u003c/p\u003e\n\u003cp\u003eG-L: degenerated dorsal view of mouthparts; After 10 h of development, the mouthparts were detached from the pupal body and remained on the pupal shell (G-L,G '-L'). Black arrow indicates mouthpiece position, and bidirectional arrow indicates mouthpiece retraction distance. Scale Bar:A-L:0.5 mm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/b115a97c03583cb6fa71e162.png"},{"id":82143742,"identity":"76c838f8-01ef-4af5-8c0f-07d6e2e9841a","added_by":"auto","created_at":"2025-05-07 06:42:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":314615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColor changes of the pupal compound eyes (side)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe compound eyes are recognizable at P10, located on both sides of the head (A); Compound eyes formed during P35-P40 without pigmentation (A-D). At P45, pigmentation began to appear in the periphery of the compound eyes (E), and from P45 to P55, the pigmentation deepened from the periphery to the middle and became orange throughout the compound eyes (E-H). From P55 to P60, the orange color was further deepened to brown color (H-J), and from P60 to P70, the brown color was transformed to red color (J-N). P70 -Emergence The color of the compound eyes gradually changes from red to red-black due to the gradual deepening of the pupal shell color (N-R). The black arrow indicates the position of the compound eye. Red arrows indicate granules in the pupae. Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/3123e8ab8abd551956e1c523.png"},{"id":82141423,"identity":"be63f8c0-8402-4cd5-a645-b99c84cf9ea2","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":365669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in wing color in the pupal stage (side view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wing color changed in the late pupal stage, from P65-P70, the lower part of the wings became gray (E-H). At P75, the whole wing became colored, the lower part of the wing became black, and the upper part became gray (H-K). There was no color change (K-N) until emergence. The blue dashed area indicates the unilateral wing position. Scale Bar:A-N:1 mm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/45b8ea2dbbfa9f08acc5e444.png"},{"id":82141427,"identity":"e6bc625c-fecf-4c07-9183-7a54fc828e91","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":253051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLeg maturation in the pupal stage (ventral view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP65-P70, appendages turned brown (A-B) and appeared segmented; At P75, leg color did not change, but segmentation became obvious (C). At 75 to 80 hours of development, the legs grow fine hair, black (C-D, D '); At 90 hours of development, the legs of males did not change (F), while the legs of females developed faster than those of males, and the fine hairs on the legs became stiff and close to those of adults (F '). The male unique structure, the sexual comb, was observed on the first tarsus of the forefoot from P75 to P80, showing black patches (C-D). The red box indicates the sexual comb; The red arrow indicates the sexual comb located on the first tarsus. Blue arrows indicate the three pairs of \u003cem\u003eDrosophila\u003c/em\u003eappendages. Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/11989d7832e02d640b3cdf2b.png"},{"id":82145072,"identity":"93a2f6c5-2287-4bb5-aca3-369e5b21ee89","added_by":"auto","created_at":"2025-05-07 06:50:19","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":285398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological changes of pupal bristles (dorsal view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBristles formation occurred in the late pupal stage, and the head bristles were first formed during P65-P70 (A-B). Secondly, thoracic bristles form between P70 and P75 (B-C), and finally abdominal bristles appear between P75 and P80 (C-D), and all three types of bristles become black and hard by P85 (E), at which point they develop into mature bristles.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/d1d99d48555a0c94cff353b3.png"},{"id":82145069,"identity":"ef36f55e-1664-4748-a701-927ead93cb78","added_by":"auto","created_at":"2025-05-07 06:50:19","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":432598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall morphological changes of pupae (back view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 10 hours of development, the pupal body first moved up as a whole (A-B), and then moved down after 10 minutes. The pupal body separated from the pupal shell to form the head, thorax and abdomen, and the outline of each part was not obvious (A-B). Within the next hour, the pupal body moves down to the upper quarter of the pupal shell, and the head, thorax, and abdomen are clearly delineated (C-G). Within an hour before emergence, the pupal abdomen elongates and the head moves upward until the pupal shell emerges as an adult (H-N). The black one-way arrow indicates that the pupal body has an overall upward trend. The black bidirectional arrow indicates the downward movement distance of the pupal body. The red bidirectional arrow indicates the pupal abdominal elongating distance. Scale Bar: 0.5 mm.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/46dc1649be9522765c4f8dda.png"},{"id":82141438,"identity":"005f4993-a5c8-49cb-aeac-f9c83f76fc12","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":562581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological changes of bubbles in pupae (back view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMovement of lateral bubbles: After 2 hours of pupation, bubbles were observed on the abdominal side of the pupal body, located under the unilateral trachea, and at this time, the diameter of the bubbles was smaller than the middle distance between the two tracheae (A). The bubble diameter increased with the development time, and the bubble diameter was close to the middle distance of the trachea at 8 hours of development (B-D). After 10 hours of development, the bubbles moved to the middle and reached the maximum diameter, which was larger than the middle distance of the trachea on both sides (E-F), and soon disappeared from the pupal body (G). Movement of the central bubble: the bubble can also initially appear from the middle of the abdomen, which is obscured by the viscera due to its small diameter (A '-C '); \"At 8 h of development, the intermediate bubble is clearly visible (D '), reaches its maximum at 10 h of development (E '), and then disappears (F '-G ').\" The red circle shows the change process of the ventral bubble. The black circle shows the process of bubble change in the abdomen. Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/3e0f1883174360c06f46730f.png"},{"id":82141447,"identity":"a738d56f-6a86-4cae-936f-ad0cb1681231","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":398206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of bubbles in pupae (back view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 10 hours of development, the outer bubble appears at the lower end of the pupal shell (A), then slowly moves upward to the pupal abdomen (B), where it moves upward from the side of the pupal (C), and the upward bubble breaks off at the pupal chest to form two bubbles, each split in opposite directions (D). The downward moving bubble then repeats the process many times over (E-F). The black curve shows the position of the outer bubble in the pupal shell. The black arrow indicates the position of the bubble edge contour in vitro. Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/d1e39eb1181878c1cb05db3f.png"},{"id":82145073,"identity":"891bc210-b2a7-4214-856a-89f74cca8642","added_by":"auto","created_at":"2025-05-07 06:50:19","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":461732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the Malpighian tubule at the pupal stage (back view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 10 h of development, shortly after the pupa dissociated from the pupal shell, A pair of Malpighian tubules appeared at the thoracoabdominal junction of the pupa (A-B). After 12-13 hours, a pair of Malpighian tubules gradually blurred to disappear and entered the white Malpighian tubules stage (B-D), which lasted for 7 to 8 hours (D-F); After 20 hours of development, a pair of Malpighian tubules reappeared from the abdomen, shorter and thicker in shape, and green in color (G). In the next 5-10 hours, the Malpighian tubules in the abdomen will move down a certain distance in a symmetric and asymmetric manner to stop. Here is an asymmetric move, a pair of Malpighian tubules in the abdomen, one in the front and one in the back (G-H); After cessation of the Malpighian tubules on the abdomen, the color changes gradually from green to black, and the Malpighian tubules are not visible to the naked eye on the abdomen shortly after black addition, about ten hours before emergence (I-N). Scale Bar:0.5 mm.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/ba9462f7454c95d484dec629.png"},{"id":82141437,"identity":"1215766f-d774-4eea-bc89-91b61bfa1c15","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":414737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecorpora allata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e at the pupal stage (dorsal view)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the 10 observed individuals, the \u003cem\u003ecorpora allata\u003c/em\u003estructure first appeared during P24-P30, above the Malpighian tubule, at the thoracoabdominal junction (A); \"It appears at the latest in individuals between P45 and P50, near the upper end of a pair of Malpighian tubules, in the abdomen (B); After P50, the color of the corpus luteal structure gradually deepened, became obvious between the Malpighian tubules, and gradually moved from the upper end to the middle of the Malpighian tubules (B-C). In all the individuals observed at P60, the Malpighian tubules moved from the upper end to the lower end of the Malpighian tubules, becoming a strip-like shape (C). Ten hours before eclosion, the \u003cem\u003ecorpora allata\u003c/em\u003e and Malparian ducts become blurred and eventually disappear (F-G). Red arrows show a pair of Malpighian structures. Black arrows indicate changes in the structure of the \u003cem\u003ecorpora allata\u003c/em\u003e. Scale Bar: 0.5 mm.