Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori

In holometabolus insects, morphology of the larval fat body is remodeled during metamorphosis. In higher Diptera, remodeling of the fat body is achieved by cell death of larval fat body cells and differentiation of the adult fat body from primordial cells. However, little is known about remodeling of the fat body at pupal metamorphosis in Lepidoptera. In this study, we found that cell death of the larval fat body in Bombyx mori occurs at shortly after pupation. About 30% of the fat body cells underwent cell death on days 1 and 2 after pupation. The cell death involved genomic DNA fragmentation, a characteristic of apoptosis. Surgical manipulation and in vitro culture of fat body cells revealed that 20-hydroxyecdysone and juvenile hormone had no effect on either initiation or progression of cell death. During cell death, a large increase in activity of caspase-3, a key enzyme of cell death, was observed. Western blot analysis of the active form of caspase-3-like protein revealed that the length of caspase-3 of B. mori was much larger than that of caspase-3 in other species. The results suggest that larval fat body cells of B. mori are removed through cell death, which is mediated by a caspase probably categorized in a novel family.     

Programmed cell death and stem cell differentiation are responsible for midgut replacement in <Emphasis Type="Italic">Heliothis virescens</Emphasis> during prepupal instar

We have analyzed midgut development during the fifth larval instar in the tobacco budworm Heliothis virescens. In prepupae, the midgut formed during larval instars undergoes a complete renewal process. This drastic remodeling of the alimentary canal involves the destruction of the old cells by programmed cell-death mechanisms (autophagy and apoptosis). Massive proliferation and differentiation of regenerative stem cells take place at the end of the fifth instar and give rise to a new fully functioning epithelium that is capable of digesting and absorbing nutrients and that is maintained throughout the subsequent pupal stage. Midgut replacement in H. virescens is achieved by a balance between this active proliferation process and cell-death mechanisms and is different from similar processes characterized in other insects. This work was supported by FAR 2006 (University of Insubria) to G.T., by a MIUR-FIRB-COFIN grant (no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).     

Cell proliferation, survival, and death in the Drosophila eye

The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a ‘Second Mitotic Wave’ cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival.     

Phospholipid synthesis in the fat body endoplasmic reticulum during primary and secondary juvenile hormone stimulation of vitellogenesis inLeucophaea maderae

Summary Juvenile hormone (JH) treatment coordinately stimulated the dose-dependent synthesis of vitellogenin and endoplasmic reticulum (ER) membrane phospholipids in fat body cells from allatectomized adult females ofLeucophaea maderae. Animals were pulse-labeled in vivo with [³²P] to simultaneously measure the rates of synthesis of the phosphorylated subunits of vitellogenin and the structural phospholipids of the ER membranes. Phospholipid synthesis in ER membranes from nontarget tissues for JH such as thoracic muscle, midgut, and larval fat body was unresponsive to hormone treatment. The proliferation of ER in response to JH treatment was thus restricted to tissue that was competent to synthesize vitellogenin.Primary and secondary vitellogenin induction was measured in allatectomized adult females treated 12 days apart with JH-III. The time-course of the primary response for vitellogenin and ER phospholipid synthesis was characterized by a 24 h latent period, a rapid increase to a maximum at 72 h, and then a gradual decline. During secondary induction, vitellogenin accumulated in the hemolymph nearly twice as fast as before and peaked at a concentration of 38 g/l. This vitellogenin titer was approximately two-fold higher than that found at the height of the primary response. During both primary and secondary stimulation with JH, ER phospholipid synthesis, as measured by [¹⁴C]choline incorporation into microsomal phosphatidylcholine, was stimulated five-fold over the untreated control animals. The amplified production of vitellogenin during the secondary response was associated with a 24 h-earlier peak of ER phospholipid synthesis in the fat body.     

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway

In this paper we have investigated the developmental–genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells.     

