Synthesis and transport of storage proteins by testes in Heliothis virescens.

The synthesis of two storage protein subunits, 76,000-Mr and 82,000-Mr polypeptides, by the testes sheath has been studied in Heliothis virescens. Like fat body, which is the primary site of synthesis for the large extratesticular pool, cells of the testes sheath secrete glycosylated storage proteins assembled into hexamers. The testis sheath differed from fat body in several important respects, including the failure to synthesize an abundant (in the hemolymph) 74,000-Mr storage protein, its relatively reduced expression of the 76,000-Mr polypeptide, and the absence of resorption of storage proteins from the lumen of the testis during pupal development. Cyst cells were also shown to import actively the 82,000-Mr storage protein by pinocytosis of testicular fluid and transfer it to the developing spermatids. Unlike other cell types that sequester storage proteins in the form of cytoplasmic granules, their localization within spermatids was exclusively mitochondrial. These observations suggest that expression of the storage protein genes is regulated tissue specifically and reveal novel pathways for their transport and, perhaps, utilization and function during development.     

Flow cytometric analysis of mature adipocytes

Flow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to greater than 120 microns in diameter. Stored fat occupies 90-98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle. These obstacles were overcome by 1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37 degrees C; 2) using a 200 microns diameter orifice; 3) using a short, 300 microns inside diameter Teflon sample delivery line; 4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and 5) using the pH-sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets. Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo-1, label both fat cells and fat droplets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of lepidopterous tissues with proteolytic enzymes: Comparison of collagenase,trypsin, and pronase

The fat body and certain other tissues of lepidopterous larvae and diapausing pupae can be completely dissociated by incubation in crude or purified collagenase. Damage to the cells is minimal. Dissociation with trypsin is incomplete, and pronase causes extensive damage. All three enzymes act principally by digesting the extracellular connective sheath that envelops the individual lobes of fat body, since the cells at this stage are not intrinsically cohesive. The experimental findings support the notion that collagen is an important structural component of insect connective membranes.     

Immunological analysis of lipophorin in the haemolymph,ovaries, and testes of the fall webworm,Hyphantria cunea (Drury)

Lipophorin (LP) was purified from haemolymph in last instar larvae of Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation and gel filtration. LP is composed of Apo-LP I and Apo-LP II with molecular weights of 230 kDa and 80 kDa, respectively. The level of haemolymph LP in early pupae was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development, whereas the level of haemolymph LP decreases in female pupae beginning at day 7, coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their pre-adult levels. Actually, LP was found in the ovary by immunodiffusion, tandem-crossed immunoelectrophoresis, and Western blotting. Location of LP in the ovary was also traced by immunogold labelling. Also, LP appeared in small amounts in protein yolk bodies of the ovary at an early stage of vitellogenesis, when nurse cells are bigger than the oocyte, but in greater amounts at those stages when the oocyte is larger than nurse cells—that is, when vitellogenesis is actively taking place. This fact clearly reveals that LP is synthesized by fat body and released into the haemolymph, and then taken up by the growing ovary during vitellogenesis. Also, LP was detected in testes by immunological analysis. Western blotting showed that LP was present in testicular fluid but not in the peritoneal sheath and cysts. To test whether LP is also synthesized in testes, testes and fat body tissues were cultured in vitro, indicating that fat body synthsizes LP but testes do not. The result showed that the haemolymph LP itself is taken up into the testes. © 1994 Wiley-Liss, Inc.     

Larvae of an endoparasitoid,Cotesia kariyai (Hymenoptera: Braconidae), feed on the host fat body directly in the second stadium with the help of teratocytes

Larvae of a gregarious endoparasitoid, Cotesia kariyai (Watanabe), grew rapidly during the second stadium in the host. The fat body of a Pseudaletia host parasitized by C. kariyai was completely consumed by 10 d, just before larval emergence. It seemed hard to explain the growth of the second instar parasitoids and the rapid consumption of the fat body only by ingestion of hemolymph converted from the fat body or other organs of the host. Paraffin sections of the parasitized host revealed that many teratocytes were attached to the surface of the fat body in many sites and destroyed the fat body tissue locally. Zymography of proteins released from the teratocytes revealed that the teratocytes 4 to 9 days after parasitization showed collagenase activity (as a gelatinase). Further, 1st instar parasitoids which were transplanted together with teratocytes into unparasitized hosts preconditioned with C. kariyai polydnavirus (CkPDV) plus venom, grew normally to the 2nd stadium. Abnormal growth of parasitoid larvae was observed when parasitoid larvae were transplanted without teratocytes. These results suggest that the teratocytes attach to the outer sheath of the fat body, secrete an enzyme that makes a hole in the matrix of the fat body, thus allowing the second instar parasitoid to ingest the content of the fat body.     