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/d90d1ecd2764fc25707526d4.png"},{"id":84681487,"identity":"0a5a3ace-5d71-4818-949d-702e152d38cd","added_by":"auto","created_at":"2025-06-16 08:24:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7986369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/ed119a75-a9b6-4340-9749-05a36d60fe3d.pdf"},{"id":82141432,"identity":"2007e4f4-daf4-438a-a1e2-639044b688c5","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2762087,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/3d316331e858737f43902f01.pdf"},{"id":82141475,"identity":"4f5a85bc-308d-4807-b194-18b1b4ee4005","added_by":"auto","created_at":"2025-05-07 06:34:23","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":54005680,"visible":true,"origin":"","legend":"","description":"","filename":"SupplymentalMaterialVideo.zip","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/5f733928ee15006e5335db17.zip"},{"id":82141435,"identity":"44a3594b-7c5f-47b5-8379-0b89098af541","added_by":"auto","created_at":"2025-05-07 06:34:19","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2289264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. Development of morphological characteristics at the pupal stage under constant temperature\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image15.png","url":"https://assets-eu.researchsquare.com/files/rs-6337002/v1/f9efc655995ffc3f89b4860f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dynamic Morphological Staging of Drosophila Pupal Development","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMetamorphosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] encompasses drastic morphological and physiological transformations across an organism\u0026rsquo;s life cycle. This process encompasses different growth stages, accompanied by drastic changes in the organism's appearance and internal structure, in order to adapt to the demands of a variable ecological environment and specific living habits. Metamorphosis is not uncommon in the biological world; it occurs in insects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], amphibians [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], certain fish [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and some marine invertebrates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among them, the existence of metamorphosis in insects is the most widespread [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is estimated that over 98% of insect species undergo varying degrees of metamorphosis during their developmental cycle [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, insects are generally classified into three main categories based on their metamorphic development: ametabolous, hemimetabolous, and holometabolous [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ametabolous insects refer to a group that undergoes only changes in body size during development, without any change in shape, such as \u003cem\u003eEntognatha\u003c/em\u003e and \u003cem\u003eApterygota\u003c/em\u003e, which continue to molt throughout their lives [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Hemimetabolous development includes three main stages: egg, nymph and adult. In hemimetabolous development, the nymph gradually approaches the adult form, but there are no significant morphological changes. At this time, the living habits and diets of nymphs and adults remain highly consistent [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Grasshoppers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and cockroaches [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] are commonly used animal models for studying hemimetabolous development. In contrast, the metamorphosis of bees [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], dragonflies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and butterflies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] begins with a fertilized egg and goes through four stages: egg, larva, pupa and adult. The larval stage focuses mainly on feeding and nutrient accumulation. The pupal stage involves extensive and intense changes in cellular organization, as well as the remodeling of body shape and organs, ultimately transforming into the adult form. After developing into an adult, it will complete the functions of reproduction and gene transmission, maintaining survival through activities such as flying and foraging. Metamorphosis that shows significant differences in morphology and behavior at each developmental stage is called complete metamorphosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong all the metamorphic development methods of insects, holometabolous development accounts for the largest proportion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Holometabolous insects are considered to be the most evolved group within the class Insecta, and they are also the most diverse in terms of species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, most research is primarily focused on complete metamorphic insects [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Within this group, \u003cem\u003eDrosophila\u003c/em\u003e has emerged as a key model organism due to its experimental tractability [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Currently, research on \u003cem\u003eDrosophila\u003c/em\u003e mainly focuses on the larval and adult stages [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In contrast, the pupal stage, which occupies nearly 100 hours in the \u003cem\u003eDrosophila\u003c/em\u003e's life cycle, has relatively little research due to the unclear details of its overall development process. However, the pupal stage is a very important phase; during this time, the nutrients acquired during the larval stage gradually take effect within the pupal body, and both the imaginal discs and nerve cells develop [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The process of imaginal disc development into adult organs can be observed through a stereo microscope, but the central nervous system cannot be directly observed. If we can grasp the development of the adult organs corresponding to the central nervous system development during the proposed research period, it will greatly enhance the accuracy of experimental sampling time.\u003c/p\u003e \u003cp\u003eAs a classic research subject among holometabolous insects, \u003cem\u003eDrosophila\u003c/em\u003e exhibits significant differences at various developmental stages, similar to other holometabolous insects. Therefore, it is essential to conduct detailed staging when performing in-depth mechanistic studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Currently, mainstream experimental protocols define the absolute time after pupation by hours after pupal formation (APF) to represent the staging of pupal development [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, considering the differences in developmental speed, genetics and microenvironment among individual pupae, it is challenging to ensure that all pupae are at the same developmental stage under absolute time conditions. As development progresses towards the later stages of pupation, the synchronization among individuals decreases [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Sole reliance on temporal staging compromises accuracy and reproducibility, highlighting the need for a continuous morphological map of pupal development. Currently, many visualization imaging techniques have been applied to the study of insect metamorphosis, some of which have also been used in \u003cem\u003eDrosophila\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Considering the advantages and disadvantages of various imaging techniques, due to the semi-transparent nature of the \u003cem\u003eDrosophila\u003c/em\u003e's cuticle, it is relatively convenient to observe the overall morphology of organ development during the pupal stage using optical microscopy after moistening the specimen. Correspondingly, there is indeed a substantial amount of research that applies morphological changes during the pupal stage to the study of metamorphic development mechanisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Compared to merely using a temporal scale to categorize the \u003cem\u003eDrosophila\u003c/em\u003e life cycle, employing developmental staging definitions based on morphological characteristics can yield better comparative results when comparing multiple individuals [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, most experiments observing the pupal stage of \u003cem\u003eDrosophila\u003c/em\u003e involve removing the experimental animals from their pupal cases for filming and recording [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], during which the documentation of the characteristics of the pupal cases may be lost.\u003c/p\u003e \u003cp\u003eCurrently, the most detailed morphological staging of the \u003cem\u003eDrosophila\u003c/em\u003e pupal stage was established in 1981, classifying 24 developmental stages from 51 observable morphological sequences In recent years, many studies have continued to use the staging method from this literature [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], but this method also studies experimental animals after the removal of the pupal shell. Additionally, due to the earlier time period, imaging equipment was relatively outdated, leading to a rough classification of the early \u003cem\u003eDrosophila\u003c/em\u003e pupal stage. In fact, during the prepupal stage, the central nervous system synapses of the \u003cem\u003eDrosophila\u003c/em\u003e have already begun to degenerate [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], but earlier literature did not provide a relevant classification for this period. Therefore, based on existing research progress, we utilized a high-resolution stereomicroscope to observe and record the complete developmental changes of the wild-type \u003cem\u003eDrosophila\u003c/em\u003e melanogaster pupal stage through continuous photography and time-lapse imaging, aiming to provide a basis for accurately defining the developmental stages of the pupa and for precise sampling. At the same time, in the central nervous system of the \u003cem\u003eDrosophila\u003c/em\u003e, transmembrane proteins encoded by the Neurexin and Neuroligins genes are located on the presynaptic and postsynaptic membranes respectively. Their interaction is significant for forming synaptic connections and maintaining the normal structure and function of synapses [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Current research has clearly shown that the nervous systems of \u003cem\u003ednrx\u003c/em\u003e and \u003cem\u003ednlgs\u003c/em\u003e are inevitably affected during their development [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, we are still unclear whether there are differences in the morphological development during the pupal stage between mutant \u003cem\u003eDrosophila\u003c/em\u003e and wild-type \u003cem\u003eDrosophila\u003c/em\u003e, and at which specific stage of the pupal phase certain homozygous lethal mutant strains are unable to continue normal development. Therefore, we used the method of observing the morphological development of wild-type \u003cem\u003eDrosophila\u003c/em\u003e pupae to simultaneously observe the morphological development of the mutant pupae, recording the characteristic morphological features of \u003cem\u003ednrx\u003c/em\u003e and \u003cem\u003ednlgs\u003c/em\u003e during their development, and further linking these features to the poor development of their nervous systems.