The transmembrane protein,Tincar, is involved in the development of the compound eye in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We previously cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. Here, we have studied the expression pattern and functions of tinc during developmental processes. tinc mRNA is expressed in the central and peripheral nervous systems, and midgut during embryogenesis. In the third-instar larval eye disc, tinc mRNA is strongly expressed in all the differentiating ommatidial cells within and in the vicinity of the morphogenetic furrow. Loss-of-function analysis using the RNA-interference method revealed severe defects of eye morphogenesis during the late developmental stages. Our results suggested that tinc may have an indispensable role in the normal differentiation of ommatidial cells.Edited by C. Desplan     

Beta-cyclodextrin facilitates cholesterol efflux from larval Manduca sexta fat body and midgut in vitro

The ability of 2-hydroxypropyl-β-cyclodextrin (HPβCD) and methyl-β-cyclodextrin (MβCD) to promote cholesterol efflux from [³H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both β-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MβCD than for HPβCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.     

Bombyxin stimulates proliferation of cultured stem cells derived from Heliothis virescens and Mamestra brassicae larvae

Summary Bombyxin stimulated proliferation of cultured midgut stem cells that were derived from two noctuiid moth larvae, Heliothis virescens and Mamestra brassicae. Bombyxin exhibited the highest activity at 10⁻¹² M. The number of cells increased for 3 d after the addition of bombyxin. Although a single addition of bombyxin did not maintain proliferation, a second addition, made 3 d after the first treatment, retained the effect. Results suggest that the decline of effect after the first addition was not due to the loss of sensitivity of the cultured cells but to the loss of effect of the growth factor added. Addition of bombyxin at more than 10⁻¹⁰ M was less effective. Bombyxin did not affect the number of cultured midgut cells without pupal fat body extract (FBX). The data suggest that FBX contains the factors that maintain sensitivity of midgut cells to proliferate in the presence of bombyxin. Bombyxin must be a unique growth factor that stimulates proliferation of midgut stem cells in vitro from lepidopteran larvae. Materials listed here are not endorsed by the U.S. Department of Agriculture.     

Histopathology of an aphid,Schoutedenia lutea,parasitized by a gall midge,Pseudendaphis sp

Pseudendaphis sp. (Barnes) [Diptera: Cecidomyiidae] is a larval endoparasitoid of the aphidSchoutedenia lutea (van der Goot) [Aphididae: Greenideinae]. A brief investigation of the biology ofPseudendaphis sp. revealed that parasitization by this species occurs only in viviparous instar III to adult life stages of the host aphid. Comparative histology of parasitized, unparasitized and starved aphids was used to evaluate proposed mechanisms of pathogenesis. Parasitization byPseudendaphis sp. was found to cause the destruction of host embryos and pathological changes in the host fat body, yet resulted in an increase in the number of host mycetocytes. There was no evidence of injury to host tissues through direct physical attack, a cytolytic enzyme or starvation-like factors. Remaining hypotheses for altered host histologies in parasitized aphids are selective nutrient depletion and production of a non-cytolytic bioactive substance by thePseudendaphis larvae.      

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

Location of ecdysteroid 22-O-acyltransferase in the larvae ofHeliothis virescens

Larvae of the tobacco budworm,Heliothis virescens, are resistant to high levels of ingested 20-hydroxyecdysone which could cause potential inhibition to the development of many other lepidopteran species. This resistance is attributed to the ability of the larvae to metabolize this molting hormone to its 22-acyl ester forms. When tobacco budworm larvae were fed large quantities of 20-hydroxyecdyone, the hormonal metabolites were found in gut and fat body tissues. When incubated with 20-hydroxyecdysone gut tissue converted 20-hydroxyecdysone into its 22-acyl ester metabolites. Lumen site of the midgut was found to be the major location of this bio-transformation. In contrast, fat body tissue failed to convert 20-hydroxyecdysone to 22-acyl ester metabolitesin vitro. After the oral injection of³H-ecdysone, the major metabolites formed were ecdysone 22-acyl esters whereas the majority of³H-ecdysone was transformed to polar metabolites after it was injected into the hemocoel of the larvae. Similar distributions of ecdysteroid 22-O-acyltransferase and alkaline phosphatase activity in subcellular fractions demonstrates the co-localization of these enzymes in plasma membrane of the gut epithelial cells. These results suggest that gut brush border membrane is the major site of ecdysteroid 22-acyl ester formation inH. virescens larvae.     

