Wandering Stage Research Articles

Purification and expression analysis of imaginal disc growth factor in the silkworm, Bombyx mori

In the present study, we purified and sequenced a homolog of the Drosophila imaginal disc growth factor (IDGF) from the hemolymph of Bombyx mori (BmIDGF). Antibodies against BmIDGF were produced and subsequently used in immunoblotting analyses. The immunoblotting analyses demonstrated an extremely high level of BmIDGF in the hemolymph throughout the period of rapid growth of the organs of B. mori. The results of RT-PCR showed that BmIDGF was predominantly expressed in fat bodies. Real-time RT-PCR revealed that BmIDGF transcripts in fat bodies were highly expressed during the feeding stage but significantly suppressed during the molting, wandering, and pupal stages. Starvation brought about a significant decline of BmIDGF mRNAs in the fat bodies and BmIDGF proteins in the hemolymph. After re-feeding, the BmIDGF transcripts in fat bodies and BmIDGF proteins in the hemolymph increased again. In addition, an immunocytochemical study revealed BmIDGF proteins on the surface of wing discs. The present findings suggest that the level of BmIDGF in the hemolymph was modulated by the fat body in response to nutritional conditions and that BmIDGF was transported to target organs through the hemolymph.

The impacts of classical insect hormones on the expression profiles of a new digestive trypsin‐like protease (TLP) from the cotton bollworm, Helicoverpa armigera

Trypsin proteinases perform important roles in the protein digestion of an insect midgut. A 1042 bp full-length cDNA was cloned from Helicoverpa armigera. The gene encoded a 32 kDa protein, with a predicted isoelectric point of 5.7. The amino acid sequence of the protein had a trypsin-like serine protease domain, and the gene was named Ha-TLP. The expression of the gene was tissue-specific and the transcript of Ha-TLP existed only in the midgut and was not found in the head-thorax, integument, fat body and haemocytes from 5th instar larvae, with similar expression levels between those in feeding larvae and in molting larvae. In the midgut, the gene transcription level declined from 6th instar 72 h after the larvae entered the wandering stage, and disappeared from 6th instar at 96 h until the pupal stage. By immunohistochemistry, Ha-TLP was detected in the cytoplasm of the midgut epithelial cells of the 6th instar feeding stage worms. The expression of Ha-TLP could be up-regulated by a juvenile hormone (JH) analog methoprene and down-regulated by 20-hydroxyecdysone (20E). These facts indicate that Ha-TLP was involved in food digestion during larval growth and probably up-regulated by JH and suppressed by extra 20E in vivo.

Switchover of protein synthesis occurs during the wandering stage in the labial glands of the sweet potato hornworm, Agrius convolvuli

Switchover of protein synthesis occurs during the wandering stage in the labial glands of the sweet potato hornworm, Agrius convolvuli

Endogenous 20-hydroxyecdysone levels in the haemolymph of non-diapause-destined and diapause-destined generations of tasar silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) and associated developmental changes

A complete profile of the 20-hydroxyecdysone (20-HE) titer, development and endocrine events from 1st instar to pupation of the larvae of non-diapause-destined (NDD) and diapause-destined (DD) tasar silkworm, Antheraea mylitta Drury (Lepidoptera: Saturniidae) was studied. Diapause is induced by short days of 11 hr photophase coupled with = 12h light, >= 26°C) and one generation is completed by August. The growth rate during the course of development of larval instars decreases and instar durations are inversely related to the body weight at the time of initiation of a larval instar. A growth compensation mechanism operates during the development of the larval instars. The growth rate was higher in early instars (1st to 4th) in both generations. The DD larvae complete the final instar in 16 days followed by a spinning stage of 13 days. The NDD larvae complete the final larval instar in 9 days followed by spinning stage of 6 days and spend 14 days in the pupal stage. The signal to release the prothoracicotropic hormone (PTTH) is related to critical body weight of larvae. From 1st to 4th instar, pre-ecdysial peaks of 20-HE were recorded in both NDD and DD generations. The programme for undergoing diapause was initiated during 3rd instar and induced by a sudden decrease in the level of 20-HE in the DD generation. Two peaks of 20-HE are required for the larval-pupal transformation, first at the wandering stage and the second at cuticle formation.

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in naïve insects and in insects manipulated with bacterial and hormonal treatments.