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Fly stocks and rearing conditions\u003c/h2\u003e \u003cp\u003eThe flies used in this experiment include \u003cem\u003eCS\u003c/em\u003e, \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ednlg2\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO70\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ednlg3\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO127\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ednlg4\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO10\u003c/em\u003e\u003c/sup\u003e. Pour 30 flies (half female and half male) into a large glass bottle or fly vial containing standard culture medium. Rear the flies at 25\u0026deg;C and expose them to a 12h:12h light-dark cycle. The rearing room was equipped with air conditioning and a humidifier to maintain constant conditions (25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, 60% relative humidity).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sampling and Photography Methods during the Pupal Stage\u003c/h2\u003e \u003cp\u003eWe began to record the characteristics of the late third instar larvae. The late third instar larvae tend to search for suitable pupation sites on the tube wall, but the shooting range of the camera is limited. They often actively avoid the light source of the camera, thus hindering the observation of the characteristic changes of the late third instar larvae entering the pupation stage. Based on this situation, we designed the following experiment utilizing the characteristic of late third-instar larvae stopping feeding and crawling up the tube walls:\u003c/p\u003e \u003cp\u003eWe initiated the experiment by inserting six to eight cleaned and dried pathological slides into the culture medium of each large glass bottle, followed by incubation under standard conditions for two to three days to facilitate pupation of the larvae on both sides of the slides. Alternatively, for sample collection, \u003cem\u003eDrosophila\u003c/em\u003e tubes containing standard culture medium could be employed. In this scenario, a transparent polypropylene film was cut into rectangles measuring 5cm \u0026times; 10cm, inserted into the culture medium, and incubated under standard conditions until the larvae migrated onto the polypropylene film. When third-instar larvae were observed to be motionless on the slides or polypropylene film, mechanical stimulation was applied using a soft-bristled brush. If the larvae resumed crawling, they were permitted to continue growing. However, if they remained motionless, they were deemed to have entered the Head-static period (HSP). Subsequently, the slides or polypropylene film were removed using tweezers, and larvae, pupae, and food residues on one side were wiped off with a wet tissue. Any excess food residues on the observation side were also cleaned off. The slide was then positioned under a high-resolution stereomicroscope (OLYMPUS SZX7), where the bottom and side light sources were adjusted, a magnification of 1.6\u0026times; was selected, and the complete process of the respiratory openings extending from the late third-instar larvae to the P0 stage was captured.\u003c/p\u003e \u003cp\u003eBased on the established morphological characteristics of the P0 stage, we collected ten typical P0 specimens with distinct morphological features and arranged them neatly on a new clean glass slide with the ventral, dorsal, and lateral sides facing up. A small amount of glycerin was applied to the surface of the samples using a brush to enhance surface contrast and reduce moisture evaporation from the pupae. The slide containing the samples was then placed on the imaging device, a magnification of 1.6\u0026times; was selected, and continuous or intermittent video recording of the samples was conducted to observe the developmental status of the fly pupae from different observation angles. Concurrently, changes in the complete morphological characteristics of the pupal stage and their appearance times were recorded, obtaining time-marked characteristic organs such as tracheae, mouthparts, compound eyes, wings, appendages, bristles, and the overall shape of the pupa, as well as bubbles, Malpighian tubules, and the \u003cem\u003ecorpora allata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFor the assembly of the shooting device, a piece of filter paper was placed on the bottom light source to prevent interference from the background color. A petri dish (90mm in diameter) was placed on a hollow support, the hollow bottom of which increases air flow and thereby reduces heating of the petri dish. Inside the petri dish, a glass slide was placed on top of a 3cm high support, and the polypropylene film containing the sample was positioned on the glass slide. The petri dish was then sealed with transparent cling film, and a 1cm \u0026times; 1cm breathable hole was cut directly above the sample to prevent water mist interference during shooting and to maintain humidity. Finally, an appropriate amount of deionized water was injected into the petri dish; the water level did not cover the glass slide but maintained humidity throughout the shooting process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Continuous video recording and time-point sampling observation during the pupal stage\u003c/h2\u003e \u003cp\u003eDue to the potential impact of transferring the P0 phase onto the slide with a brush on the development of the pupae, we validated the characteristics of the pupal development of fruit flies in the fly culture tubes based on the preliminary data obtained from the aforementioned experiments.\u003c/p\u003e \u003cp\u003eWe began the experiment by introducing more than 15 adult flies, with an equal ratio of males to females, that had eclosed for 3\u0026ndash;7 days into culture tubes, with approximately 20\u0026ndash;30 tubes used in total. These tubes were cultured under standard conditions for 2 days, after which the adult flies were extracted, and the tubes were cultured for an additional 3\u0026ndash;4 days. During this period, we observed the presence of stationary white pupae, brown pupae, and larvae on the walls of the tubes.\u003c/p\u003e \u003cp\u003eTo systematically track the development, we used a marker to number each culture tube (A1, B1, C1, etc.) and marked the white pupae on the walls of each numbered tube, recording the initial marking time as T1. The marked tubes were then placed back into the incubator. After 1 hour, the tubes were removed, and new white pupae that had appeared on the tube walls were marked with a marker. The marking time was recorded as T2 (T2\u0026thinsp;=\u0026thinsp;T1\u0026thinsp;+\u0026thinsp;1h), along with the corresponding number of newly marked pupae. This process was repeated every hour, with each marking session kept within a 10-minute window, until the required quantity of samples for the experiment was reached.\u003c/p\u003e \u003cp\u003eIt is important to note that the white pupae (P0) collected at T1 were not used for the experiments. Instead, the culture tubes containing different batches of P0 collected after T1 were placed in an incubator for continued cultivation. Pupae that had been cultivated for 2 hours were marked as P2, and similarly, those cultivated for 4 hours were marked as P4. The entire pupal period was divided into 24 stages: P0, P2, P4, P6, P8, P10, P12, P14, P16, P20, P24, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, and P90.\u003c/p\u003e \u003cp\u003eBased on the time records, we collected samples from the tube walls using a brush at the corresponding time points and arranged them sequentially on a slide. By examining the consistent developmental characteristics observed in more than half of the flies at the same time point, we made judgments and corrections on the recorded time-marked morphological features.\u003c/p\u003e \u003cp\u003eFinally, we compared the appearance time and characteristics of various morphological features, including tracheae, mouthparts, compound eyes, wings, appendages, bristles, and the overall shape of the pupa, as well as bubbles, Malpighian tubules, and the \u003cem\u003ecorpora allata\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Definition of the Head-movement phase, Head-static period, and White Pupa Stage\u003c/h2\u003e \u003cp\u003eAccording to our aforementioned method, individuals at different developmental stages can be observed within the same field of view (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E, Video S1). In addition to the wandering third instar larvae (which we refer to as L3w) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and the stationary brown pupae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), three other types of developmental individuals can also be seen. Two of these exhibit a shortened larval form, which we tentatively name the head-moving stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and the head-stationary stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) based on their external morphology and behavioral characteristics. The third type resembles the brown pupa but has a white exterior, which we call the white pupa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHead-movement phase\u003c/strong\u003e \u003cp\u003eIn the L3w stage before the head movement stage, feeding has stopped, and individuals crawl around on the slide and the walls of the tube, aiming to find a suitable position to pupate. During the head movement stage, the body of the individual shortens, and the cuticle on the surface of the abdomen hardens and adheres to the slide. The mouthparts of the thorax extend and retract, causing the individual to sway back and forth in place (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), with the mouthparts not fully retracted into the body. When illuminated with a light source or moved with a brush, individuals in the head movement stage will crawl again on the slide, relocating to other positions to pupate.