Cloning of HumanENC-1and Evaluation of Its Expression and Regulation in Nervous System Tumors

We recently identified and characterized a novel murine gene,ENC-1,that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role forENC-1gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue ofENC-1.The humanENC-1gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels ofENC-1mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of theENC-1gene during neural crest cell differentiation. We found that the expression ofENC-1increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest thatENC-1expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.     

Complexity in evolved regulatory variation for alcohol dehydrogenase genes in HawaiianDrosophila

Summary The alcohol dehydrogenase gene (Adh gene) ofDrosophila affinidisjuncta is expressed at a higher level in the larval midgut and Malpighian tubules than the homologous gene fromDrosophila hawaiiensis. This study analyzed thecis-acting sequences responsible for these regulatory differences in larval tissues ofDrosophila melanogaster transformants. A series of 10 chimeric and deletedAdh genes was introduced into the germ line ofD. melanogaster, and tissue-specific expression levels were quantified by gel electrophoresis of tissue extracts. Sequences in the upstream region of the two genes had the strongest influence on enzyme production in the midgut and Malpighian tubules. Other sequence elements also showed effects, some of which were tissue specific. Most gene fragments displayed context-dependent effects, thus supporting the proposed model of polygenic regulation ofAdh gene expression.     

Pulses of ammonia and methylamine induce down-regulation of nematocyte and nerve cell populations in Hydrozoa (Hydra; Hydractinia)

Summary Hydrozoa replace used-up nematocytes (cnidocytes) by proliferation and differentiation from interstitial stem cells (i cells). Repeated pulsed exposure ofHydra to elevated levels of unprotonated ammonia leads to successive loss of the various types of nematocytes: first of the stenoteles, then of the isorhizas and finally of the desmonemes. The loss is due to deficits in supply; the number of nematoblasts and differentiating intermediates is reduced. In the hydroidHydractinia the main process leading to numerical reduction was observed in vivo: mature nematocytes as well as precursors emigrate from their place of origin into the gastrovascular channels where they are removed by phagocytosis. This is a regular means by which these animals down-regulate an induced surplus of nematocytes. With lower effectiveness, pulses of methylamine, trimethylamine and glutamine also induce elimination of the nematocyte lineages. In the long term the population of nerve cells, which are permanently but slowly renewed from interstitial neuroblasts, decreases, too. After 2 months of daily repeated treatment the density of the Arg-Phe-amide-positive nerve cells was reduced to 50% of its normal level. Thus, ammonia induces down-regulation of all interstitial cell lineages. The temporal sequence of the ammonia-induced loss reflects the diverse rates with which the various i cell descendants normally are renewed.     

DSP toxin contents inDinophysis fortii and scallops collected at Mutsu Bay,Japan

Cell densities of toxic phytoplankton species responsible for diarrhetic shellfish poisoning (DSP) were monitored at a sampling site in Mutsu Bay, Japan, in 1995.Dinophysis fortii almost completely dominated the toxic phytoplankton community. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) contents in bothD. fortii cells and midgut glands of scallops collected at the same sampling site were determined by HPLC — fluorometry. DTX1 was detected fromD. fortii and scallops. The contents of DTX1 inD. fortii changed markedly during the experimental periods (5–252 pg cell^–1). The highest concentration of DTX1 in the midgut glands of scallops coincided with the period of relatively high cell densities ofD. fortii with the highest content of DTX1 (252 pg cell^–1). The results demonstrate that toxin content in the cells is an important factor affecting the toxicity of shellfish.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Ultrastructural changes in the muscles,midgut, hemopoietic organ,imaginal discs and Malpighian tubules of the mosquito Aedes aegypti larvae infected by the fungus Coelomomyces stegomyiae

Fungi belonging to the genus Coelomomyces can infect mosquito larvae and develop within the larval hemocoel. To examine fungal development, Aedesaegypti larvae infected with Coelomomyces stegomyiae Keilin were fixed, embedded and sectioned for both light and electron microscopy. While fungal hyphae of C. stegomyiae did not invade cells other than the cuticular epithelial cells, they did penetrate a number of tissues including muscles, midgut, hemopoietic organ, imaginal discs, and Malpighian tubules. This revised version was published online in July 2006 with corrections to the Cover Date.