SNF4Aγ, the Drosophila AMPK γ subunit is required for regulation of developmental and stress-induced autophagy

In holometabolous insects including Drosophila melanogaster a wave of autophagy triggered by 20-hydroxyecdysone is observed in the larval tissues during the third larval stage of metamorphosis. We used this model system to study the genetic regulation of autophagy. We performed a genetic screen to select P-element insertions that affect autophagy in the larval fat body. Light and electron microscopy of one of the isolated mutants (l(3)S005042) revealed the absence of autophagic vesicles in their fat body cells during the third larval stage. We show that formation of autophagic vesicles cannot be induced by 20-hydroxyecdysone in the tissues of mutant flies and represent evidence demonstrating that the failure to form autophagic vesicles is due to the insertion of a P-element into the gene coding SNF4Aγ, the Drosophila homologue of the AMPK (AMP-activated protein kinase) γ subunit. The ability to form autophagic vesicles (wild-type phenotype) can be restored by remobilization of the P-element in the mutant. Silencing of SNF4Aγ by RNAi suppresses autophagic vesicle formation in wild-type flies. We raised an antibody against SNF4Aγ and showed that this gene product is constitutively present in the wild-type larval tissues during postembryonal development. SNF4Aγ is nearly absent from the cells of homozygous mutants. SNF4Aγ translocates into the nuclei of fat body cells at the onset of the wandering stage concurrently with the beginning of the autophagic process. Our results demonstrate that SNF4Aγ has an essential role in the regulation of autophagy in Drosophila larval fat body cells.

Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.

Characters and expression of the gene encoding DH, PBAN and other FXPRLamide family neuropeptides in Antheraea pernyi

AbstractThe full‐length cDNA encoding diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) in Antheraea pernyi (Anp‐DH‐PBAN) were cloned and sequenced by rapid amplification of cDNA ends methods. The Anp‐DH‐PBAN cDNA encodes a 196‐amino acid (aa) prehormone that contains a 24‐aa DH‐like peptide, a 33‐aa PBAN and three other neuropeptides, all of which share a common C‐terminal pentapeptide motif FXPR/KL (X = G, T, S). The Anp‐DH‐PBAN shows highest homology (82%) to that of Samia cynthia ricini at amino acid level. Northern blots demonstrate the presence of a 0.8‐kb transcript in the brain and suboesophageal ganglion (SG) complex. During the early pupal stages, the Anp‐DH‐PBAN mRNA contents increase consistently in both diapause‐ and non‐diapause‐destined pupae. From day 7, mRNA in diapause pupae dropped promptly and maintained a lower level than that in the non‐diapause type. The Anp‐DH‐PBAN mRNA was expressed mainly in SG, at much lower levels in brain and thoracic ganglia (TG), but not in non‐neural tissues. FXPRLamide (Phe‐X‐Pro‐Arg‐Leu) peptide immunoreactivity was detected in the SG, TG and terminal abdominal ganglion of A. pernyi by whole‐mount immunocytochemistry. The titres of FXPRLamide peptides in the haemolymph in diapause type are consistently lower than in non‐diapause insects. In non‐diapause individuals, there are two peaks of FXPRLamide titres, day 2 in the wandering stage and day‐5 in the pupal stage respectively.

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.

Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.

Psidin Is Required in Drosophila Blood Cells for Both Phagocytic Degradation and Immune Activation of the Fat Body

Phagocytic blood cells are critical to innate immune defense: They internalize and destroy microbial invaders and produce signals that trigger other immune responses. Despite this central role, the in vivo contributions of phagocytosis to systemic immune activation are not well understood. Drosophila has proven a fruitful model for the investigation of evolutionarily conserved innate immune mechanisms, including NF-kappaB-dependent transcriptional induction, RNAi in antiviral responses, and phagocytosis. The phagocytes of Drosophila encounter bacterial invaders early in infection and contribute to survival of infection. Phagocytosis in flies and mammals is highly homologous: Both rely on scavenger receptors, opsonins, and actin rearrangements for engulfment; have phagosomal cysteine proteases active at low pH; and can be subverted by similar intracellular pathogens. Although the role of Drosophila phagocytes in the activation of other immune tissues has not been clear, we show that induction of the antibacterial-peptide gene Defensin in the fat body during infection requires blood-cell contributions. We identify a gene, psidin, that encodes a lysosomal protein required in the blood cells for both degradation of engulfed bacteria and activation of fat-body Defensin. These data establish a role for the phagocytic blood cells of Drosophila in detection of infection and activation of the humoral immune response.