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHead-static period\u003c/strong\u003e \u003cp\u003eDuring this period, the entire epidermal stratum corneum of the individual hardens and shrinks, completely adhering to the slide, with both the head and tail unable to move (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the mouthparts fully retracted into the body. When subjected to slight brush stimulation or weak light stimulation, the head and tail still do not move. If water is dripped and the stimulation time or intensity is extended, the head will first exhibit forward and backward movements, while the tail remains adhered to the slide. Intense stimulation can cause some individuals to crawl a short distance.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eWhite pupal stage (P0)\u003c/strong\u003e \u003cp\u003eThe epidermis has completely hardened into a milky white pupal shell; the mouthparts retract into the pupal shell's rostrum, and the spiracles extend from the body and protrude on both sides of the rostrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). There is no extension or crawling response to light or brush stimuli. Since the duration of the white pupal stage lasts for about 1 hour (according to literature), this paper considers the white pupal stage as the beginning of the pupal stage, marked as P0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Morphological changes of pupal shell\u003c/h2\u003e \u003cp\u003eThe hallmark characteristic changes of pupal stage included changes in pupal shell morphology and pupal tissue, among which the pupal shell morphology mainly included changes in spiracle and pupal shell color.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Spiracles change\u003c/h2\u003e \u003cp\u003eThe spiracles were branched, hidden in the body during the head movement and head static stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C), and protruded on both sides of the snout during the white pupal stage (P0), with 4\u0026ndash;7 branches of about 0.05 mm in length on each side. Thereafter, no significant morphological changes were observed in the spiracles until the pupal stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). The changes in the location characteristics of the spiracle can provide a reference for distinguishing the white pupal stage from the head static stage and head movement stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Pupal shell color change\u003c/h2\u003e \u003cp\u003eIn the white pupal stage, the pupal shell color can change significantly within 2 hours. At the white pupal stage, the pupal shell was milky white (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As development progressed, the pupal shell color gradually deepened from the posterior spiracle of the tail. As observed by serial photography, the pupal shell color changed to light brown about 1 hour after the fly entered the white pupal stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After 2 hours, the pupal shell color deepens to brown (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and enters the late pupal stage. With the appearance and maturation of bristles on the pupal surface inside the shell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-K), the pupal shell surface gradually deepens from brown to tan (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Once the pupal body emerges as an adult, the remaining pupal shell loses the foil of pupal body and bristles, and appears brown again under the stereoscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Pupal shell color changes rapidly in the early stage of pupal development and is an important basis for judging pupal P0 stage.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Morphological changes of pupal tissues\u003c/h2\u003e \u003cp\u003eIn the pupal stage, the shape of pupal shell changes less, but the pupal tissue morphology in pupal shell changes significantly with the development process. During this period, most tissues and organs of larva will gradually disintegrate and fade in the pupal stage, and tissues and organs of adult will gradually develop or rebuild. The pupal tissues and organs were divided into static and dynamic morphological changes according to the criterion of whether they underwent obvious positional migration during metamorphosis. The former refers to the tissues and organs that changed morphological characteristics at the same position in the pupal body during the whole pupal stage, including tracheae, mouthparts, compound eyes, wings, appendages and bristles. The latter were the tissues and organs that showed characteristic changes during the whole pupal stage and dynamic position migration, including pupal shape, bubbles, Malpighian tubules and \u003cem\u003ecorpora allata\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Degeneration of the dorsal tracheae of the larva\u003c/h2\u003e \u003cp\u003eThe tracheae is a structure for gas exchange between \u003cem\u003eDrosophila\u003c/em\u003e and the outside world, and the dorsal tracheae of \u003cem\u003eDrosophila\u003c/em\u003e larvae will show obvious morphological changes during the pupal stage. In the larval and white pupal stages (P0), the tracheae is curved in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B '), but the curved tracheae will straighten out after the P10 stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C,B '), and then complete the extinction process in two ways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the first way, after the tracheae became straight, the tracheae were curved again, and the bending position was random, which might be the upper end, middle end or lower end (the bending position of the sample was the lower end again). The degree of bending was different in different individuals, and this re-bending could be observed more than one hour after entering P0 stage. Maximal bending was observed after approximately 2 hours of development (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The time it takes to straighten again varies from individual to individual (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and after P10 the tracheae become very blurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These blurred tracheae are disturbed by the movement of bubbles within the pupa shell within 10 to 20 minutes, and most of them disappear within a short time. Only a small segment of the upper and lower end of the tracheae of the original larvae were preserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). The preserved upper and lower end tracheae are connected to the spiracle and posterior spiracle exposed outside the pupal shell, respectively, giving a relatively uniform appearance of the pupal shell throughout the pupal stage.\u003c/p\u003e \u003cp\u003eIn the second way, after the tracheae become straight, it will remain straight until P10, until the blur disappears (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC'-H'). The first type of tracheal degeneration accounted for only 20%, but most pupae showed the second type of tracheal degeneration (the number of samples observed was 10). The time of tracheal disappearance can be used as a reference for P10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Pupal stage mouthparts retraction and degeneration\u003c/h2\u003e \u003cp\u003eThe black mouthparts of \u003cem\u003eDrosophila\u003c/em\u003e larvae begin to retract after entering the head quiescent stage, and will completely retract and degenerate within 12 hours after entering the pupal stage. The process can be observed from the dorsal and ventral sides, which can be divided into four stages: slowing down of the mouth parts, shrinking of the mouth parts, immobile of the mouth parts, and abscission of the mouth parts. At L3w, the black mouthpiece at the top of the larva's head expands and expands to feed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,A'). After entering the head movement stage, the mouthparts were still above the snout, continuously stretching back and forth and driving the chest movement, but the expansion of the mouthparts slowed down significantly during this period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,B '). Here, we carried out a supplementary experiment, taking 10 third instar larvae (non-head movement stage, head static stage) and head movement stage, and counting the average number of mouth retraction per minute in each stage. According to the statistics, the third instar larvae (non-head movement phase, head-static phase) on average expand 67 times per minute and head movement stage larvae on average expand 33 times per minute. (Statistics are provided in the Supplementary Data) During the cephalostat phase, the mouthparts were retracted into the worm body (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,C '). Then, during the development from the ab static stage to the white pupal stage (P0) and 30 minutes into P0 stage, the mouthparts in the pupal body would continuously recluse, and the distance from the pupal shell to the rostral end gradually increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E,C '-E'). The mouthparts stopped constricting 30 min after entering the pupal stage and were quiescence after a period of in situ fibrillation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F,E '-F'). After stage P10, the black mouth parts separated from the newly formed pupae to complete abscission, and thereafter remained in the same position in the pupal