Apoptosis in the fat body tissue of the beetle Tenebrio molitor parasitised by Hymenolepis diminuta

Many insects experience a decrease in reproductive output when parasitised. We are investigating mechanisms underlying this fecundity reduction using the rat tapeworm, Hymenolepis diminuta infection of Tenebrio molitor beetles. These include an increase in the resorption of developing ovarian follicles and a decrease in fat body synthesis of vitellogenin. The latter is the direct effect of a molecule produced by the parasite. Here we report a study to determine whether vitellogenin synthesis and follicle resorption are the result of parasite-induced apoptosis in the respective tissues and whether the parasite molecule acts directly on the fat body by inducing apoptosis. In vivo, the number of fat body cell nuclei with chromatin condensation are significantly elevated in parasitised females at all days examined and peaked at day 7 post-infection. A TUNEL assay to detect DNA fragmentation confirmed these observations of apoptosis. However, when fat body from uninfected females was co-cultured with live metacestodes they did not cause cells to die by apoptosis, showing that the induction signal does not come directly from the parasite. The follicle resorption observed in the ovaries of infected beetles was not associated with apoptosis of the epithelial cells. The possibility of several mechanisms underlying fecundity reduction is discussed.     

The mechanism of sensory transduction in the sensilla of the trochanteral hair plate of the cockroach,Periplaneta americana

Summary The trochanteral hair plate of the cockroach leg contains approximately 60 hair sensilla that are deflected by a joint membrane during flexion of the leg. Previous work has shown that the organ is a mechanoreceptor which limits leg flexion during walking by reflex connections to flexor and extensor motoneurons. Functional analysis of the largest sensilla has shown that their behaviour may be well approximated by a velocity detector followed by a unidirectional rectifier.We report here the results of an examination of the largest sensilla by scanning and transmission electron microscopy in an attempt to correlate the structure with the known functional elements. Each hair is innervated by a single sensory dendrite which is surrounded by an electron dense dendritic sheath. The dendrite terminates below the hair shaft in a tubular body containing a parallel array of microtubules embedded in an electron dense matrix, while the dendritic sheath extends beyond the tubular body to form the walls of the ecdysial canal. At the proximal end of the tubular body the dendritic sheath and sensory dendrite are anchored to the cuticular socket by a fibrous dome which seems to form a fulcrum around which the tubular body can be deflected by movements of the hair. We suggest that the basis for the detection of velocity may be mechanical differentiation by a fluid space between the dendritic sheath and the tubular body. The structure is also discussed with relation to the mechanism of sensory transduction and the possible causes of the unidirectional sensitivity.Supported by the Canadian Medical Research Council. The authors gratefully acknowledge the expert technical assistance of Sita Prasad     

Studies on the cytophysiology of the fat body of the American silkmoth

Summary A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.Studies on the influence of egg maturation on the morphology of the fat body of Hyalophora gloveri (L.) revealed that ovariectomy of pupae yielded adults having more fat body than normal females, and that the fat body cells of the ovariectomized animals contained more glycogen, lipid and protein. Male pupae receiving ovarian implants developed into adults containing eggs and possessed more fat body than normal females but less than normal males. Very few glycogen granules were found in the fat body cells of normal males or males with implanted ovaries.Supported by grant AM-02818 from the National Institutes of Health.We thank Dr. James Oschman for his helpful suggestions and constructive criticisms.     