The control of growth and differentiation of the wing imaginal disks of Manduca sexta

During the larval feeding period, the growth of the wing imaginal disks of Lepidoptera is dependent on continuous feeding. Feeding and nutrition exert their effect via the secretion of bombyxin, the lepidopteran insulin-like hormone. When larvae stop feeding and enter the wandering stage in preparation for metamorphosis, the control of imaginal disk growth becomes feeding and nutrition-independent. Growth of the wing imaginal disks of non-feeding wandering stage Manduca sexta can be stopped by removal of the brain, indicating that a brain-derived factor is required for continued disk growth. Isolated wing disk growth in vitro requires both 20-hydroxyecdysone (20E) and either brain extract or bombyxin to achieve normal growth. In vitro, brain extracts and synthetic bombyxin have little or no effect in stimulating disk growth, but they greatly enhance the effect of 20E, indicating that 20E and bombyxin act synergistically to modulate growth of the imaginal disk. Brain extract and bombyxin induce a suite of insulin-response events in cultured wing disks, which indicate that bombyxin and 20E act through separate and synergistic pathways. The dose-response to 20E reaches a plateau at about 0.1 μg/ml. Tracheal differentiation of the wing disks can be induced to initiate in vitro by a low concentration of 20E, whereas higher concentrations of 20E only stimulate growth.

Membrane-bound sorbitol 6-phosphatase in fat body cells controls the dynamics of sorbitol 6-phosphate, a major hemolymph sugar in the silkworm

Sorbitol 6-phosphate (S6P) is one of two major sugars (another is trehalose) in the larval hemolymph of Bombyx mori, and its amount dramatically decreases concomitantly with the onset of prepupal period. In the last (fifth) instar larvae, the amount of S6P is approximately 30 μmol/larva at its maximum and decreases to less than 1 μmol at the wandering stage. Incubation of fat bodies of wandering larvae with S6P generates sorbitol in the medium, while S6P in the medium decreases, indicating that fat body possesses sorbitol 6-phosphatase (S6Pase) activity. S6Pase activity in the fat body remains low during the feeding period, abruptly increases at the wandering and decreases to a low level after gut purge. 20-Hydroxyecdysone (20E) increases S6Pase activity in the fat body of feeding larvae, and the activation is dose-dependent. Cell fractionation studies show that S6Pase is mainly associated with the membrane and the optimal pH for membrane-bound S6Pase is 5.5, which is different from that for soluble acid phosphatase (pH 4). Present findings indicate that the S6Pase responsible for a decrease in hemolymph S6P is membrane-bound, and its activity is controlled by a rise of hemolymph ecdysteroid titer at the onset of the wandering stage.

Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda

The focus of this study was on the characterization and expression of genes encoding enzymes responsible for the synthesis and degradation of chitin, chitin synthase ( SfCHSB) and chitinase ( SfCHI), respectively, in the midgut of the fall armyworm, Spodoptera frugiperda. Sequences of cDNAs for SfCHSB and SfCHI were determined by amplification of overlapping PCR fragments and the expression patterns of these two genes were analyzed during insect development by RT-PCR. SfCHSB encodes a protein of 1523 amino acids containing several transmembrane segments, whereas SfCHI encodes a protein of 555 amino acids composed of a catalytic domain, a linker region and a chitin-binding domain. SfCHSB is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are expressed along the whole midgut. Chitin staining revealed that this polysaccharide is present in the peritrophic membrane (PM) only when SfCHSB is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHSB is responsible for PM chitin synthesis during the larval feeding stages and SfCHI carries out PM chitin degradation during larval–pupal molting, suggesting mutually exclusive temporal patterns of expression of these genes.

Gene structure and expression profile of Manduca sexta prophenoloxidase‐activating proteinase‐3 (PAP‐3), an immune protein containing two clip domains

Prophenoloxidase-activating proteinase-3 (PAP-3) is a component of the defence system in Manduca sexta. We have isolated genomic clones and elucidated the organization of this gene. The 3' end of exon 2, the entire exon 3 and the 5' end of exon 4 encode the two amino-terminal clip domains. Southern blot analysis suggested a single copy of the PAP-3 gene in the genome. We identified several putative immune-responsive elements in the upstream region. The PAP-3 gene is not highly expressed in the fat body during larval development until the wandering stage begins. The mRNA level is high in the epithelium, fat body and haemocytes. Tissue-specific alternative splicing occurs in the fat body and trachea. A bacterial injection markedly induced the gene expression in the fat body and haemocytes.

Hemocyte differentiation in the hematopoietic organs of the silkworm, Bombyx mori: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%-70%) and oenocytoids (30%-40%). Granulocytes comprised only about 0.5%-1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10-30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.