shell until emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-L, G '-L'). The changes of mouthpiece retraction during the pupal stage provided the basis for distinguishing the head movement stage from the head static stage, and was also a major feature of P10 in the pupal stage under standard culture conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Compound eye formation and eye color changes\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDrosophila\u003c/em\u003e melanogaster has compound eyes. After 10 hours of development from P0, with the formation of the head, thorax and abdomen of the pupal body, the position of the compound eyes can be determined by the pupal shell, which is located on both sides of the head (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,A '). At this time, the pupal shell was removed, and it was found that the pupal body was full of particles. When the pupal body was puncted with forceps, a large number of particles emerged, and adult tissues such as compound eyes did not form at this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,B '). During the period from P35 to P40, the pupal shell of the sample from this period was removed, and the particles could not be observed. Adult tissues such as compound eyes formed, and a transparent film covered the tissues, which was called pupal cuticle. During this period, there was no pigment deposition in the compound eyes, and the eye color was consistent with the pupal shell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D,C '-D'). Pigmentation only begins to appear around the contour of the compound eye (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E,A '-E'). During the next 10 hours, the pigment gradually deepened from the periphery to the middle of the compound eye until the entire compound eye became orange (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H,E '-H'). At this time, the pupa developed for about 55 hours. During the next 5 hours, the orange compound eye darkening to brownish brown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J,H '-J'); After 10 hours of development to P70, the eye color changes from brown to red (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-N,J '-N'); Then it enters the late pupal stage. Due to the gradual deepening of the pupal shell color, the color of the compound eye observed through the pupal shell also gradually deeps until it becomes red and black before emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN-R,N '-R').\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4 Wing formation and color change\u003c/h2\u003e \u003cp\u003eThe formation of wings was in the same period as the compound eye, about P35-P40. The pupal shell could not be observed through the pupal shell. When the pupal shell was removed, the wings were located under the pupal cuticle, close to the sides of the body, and the wings were transparent. Wing color change occurs during the late pupal stage and lasts about 10 hours. From P65-P70, the ends of the wings showed color and gradually deepened to gray, while the other parts of the wings showed no color change (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H). With the development, the wing color gradually deepened. At 75 hours of development, the end of the wing color deepened to black, and the root of the proximal end of the wing became gray (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-K). After that, the color of the wings does not change again until emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK-N).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5 Appendage formation and maturation\u003c/h2\u003e \u003cp\u003eThe newly formed appendages were difficult to observe through the pupal shell. During P35-P45, when the pupal shell was removed, the three pairs of appendages were closely juxtaposed from top to bottom on the ventral side of the pupal body. The development of appendages was slow, and the landmark morphological changes of legs did not occur until after the P65 stage. Between P65 and P70, legs became brown in color and began to appear segmented (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B); At P75, the leg color was still brown, but the segments became prominent (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). From P75 to P80, the legs grew black fine hair, and the leg color was black at this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD,D '). During this period, the sexual comb structure was observed on the first tarsus of the forefoot of the male individual (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), which was black patches. After development to P80, the sex of \u003cem\u003eDrosophila\u003c/em\u003e can be distinguished by sexual comb. At P90, the legs of males and females developed differently, and the legs of males did not show obvious changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F). However, at this time, the legs of females developed faster than those of males, and the fine hairs on the legs became hard (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD '-F'). If the pupae shell was removed at this time, the females could walk.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.6 Formation and hardening of bristles\u003c/h2\u003e \u003cp\u003ePupal bristles were classified into three categories based on the position of adult bristles: head bristles, chest bristles, and abdominal bristles. The morphological changes of the three types of bristles occurred in the late pupal stage, about P65-P85. Head bristles were first observed between P65 and P70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B); Chest bristles were observed from P70 to P75 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-C). Abdominal bristles were last observed between P75 and P80 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-D); All three types of bristles develop and mature within the next 5 hours. The bristles are black and hard (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.3.7 Pupal head, thorax and abdomen segments and their morphological displacement changes\u003c/h2\u003e \u003cp\u003eAt P0 stage, the newly formed pupal body was not separated from the pupal shell and retained most of the larval structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). After development to P10, the pupal body first moved to the snout as a whole under the action of bubbles at the lower end of the shell (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Within 10 minutes, the pupal body moved to the tail to complete the separation of the pupal body from the pupal shell and form the head, thorax and abdomen. At this time, the outline of each part was not obvious (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB-C). The whole pupal body gradually moved downward until it was about 1/4 from the upper end of the pupal shell, during which the outline of the head, thorax and abdomen gradually appeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC-G). One hour before emergence, the pupal abdominal segment gradually elongates, causing the head to move up to the front of the pupal shell. The pupal shell is poked and the pupal shell is left behind by the emerging adult (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eH-N). The separation of the pupal body from the pupal shell to form the head, thorax and abdomen was one of the most significant temporal morphological changes in the pupal stage. Based on this morphological change, it could be quickly inferred whether the pupal development time was before or after P10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.8 Appearance and displacement of bubbles in pupae\u003c/h2\u003e \u003cp\u003eBubbles were observed both in the pupal stage and in the pupal stage, and both of them had obvious characteristics of expansion and migration. Bubbles in the pupal body appeared earlier and were divided into lateral and central pupal bubbles according to the location of their appearance. Pupal side bubbles appear below the abdominal unilateral (or left or right) tracheae shortly after the pupal shell color darkens to brown and after P2 development, at which point the bubble diameter is smaller than the spacing of the two tracheae (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA). In the subsequent development, the bubbles will gradually expand, and the diameter of P8 bubbles will be close to the middle distance between the two sides of the tracheae (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB-D). Over the next 2 hours, the bubble moves toward the middle and reaches its maximum diameter, which is larger than the middle distance between the tracheae on each side (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD-F). At P10-P12, the bubbles disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen the pupal central bubble developed to P2, it appeared in the central position of the pupal body. Because the initial bubble had a small diameter and would be blocked by the filth in the pupal body, only when the pupal shell was punctured, the small bubbles would escape from the pupal body and float on the dissection fluid. Therefore, the central bubbles in the pupa are observed through the pupal shell more slowly than the ventral bubbles appear. There were differences in the time when the central bubbles were observed among different individuals, but at P8, due to the large size of the central bubbles (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD '), large central bubbles could be observed through the pupal shell in all the samples observed. The bubbles in the central and side of the pupal body disappeared about 11 hours after development (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eG ').