Fine structural alterations with age in the fat body of the adult male housefly,Musca domestica

Summary The fat body of the adult housefly is composed of two types of cells, the lipid-and glycogen-rich fat body cells and the oenocytes. A comparison of the fine structure of the abdominal fat body in 4-day old and 31–35 day old male houseflies indicated an increase in lipid and a decrease in glycogen content in the fat body cells of old flies. Oenocytes of old flies exhibit deteriorative alterations with an accumulation of secondary lysosomes. Both fat body cells and oenocytes in senile flies are ingested by hemocytes.Supported by a grant from the National Science Foundation.     

A peculiar arteriovenous leptomeningeal complex in the guinea pig

In most areas of the body, arteries and veins run close together, often sharing a common connective tissue sheath. One exception to this is observed in the brain, where arteries come in from the base and veins collect over the convexity. Classically the larger blood vessels are formed by three coats: intima, media, and adventitia. Leptomeningeal vessels are further reinforced by a monolayer of pial cells. In the guinea pig, however, above the corpus callosum we found a group of blood vessels (an artery and several veins) enclosed in a common leptomeningeal sheath. The artery arises at the confluence of the anterior cerebral arteries; the veins drain into the straight sinus. The epithelial nature of the sheath is evident by the close apposition of cell membranes, the presence of junctional devices, and the existence of a basal lamina. The ultrastructural features of this epithelium are similar to those of the arachnoid-dural membrane. Whether this peculiar vascular complex has any specific function needs to be investigated further. The presence of these vessels apparently 'isolated' within a leptomeningeal subcompartment may provide a suitable model to study vascular-extravascular-cerebrospinal fluid substance exchange.     

Control of pupal fat body disappearance in the female black blow fly,Phormia regina (Meigen) by the brain and the corpus allatum

Neck-ligation, brain implantation, allatectomy, methoprene treatment, and ovariectomy indicated that the disappearance of pupal fat body cells in newly emerged adult female blow fly, Phormia regina, is controlled by the brain and the corpus allatum (CA). Absence of brain or CA greatly lowered the rate of fat body cell disappearance (i.e. death). Dependency on the CA decreased from 0 to 36h post-emregence, indicating that the CA was active during the earlier part of this timespan. Methoprene treatment enhanced pupal fat body cell disappearance in allatectomized females. Brain implantation restored the rate of pupal fat body cell disappearance in neck-ligated flies. Brains from day 1 sugar-fed flies proved to be more effective than those from day 2 sugar-fed flies, indicating that there may be a window after adult emergence that allows the brain to act directly or indirectly on the death of pupal fat body cells. Ovariectomy did not alter the rate of pupal fat body cell death in test animals. Dying pupal fat body cells were smaller in size, less dense (i.e. did not sink in saline like normal pupal fat body cells), and stickier (i.e. attached to other tissues tighter) than the healthy cells. A possible role played by ecdysteroids is also discussed.     

Unique intertesticular tissue complex in larvae of Heliothis virescens (F.) (Lepidoptera : Noctuidae)

A unique heart-testis complex has been found in the last instar of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera : Noctuidae). This complex consists of a previously undescribed muscle system, herein designated the cardiotesticular muscle, that attaches the heart to the testes, and a ubiquitous connective tissue sheath that overlays this muscle and surrounding fat body and pericardial cells. These interconnected organs form a sieve-like septum enclosing a sinus that contains clusters of hemocytes and lipid droplets. Lipid droplets appear to be released from the surface of the testes sheath. The cardiotesticular muscle is innervated by neuroendocrine nerves that may be the route by which the brain peptide, testis ecdysiotropin, reaches the testes. The cardiotesticular muscle and products sequestered in the cardiotesticular sinus may be involved in the fusion of the paired testes and maturation of the male reproductive system, because they are present only during this stage of development.     

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.     

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.     

Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization

In teleosts, such as medaka, ossification of the vertebral column starts with the mineralization of the notochordal sheath in a segmental pattern. This establishes the chordal centrum, which serves as the basis for further ossifications by sclerotome derived osteoblasts generating the vertebral body. So far, it is unclear which cells produce the notochordal sheath and how a segmental pattern of mineralization is established in teleosts. Here, we use a transgenic medaka line that expresses nlGFP under the control of the col10a1 promoter for in vivo analysis of vertebral body formation. We show that col10a1:nlGFP expression recapitulates endogenous col10a1 expression. In the axial skeleton, col10a1:nlGFP cells appear prior to the mineralization of the notochordal sheath in a segmental pattern. These cells remain on the outer surface of the chordal centra during mineralization as well as subsequent perichordal ossification of the vertebral bodies. Using twist1a1:dsRed and osx:mCherry transgenic lines we show that a subset of col10a1:nlGFP cells is derived from sclerotomal precursors and differentiates into future osteoblasts. For the first time, this shows a segmental occurrence of putative osteoblast precursors in the vertebral centra prior to ossification of the notochordal sheath. This opens the possibility that sclerotome derived cells in teleosts are implicated in the establishment of the mineralized vertebral column in a similar manner as previously described for tetrapods.     

Inhibition of the formation of protein storage granules by glutaurine in the larval fat body cells of Mamestra brassicae (Insecta, Lepidoptera)

Correlative changes in the protein contents of haemolymph and fat body and the accumulation of protein storage granules in the fat body cells of Mamestra brassicae were investigated during the last larval stage in normally developing larvae and following administration of glutaurine (1 X 10(-4) mg/g body weight). The protein content of the haemolymph of untreated larvae increased up to the 4th day of the stage, declined during days 5 and 6, and increased again before pupation. In the glutaurine-treated larvae the amount of proteins in the haemolymph was as high as in the controls during the first four days but continued to rise up to the end of the stage. The protein content of the fat body started to increase from the 3rd day and heavy accumulation of protein storage granules in the cells of fat body was observed on the 5th and following days. The protein content of the fat body of glutaurine-treated larvae remained at a low level and the protein storage granules were absent in the cells. The inhibition of the selective uptake of haemolymphatic storage proteins by fat body following glutaurine treatment is suggested.     

Evidence for the participation of arachidonic acid metabolites in trehalose efflux from the hormone activated fat body of the cockroach (Periplaneta americana)

The hypertrehalosemic hormones, HTH-I and HTH-II, activate trehalose synthesis and increase the rate of sugar efflux from Periplaneta americana fat body in vitro. These processes are unaffected by the diacylglycerol, 1-oleyl-2-acetyl-sn-glycerol, an activator of protein kinase C. Similarly, H-7 and spingosine, inhibitors of protein kinase C, are also inactive against trehalose efflux. The possibility that diacylglycerol lipase might generate an active fatty acid species was ruled out because of the failure of the inhibitor RHC-80267 to inhibit trehalose efflux. Activation of trehalose efflux from the intact fat body by HTH-I was strongly inhibited in a concentration dependent manner by the cyclooxygenase inhibitors indomethacin and diclofenac, but not by acetylsalicylic acid. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, also blocked HTH-I activated trehalose efflux in a concentration dependent fashion. The phospholipase A(2) inhibitors mepacrine and 4'-bromophenacyl bromide were also effective in decreasing the efflux of trehalose from HTH-I challenged fat body. The data suggest possible roles for arachidonic acid metabolites in the regulation of trehalose synthesis and in the efflux of the sugar from the fat body.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

Regional differentiation of fat bodies in larvae of the indianmeal moth,Plodia interpunctella

Larvae of the Indianmeal moth, Plodia interpunctella, contain two morphologically distinct fat bodies. Tan-colored, highly tracheated fat body located posteriorly in the abdomen was the predominant fat body tissue during the early larval instars. White, sheet fat body located more anteriorly became the predominant type during the fifth (last) larval instar and eventually occupied most of the space of the hemocoel. Ultrastructural morphology of tan fat body showed the tissue to be composed of cells containing numerous, large, spherical mitochondria, with only few lipid, glycogen, or protein storage structures. In contrast, white fat body was composed of cells that in later larval stages had organelles typical of storage functions. Both fat bodies produced storage proteins during the late fifth instar, whereas only white fat body accumulated the storage proteins. Tan fat body dispersed and apparently autolyzed in pharate pupae, whereas the white fat body metamorphosed and persisted into the adult stage. These observations indicate that fat body of the Indianmeal moth is functionally and morphologically differentiated along the anterior-posterior axis into two regional subgroups of cells.