The role of 20-hydroxyecdysone in the CNS metamorphosis in flesh fly (Neobellieria bullata) larvae (Diptera: Sarcophagidae)

The role of 20-hydroxyecdysone (20E) in the metamorphosis of the central nervous system was investigated by recording feeding, wandering and pupariation behaviour as the measures of hormonal effects on the neural tissues in the flesh fly Neobellieria (Sarcophaga) bullata. The minimum amount of food essential for the commitment to metamorphosis is rather small and constant and the larvae ingest it during the first 4 to12 h of the last instar. Underfed larvae maintain food appetency for a certain time and this period of hunger can be considerably shortened by the application of 20E. Application of 20E also significantly shortens or abolishes the intake of additional food offered to starving larvae. Thus 20E has an effect on neural mechanisms controlling both termination of the period of hunger and suppression of feeding behaviour. Application of 20E at any time during the wandering stage stimulates the precocious onset of pupariation. The central neurons controlling motor patterns of pupariation behaviour are committed to perform properly their programme some 15 h earlier than the larval integument is ready to transform into normal puparium. As a result of this temporal shift of tissue determination, some precociously formed puparia are not properly shaped despite the fact that the larva is exhibiting normal patterns of pupariation behaviour.

Regulation of chitin synthesis in the larval midgut of Manduca sexta

In insects, chitin is not only synthesized by ectodermal cells that form chitinous cuticles, but also by endodermal cells of the midgut that secrete a chitinous peritrophic matrix. Using anti-chitin synthase (CHS) antibodies, we previously demonstrated that in the midgut of Manduca sexta, CHS is expressed by two cell types, tracheal cells forming a basal tracheal network and columnar cells forming the apical brush border [Zimoch and Merzendorfer, 2002, Cell Tissue Res. 308, 287–297]. Now, we show that two different genes, MsCHS1 and MsCHS2, encode CHSs of midgut tracheae and columnar cells, respectively. To investigate MsCHS2 expression and activity in the course of the larval development, we monitored chitin synthesis, enzyme levels as well as mRNA amounts. All of the tested parameters were significantly reduced during molting and in the wandering stage when compared to the values obtained from intermolt feeding larvae. By contrast, MsCHS1 appeared to be inversely regulated because its mRNA was detectable only during the molt at the time when tracheal growth occurs at the basal site of the midgut. To further examine midgut chitin synthesis, we measured enzyme activity in crude midgut extracts and different membrane fractions. When we analysed trypsin-mediated proteolytic activation, a phenomenon previously reported for insect and fungal systems, we recognized that midgut chitin synthesis was only activated in crude extracts, but not in the 12,000 g membrane fraction. However, proteolytic activation by trypsin in the 12,000 g membrane fraction could be reconstituted by re-adding a soluble fraction, indicating that limited proteolysis affects an unknown soluble factor, a process that in turn activates chitin synthesis.

A baculovirus-encoded protein tyrosine phosphatase gene induces enhanced locomotory activity in a lepidopteran host.

Enhanced locomotory activity (ELA), such as wandering, is a normal behavior that occurs at the end of the larval stage in lepidopteran (butterflies and moths) insects. Baculovirus infection can also induce ELA in lepidopteran larvae. The belief is that the virus induces this behavior to increase its transmission [Goulson, D. (1997) Oecologia 109, 219-228]. Here we show that a baculovirus-encoded protein tyrosine phosphatase (PTP) gene (ptp) induces ELA that is activated by light. ELA was induced in silkworm Bombyx mori infected with the baculovirus B. mori nucleopolyhedrovirus (BmNPV) beginning at approximately 3.75 days postinfection (p.i.) and continued until 4.75 days p.i. The intensity of the ELA was dramatically reduced immediately before death at 5.25 days p.i. Light activated the intensity of the ELA by approximately 3-fold, and larvae with ELA showed positive phototropism. ELA was not induced in larvae of B. mori infected with a BmNPV ptp knockout mutant (BmPTPD). However, when a silkworm-derived ptp gene (Bmptp-h) was inserted into BmPTPD, ELA was partially recovered. Bmptp-h was identified from silkworms at 2 days after the start of the natural wandering stage. The deduced amino acid sequence of Bmptp-h showed 48.2% identify (80.7% similarity) to the deduced amino acid sequence of BmNPV ptp. On the basis of the high homology and larval stage at which Bmptp-h was isolated, we postulate that the modern baculovirus may have acquired its ptp gene from an ancestral host and that this gene was selectively maintained because it increases virus transmission.

Development of the adult leg epidermis in Manduca sexta: contribution of different larval cell populations

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.