\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.9 Outer pupal bubble formation and displacement\u003c/h2\u003e \u003cp\u003eThe outer pupal bubble refers to the air bubble between the pupal shell and the pupal body, which formed later. The bubble diameter reached the maximum in the body, and appeared on the day before the pupal body separated from the pupal shell, at which time the development was about 10\u0026ndash;12 hours. The specific process is as follows (Video S2): after the air bubbles inside the pupa expand to the maximum, the outer air bubbles appear in the pupa shell and the pupa tail and expand and move rapidly, pushing the pupa body to the head as a whole, so that the pupa tail and the pupa shell are separated (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). Then the outer pupal bubble moves forward and continues to expand from the side (or left or right) of the pupal body, moves upward and breaks off at the pupal chest to form two bubbles. One bubble continues to move toward the head and disappears at the snout, and the other bubble moves downward back to the tail of the pupal shell (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eC-E), continues to expand forward and move, and divides into two bubbles at the chest again. The two bubbles again flow toward the rostral and caudal portion of the pupal shell, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eE-F). The repeated migration and rupture of bubbles outside the pupal body, and the repeated flow through the head, thorax and abdomen of the pupal body are the necessary conditions for the separation of the pupal shell from the pupal body to form the head, thorax and abdomen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.10 Appearance and migration of Malpighian tubules\u003c/h2\u003e \u003cp\u003eMalpighian tubules are excreting organs in \u003cem\u003eDrosophila\u003c/em\u003e, which contain a pair of tubular structures. During metamorphosis, a pair of Malpighian tubules will show obvious morphological changes and positional migration. Malpighian tubules appear at the thoracoabdominal junction within 10\u0026ndash;20 minutes after the pupal body separates from the pupal shell to form the head, thorax and abdomen, and a pair of Malpighian tubules are elongated in morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA-B). As development progresses, the elongated Malpighian tubules gradually blur at the thoracoabdominal junction and develop to P13, when the Malpighian tubules are not visible through the pupa shell. According to the literature, the Malpighian tubules are obscured by internal tissues at this time and enter the white Malpighian tubule stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB-D), which lasts for 7 to 8 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eD-F). In some of the individuals observed during this period, the occlusion of the Malpighian tubule was incomplete and still indistinct. After P20, the occluded Malpighian tubules gradually appeared from the abdomen (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eG). Over the next 5\u0026ndash;10 hours, a pair of Malpighian tubule will move either symmetrically down or asymmetrically toward the tail in different individuals. Symmetrically down means that in some individuals a pair of Malpighian tubule will move down a certain distance at the same time and stop, while asymmetrically down means that in some individuals a pair of Malpighian tubule will move down first and then the other pair will move down. The two Malpighian tubules are placed one up and the other down on the abdomen, but the two Malpighian tubules eventually show symmetry (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eM) in the late stage of development. Malpighian tubules appeared again after P20, becoming shorter and thicker in morphology with positional shift, and gradually becoming darker and more obvious in color (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eH-M). Malpighian tubules disappeared from the abdomen about ten hours before emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.11 Appearance and migration of \u003cem\u003ecorpora allata\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn the observed samples (N\u0026thinsp;=\u0026thinsp;10), the appearance time of the \u003cem\u003ecorpora allata\u003c/em\u003e structure varied greatly among different individuals. The \u003cem\u003ecorpora allata\u003c/em\u003e structure was first observed in the individuals during the period of P24 to P30, located above A pair of Malpighian tubules, at the thoracoabdominal junction, and was punctate (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eA,A '). It is observed in individuals between P45 and P50 at the latest, at the middle and upper end of a pair of Malpighian tubules, ventrally, and punctate (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eB,B '). After P50 stage, the color of the \u003cem\u003ecorpora allata\u003c/em\u003e in all observed samples deepened, gradually became obvious, and moved down from the middle and upper end of the Malpighian tubule to the median position (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eB-C,B '-C'). The time required for different individuals to move to the median position also varied greatly. A strip-like shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eC,C '); According to the results of continuous photography, the emerging \u003cem\u003ecorpora allata\u003c/em\u003e will move up and down in the middle of the Malpighian tubule with a small amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eC-F,C '-F'), and become blurred with Malpighian tubule within ten hours before emergence and finally disappear from the abdomen (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eF-G,F '-G'). The appearance and migration of \u003cem\u003ecorpora allata\u003c/em\u003e vary greatly in time among individuals, and generally it is not used alone to judge and distinguish pupal development, but usually plays an auxiliary role in combination with other features such as Malpighian tubules.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Differences in morphological changes between mutant and wild-type fruit flies during the pupal stage\u003c/h2\u003e \u003cp\u003eWe selected four mutant fruit flies and the wild-type \u003cem\u003eDrosophila\u003c/em\u003e CS, all at the P0 stage, and observed their pupal development under a stereomicroscope. We found differences in developmental time and pupal tissue morphology between the mutants and the wild type.\u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Differences between \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCS\u003c/em\u003e during pupal development:\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e(The genotype is homozygous.) exhibited a short-body phenotype and only had larval and pupal stages, with no adult form. When we synchronously observed the development of the \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e(The genotype is homozygous.) and the \u003cem\u003eCS\u003c/em\u003e, both exhibited similar pupal tissue changes such as mouthpart retraction and tracheal bending. At the P8 stage, the internal bubbles in both expanded to their maximum size, then gradually moved to the center, and external bubbles began to form. In the wild-type \u003cem\u003eDrosophila\u003c/em\u003e, external bubbles flowed between the rostrum and the pupal case tail, rapidly completing the separation of the pupal case from the pupal body and the formation of the head, thorax, and abdomen. However, in the \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e(The genotype is homozygous.), the head, thorax, and abdomen failed to form, and the external bubbles expanded from the rostrum to the tail, with the pupal contents continuously leaking out (Video S3). As the \u003cem\u003eCS\u003c/em\u003e continued to develop, the \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e(The genotype is homozygous.) pupae showed no further changes. Therefore, we concluded that the \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e(The genotype is homozygous.) is lethal during the pupal stage, with death occurring approximately between P8 and P12.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Differences between \u003cem\u003ednlg2\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO70\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCS\u003c/em\u003e during pupal development:\u003c/h2\u003e \u003cp\u003eCompared to the \u003cem\u003eCS\u003c/em\u003e, the \u003cem\u003ednlg2\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO70\u003c/em\u003e\u003c/sup\u003e exhibited similar timing in head, thorax, and abdomen segmentation, and in some cases, the time from P0 to segmentation was even shorter. However, the developmental timing of various tissues within the pupa was inconsistent. The appearance of Malpighian tubules in \u003cem\u003ednlg2\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO70\u003c/em\u003e\u003c/sup\u003e was delayed, making them more difficult to observe from outside the pupal case, and the \u003cem\u003ecorpora allata\u003c/em\u003e within the tubules was also hard to detect. Additionally, the timing of compound eye and eye color changes was prolonged in \u003cem\u003ednlg2\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO70\u003c/em\u003e\u003c/sup\u003e. As the three types of bristles appeared sequentially, the eye color gradually deepened to a vermilion hue, whereas the wild-type \u003cem\u003eDrosophila\u003c/em\u003e exhibited a deep red color (Video S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 Differences between \u003cem\u003ednlg3\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO127\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCS\u003c/em\u003e during pupal development:\u003c/h2\u003e \u003cp\u003eIn the early pupal stage, the slender Malpighian tubules were difficult to observe in \u003cem\u003ednlg3\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO127\u003c/em\u003e\u003c/sup\u003e. However, after P20, the tubules and \u003cem\u003ecorpora allata\u003c/em\u003e became clearly visible. Along the Z-axis, the \u003cem\u003ecorpora allata\u003c/em\u003e in \u003cem\u003ednlg3\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO127\u003c/em\u003e\u003c/sup\u003e was positioned closer to the dorsal side compared to the Malpighian tubules. As development progressed, the \u003cem\u003ecorpora allata\u003c/em\u003e moved from the side of the tubules near the rostrum to the side near the tail, with vigorous shaking (Video S5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.4.4 Differences between \u003cem\u003ednlg4\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO10\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCS\u003c/em\u003e during pupal development:\u003c/h2\u003e \u003cp\u003eCompared to the \u003cem\u003eCS\u003c/em\u003e, the \u003cem\u003ednlg4\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO10\u003c/em\u003e\u003c/sup\u003e developed more slowly, with a longer developmental time. Its compound eyes exhibited an orange-red color. In the \u003cem\u003eCS\u003c/em\u003e, eye color changes were observed before the Malpighian tubules became clearly visible. In contrast, in \u003cem\u003ednlg4\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO10\u003c/em\u003e\u003c/sup\u003e, eye color deepened only after the appearance of head bristles, eventually turning orange-red (Video S6).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Definition of head-movement phase and head static phase\u003c/h2\u003e \u003cp\u003eIt is known that the insect pupal stage will undergo drastic morphological degeneration, and thus this transitional period may also have a corresponding continuity of morphological degeneration. The pupal stage of insects can be divided into prepupal stage, cryptocephalous pupa stage, and cephalous pupa stage, such as Eristalinus aeneus and Eristalis tenax [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The prepupal stage refers to the stage when the final instar larvae of whole insects shorten their body, stop feeding, search for a suitable place, and enter the stage of imactivity before pupation. In the cryptocephalic pupa, there are bubbles in the pupa to maintain a constant body volume, and the hidden head can be observed through the transparent cuticle. When the pupa emerges, the head of the worm has broken through the shell, and thereafter, other physical features gradually emerge.\u003c/p\u003e \u003cp\u003eHowever, in \u003cem\u003eDrosophila\u003c/em\u003e, some researchers divided the pupal stage into the prepupal stage and the head pupal stage according to the morphology [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the prepupal stage, it was further divided into four stages according to the pupal shell color and the bubbles in the pupal shell. The first stage was the white pupal stage, when the flies had completely stopped peristalsis, and the pupal shell was white in color. From the white pupal stage to the bubble moving to the head, the head everting, and all the tracheal connection with the pupa outside were disconnected, the stage was considered as the prepupal stage.\u003c/p\u003e \u003cp\u003eIn fact, there is still a transition period of about 20 hours from larval to pupal stage, during which there are still obvious morphological changes in \u003cem\u003eDrosophila\u003c/em\u003e. Therefore, according to the characteristics of the head mouthparts, we divided the transition period into the head movement period and the head-static period, which had obvious morphological characteristics. The characteristics and differences of the two are briefly described as follows:\u003c/p\u003e \u003cp\u003eHead-movement phase: the body of the individual is partially shortened, the cuticle on the surface of its ventral segment stiffens and adheres to the slide, and the mouthparts are still protruding outside the head. The individual swayed and wriggled back and forth in place under the telescopic drive of the thoracic articulator. Moving individuals with a light source or a brush, they will swim and crawl again on the slide and move to other locations to pupate.\u003c/p\u003e \u003cp\u003eHead static phase: the cuticle of the body surface of the individual is hardened and shriveled, completely adheres to the slide, and neither head nor tail can move. When slight brush stimulation or weak light stimulation was given, the head and tail still did not move. If the stimulation time or intensity was prolonged, the head would first expand back and forth along the direction of the body, and the tail would still adhere to the slide. If the activity was too intense, the whole individual might move forward for a small distance.\u003c/p\u003e \u003cp\u003eHowever, the time from the head-movement phase to the head static phase is uncertain, and both brushing and light can stimulate the head quiescence phase to resume crawling. According to our observation and experimental verification, under normal circumstances, the duration from the head-movement phase to P0 phase is generally 1\u0026ndash;2 hours. If the brush is used continuously to stimulate, the transition time of the two will be prolonged, and there will be no head static phase in the normal growth state, and the behavior of mouth organ retraction and stomatal extension will occur simultaneously.\u003c/p\u003e \u003cp\u003eTherefore, we inferred that the third instar larvae would successively experience the head movement stage, head static stage, and white pupal stage (P0) after entering the late stage. The head movement stage and head quiescence stage were regarded as the transition states of the third instar larva and before pupation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Definition and collection of P0 stage (high synchronization, 1 hour duration).\u003c/h2\u003e \u003cp\u003eThe pupal shell was milky white and the epidermis was completely indurated; the spiracles extended from the body surface and protruded on both sides of the snout; there was no stretching and peristalsis of the body when stimulated (such as light stimulation and brush stimulation). In this article, we also define white pupae as pupa 0 hours, which is P0. The characteristics of P0 that we observed are consistent with, and supplemented by, multiple literatures that have identified the white pupae with eversion of stomata and shortening of the body [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The white pupa is generally considered to be the earliest stage of pupal development [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. At this time, it has already formed a hardened cuticle, but the internal structure of pupa is similar to that of larva and has not yet begun to undergo drastic changes. Therefore, we also determined P0 as the starting point of pupation, and the determination of the starting point of pupation is crucial for the fine division of pupal stage.\u003c/p\u003e \u003cp\u003eThe experiment was divided into continuous video recording and time-point sampling observation. The experimental methods used in each observation mode are innovative.\u003c/p\u003e \u003cp\u003eFor continuous video recording, we took advantage of the characteristic that the third instar larvae will climb up the tube wall in the late stage to find a suitable position for pupuium formation. A pathological slide or polypropylene film was inserted into the medium, so that the larvae could climb up the support by themselves, which was conducive to the subsequent removal of the support and the observation of the individual larvae distributed on the support through the bottom light source. This method has not been reported in the literature before. Compared with directly selecting white pupae from the tube wall [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the intelligent use of support reduces the physical stimulation of the brush to the experimental animals and the possibility of contamination of the culture tube. At the same time, it is more beneficial to the observation of the late third instar larvae to the white pupal stage, which lays a foundation for us to more carefully distinguish the emergence and movement stage from the head static stage.\u003c/p\u003e \u003cp\u003eFor the observation of time-point sampling, considering that the operation performed during the transfer of P0 stage individuals may affect the ontogenesis of the pupal stage, we cultured the individuals in the pupal stage at the same time interval in the \u003cem\u003eDrosophila\u003c/em\u003e observation tube, and the 2h of culture was marked as P2. Will start from P0, a total of 24 The patients were divided into two stages (P0, P2, P4, P6, P8, P10, P12, P14, P16, P20, P24, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, P90) and those observed on the above slide The appearance time and characteristics of the tracheae, mouthparts, compound eyes, wings, appendages, bristles and pupal body, bubbles, Malpighian tubules, and \u003cem\u003ecorpora allata\u003c/em\u003e were compared and verified. By selecting a large number of pupal flies at the same moment, the recorded morphological characteristics of pupal stage at a specific moment were verified, which increased the accuracy and scientificity of the pupal developmental appearance described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.3 The guiding significance of easily observable physical features in development\u003c/h2\u003e \u003cp\u003eDifferent developmental characteristics of organs are helpful for the study of their own and even the study of the nervous system. We take the post-pupal development time as a certain reference to divide the morphological-developmental characteristics at different time points, and on the premise of basically referring to the post-pupal development time, we provide morphological evidence to support it, which can effectively improve the efficiency and accuracy of the experiment.\u003c/p\u003e \u003cp\u003eThe pupal shell of \u003cem\u003eDrosophila\u003c/em\u003e serves as a protective and developmental environment during its life cycle. During the white pupal stage, the pupal shell gradually hardened and became milky white, and gradually deepened after 1 h until it became tan at the late pupal stage. The gas exchange between flies and the outside world is done by the respiratory system. During the development from the silent stage to the white pupa stage, branched spiracles developed on both sides of the snout, one end extending from the body surface, and the other end connected with the internal tracheae. As the development progressed, the tracheae began to become blurred at about P10. During the next 30 minutes to 1 hour, the connection between the tracheae and the breathing hole is gradually disconnected and the tracheae is eliminated. In the larval stage, fruit flies mainly feed on rotten fruits and other organic matter, and the mouthparts are mainly masticatory. After the head quiescence stage, the black mouthparts of the larva begin to retract. After experiencing four stages of retraction, retraction, immobile and abscission, the degeneration of the larval mouthparts is completed in P10.\u003c/p\u003e \u003cp\u003eThe three-segment outline of head, thorax and abdomen of \u003cem\u003eDrosophila\u003c/em\u003e adult was obviously formed after P10. After that, many structures also underwent dramatic changes in the interior of \u003cem\u003eDrosophila\u003c/em\u003e: Malpighian tubules for excretion appeared at the thoracoabdominal junction within 10\u0026ndash;20 minutes after the adult outline was formed, underwent white Malpighian tubules and yellow Malpighian tubules, and disappeared from the abdomen about ten hours before eclosion. During this period, the \u003cem\u003ecorpora allata\u003c/em\u003e, which supplies nutrients to the pupae, can be observed above the Malpighian tubule, but its appearance and migration vary greatly among individuals. It will become blurred along with Malpighian tubule within ten hours before emergence and eventually disappear from the abdomen. From P35, adult tissues such as compound eyes were formed, and the pupal cuticle covered the tissue. The two wings were located under the pupal cuticle and were transparent in color. The three pairs of appendages were closely juxtaposed from top to bottom on the ventral side of the pupa. Both compound eyes and wings become progressively darker as development progresses. After P65, the legs turned brown and began to segment. After P80, the sex of flies could be distinguished by whether the sexual comb structure was observed on the first tarsus of the forefoot, and bristles began to form on the head, chest and abdomen. After P90, the female flies were able to walk after removing the pupal shell.\u003c/p\u003e \u003cp\u003eThanks to these organs and structures, \u003cem\u003eDrosophila\u003c/em\u003e can adapt to various environments and complete various stages of its life cycle. Our demonstration of the pupal stage enables researchers to use this ideal model organism to explore biological questions such as gene expression, development and behavior.\u003c/p\u003e \u003cp\u003eBased on the published literature, there are relatively many studies on the P8-P12 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], P40-P50 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and P70 stages [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] in the pupal stage of \u003cem\u003eDrosophila\u003c/em\u003e. In order to make the selection of stages more accurate in the future research, the typical developmental characteristics of these stages are briefly described as follows:\u003c/p\u003e \u003cp\u003eP8-P12: pupal segmentation is the most significant change in this period, and the overall morphology of the larval stage is gradually divided into head, thorax and abdomen. Whether there is segmentation can be clearly seen can be used as a marker to determine the P10 stage. The bubbles in pupae developed gradually from P8 to P10 to the largest volume. At P8, the bubble diameter was slightly smaller than the distance between the two sides of the tracheae, and was between P6 and P10. At P10, the bubble moved to the middle and reached the largest diameter, which was larger than the middle distance between the two sides of the tracheae. The outer pupal air bubbles began to appear at P10 to make up for the reduction in pupal shell volume after segmentation.\u003c/p\u003e \u003cp\u003eP40-p50: In this period, for non-white-eyed flies, eye color will be a good characteristic observation point. At P40, adult tissues such as compound eyes have been formed, the compound eyes are covered with a transparent film, and transparent wings have appeared. From P45, there is pigmentation around the compound eyes, and at this time, the color of compound eyes and wings is gradually deepened.\u003c/p\u003e \u003cp\u003eP70: At this point, the form of the adult worm in the pupal shell of the \u003cem\u003eDrosophila\u003c/em\u003e becomes more pronounced. The color of the compound eyes had deepened to red, and the ends of the bilateral wings were gray. The legs were brown in color and already segmented. Head bristles can already be observed and chest bristles are beginning to appear.\u003c/p\u003e \u003cp\u003eDuring the concurrent observation of morphological development in mutant and wild-type \u003cem\u003eDrosophila\u003c/em\u003e pupae, we made several notable discoveries. In 1996, researchers had already identified that \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e exhibited homozygous lethality, although the underlying cause of death was not reported[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our study, we observed that the homozygous lethality of \u003cem\u003ednrx\u003c/em\u003e\u003csup\u003e\u003cem\u003e273\u003c/em\u003e\u003c/sup\u003e occurred approximately between pupal stages P8 and P12. During this period, wild-type fruit flies exhibit characteristic morphological changes, including the gradual disappearance of internal bubbles, separation of the pupal case from the pupal body, and segmentation of the head, thorax, and abdomen. Based on these observations, we preliminarily hypothesize that the function of the \u003cem\u003enrx\u003c/em\u003e is most critical during this specific stage and may be closely related to cellular differentiation, tissue remodeling, or organ formation occurring at this time. Additionally, these findings provide a more precise basis for developmental stage classification in subsequent studies and facilitate targeted genetic modifications during specific stages.\u003c/p\u003e \u003cp\u003eFurthermore, we found that \u003cem\u003ednlgs\u003c/em\u003e generally exhibited prolonged overall development time, most notably characterized by delayed color changes in the compound eyes. Although the protein encoded by the Neuroligin gene is primarily associated with the development and function of the nervous system, abnormalities in the nervous system may indirectly affect the synchrony and coordination of morphological changes through various mechanisms. This is particularly significant during the pupal stage, a critical phase of metamorphosis, where precise intercellular signaling is required for the formation of specific structures.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eAccording to the above characteristic changes of the time signature of the pupal stage under the constant temperature condition and the observation results of the gap sampling of the pupal stage (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S5, Video S7), the time differential characteristics of the 24 stages of the pupal stage were summarized in the following table(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), so as to provide a judgment basis for the subsequent sampling of the pupal stage.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eNo potential conflict of interest was reported by the authors.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China Grant 32070811(G,G) and 32271013(G,J).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGan guangming, Chen Mei, Xu Liwen contributed to the conception of the study;Chen Mei, Xu Liwen, Pang Xuan, Zhang Yumeng performed the experiment;Xu Liwen, Chen Mei, Tang Jiaxu performed the data analyses and wrote the manuscript;Zhang Chenchen, Geng junhua, Xie Wei, Gan Guangming helped perform the analysis with constructive discussions.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eAll datasets presented in the study are included in the article/supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNielsen C. 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Cell. 1996;87(6):1059\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Drosophila, Metamorphosis, Pupal Development, Morphological Staging","lastPublishedDoi":"10.21203/rs.3.rs-6337002/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6337002/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study provides a comprehensive analysis of the morphological-developmental characteristics of the pupal stage in \u003cem\u003eDrosophila\u003c/em\u003e, a model organism for metamorphosis research. We have delineated the critical stages of pupal development, from the late third-instar larvae to the adult emergence, through continuous photography and time-lapse imaging. Our findings include the identification of the head-movement phase and head-static period as distinct transitional states preceding the pupal stage. We describe the dynamic changes in the pupal shell, including color transitions and the development of spiracles, which serve as reliable markers for staging. Additionally, we detail the morphological transformations of internal tissues and organs, such as the degeneration of larval tracheae, the retraction and degeneration of mouthparts, the formation of compound eyes and wings, and the development of appendages and bristles. Our research also highlights the significant morphological changes in pupal tissues, including the movement and displacement of bubbles, the appearance and migration of Malpighian tubules and \u003cem\u003ecorpora allata.\u003c/em\u003e In conclusion, we have identified several critical periods: P10 serves as a definitive marker for the onset of segmentation during the pupal stage, P40-P50 represents an optimal period for observing the initiation of changes in eye and wing coloration, and P70 marks the beginning of the investigation into the morphological changes of the adult within the \u003cem\u003eDrosophila\u003c/em\u003e pupal case. We also found that compared with wild-type \u003cem\u003eDrosophila\u003c/em\u003e, mutant fruit flies showed some differences in development time and pupal tissue organization. These observations offer a detailed developmental map of the \u003cem\u003eDrosophila\u003c/em\u003e pupal stage, which is essential for accurate experimental staging and sampling. This study's findings not only contribute to the fundamental understanding of insect metamorphosis but also provide a valuable resource for researchers utilizing \u003cem\u003eDrosophila\u003c/em\u003e as a model system to study gene expression, development, and behavior.\u003c/p\u003e","manuscriptTitle":"Dynamic Morphological Staging of Drosophila Pupal Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:34:14","doi":"10.21203/rs.3.rs-6337002/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2202017b-7115-46c7-931a-d5d41e7e89ed","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-09T17:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 06:34:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6337002","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6337002","identity":"rs-6337002","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}