Marcia J. Loeb

Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells in vitro

Abstract Fluorescent brighteners significantly lower the LC50 and LT50 in a variety of nucleopolyhedrovirus–insect host systems. In larvae of the gypsy moth, Lymantria dispar (L.), a European NPV strain of virus (LdMNPV) does not normally replicate in the midgut, but addition of a fluorescent brightener (Calcofluor M2R) to the virus suspension results in productive infections. In the current study, we show that LdMNPV also does not replicate in a larval midgut primary cell culture system unless a fluorescent brightener (Blankophor P167) is added. Morphological and cellular changes characteristic of apoptotic cell death were noted in infected midgut cells in vitro. We used the TUNEL assay to measure apoptosis in virus-challenged midgut cell cultures at 24–48 h post-inoculation. A significant decrease in apoptotic midgut cells was noted in the presence of 0.01 M brightener. The inhibition of apoptosis and presumptive inhibition of shedding of infected midgut cells in the presence of fluorescent brightener in the insect midgut appeared to promote virus replication and are likely to be partly responsible for enhancement of LdMNPV activity that is observed in gypsy moth larvae.

Stimulation of Midgut Stem Cell Proliferation and Differentiation by Insect Hormones and Peptides

Abstract: Stem cells derived from midguts of the caterpillar, Spodoptera littoralis, can be induced to multiply and differentiate in vitro. Ecdysone (E) and 20‐hydroxyecdysone (20E) had a concentration‐dependent effect: E was more active in cell proliferation and 20E in differentiation. Ecdysteroid receptors in midgut stem cell nuclei were stained with the antibody 9B9. In addition, α‐arylphorin and four midgut differentiation factors (MDF) specifically stimulated proliferation and differentiation of stem cells, respectively. The activity of a panel of peptide growth factors and hormones on growth and metamorphosis of the insect midgut is discussed.

Primary culture of insect midgut cells

This protocol describes the preparation of primary cell cultures from Lepidopteran midgut. These cultures have been used to identify factors that control midgut growth and differentiation, cell responses to these factors, effects of toxins on midgut growth, and the regulation of cell physiology. The protocol is divided into (1) procedures for cell collection, (2) composition of the culture, and (3) assay methods used for cell health, proliferation, and differentiation. Collection and setup require 4–6xa0h. Once established, a culture can survive several months at 25°C, be kept a year or longer at 4°C, or be frozen for indefinite storage.

Rapid Isolation of Nanogram Amounts of Crustacean Erythrophore Concentrating Hormone from Invertebrate Nerve Tissue by RP-HPLC

Abstract A reverse phase-high performance liquid chromatography (rp-hplc) method was developed for the rapid isolation of nanogram amounts of crustacean erythrophore concentrating hormone (CECH) from invertebrate nerve tissue. Tissue homogenates from the shrimp, Paleomenetes pugio, were subjected to a multistep work-up to remove proteins and lipids prior to analysis by rp-hplc. Samples were eluted with a concave gradient of 0.1% trifluoroacetic acid (TFA) verses acetonitrile. Detection at 210 and 254 nm combined with the use of highly efficient and end-capped columns permitted the determination of less than 5 ng of CECH. Pure CECH was isolated from the columns by fraction collection followed, by lyophilization of the volatile TFA buffer.

EFFECTS OF A FAT BODY EXTRACT ON LARVAL MIDGUT CELLS AND GROWTH OF LEPIDOPTERA

SummaryTreatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentraton-dependent manner. This effect was compared with those of insect molting hormones, ecdystone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.

INHIBITORY EFFECTS OF PARASITISM BY THE GREGARIOUS ENDOPARASITOID COTESIA CONGREGATA ON HOST TESTICULAR DEVELOPMENT

Cotesia congregata is a gregarious larval endoparasitoid of the tobacco hornworm, Manduca sexta. Parasitized larvae exhibit a variety of physiological and developmental aberrations, the most obvious of which is the induction of developmental arrest characterized by the absence of wandering behavior and suppression of pupation. This arrest appears attributable to continued maintenance of an elevated titer of juvenile hormone and reduced levels of hemolymph juvenile hormone esterase activity. Injection of the wasps polydnavirus into nonparasitized larvae also causes arrest and the larvae eventually form larval-pupal intermediates instead of normal pupae, indicating the virus may be partially responsible. Aside from causing arrested host development, parasitism also inhibits the normal development and differentiation of testes in male host larvae, so that the testes atrophy instead of growing synchronously with other larval tissues. Here we report that parasitism has pronounced disruptive cytological effects on the developing reproductive organs of male hosts, in addition to causing them to atrophy. Parasitism results in a reduction in testicular volume attributable to a reduction in the number of developing germ cells. Microscopy revealed that the structural integrity of the sheaths surrounding the testicular follicles also is disrupted, so that the tissues appear grossly abnormal compared to those of nonparasitized larvae. Intrahemocoelic injection of purified C. congregata polydnavirus in combination with venom into nonparasitized fourth instar larvae, or topical application of 100 μg of methoprene to fourth instar larvae, also alters sheath integrity and reduces the numbers of developing germ cells, but not to the same degree as the pattern observed in truly parasitized hosts. The occurrence of cell death in the male gonad was documented using the vital dyes acridine orange and ethidium bromide. Arch. Insect Biochem. Physiol. 36:95–114, 1997.

In vitro rearing of Edovum puttleri, an egg parasitoid of the Colorado potato beetle – development from egg through the pupal stage

A variety of semi-defined artificial diets were developed and tested for their ability to support the in vitro development of Edovum puttleri. In the most effective diet, 2.6% of E. puttleri pupated. This diet contained high levels of hen egg yolk combined with Manduca sexta larval hemolymph, or with a mixture of M. sexta egg homogenate and larval hemolymph. Egg homogenate alone (without the addition of hemolymph) was not capable of supporting the parasitoids development. Thus, hemolymph appears to contain unidentified factor(s) important for inducing pupation of the wasp. Addition of M. sexta pupal fat body tissue extract (in place of hemolymph) also promoted pupation of E. puttleri. Gypsy moth (Lymantria dispar) larval hemolymph could not replace M. sexta larval hemolymph. Fractionation irreversibly reduced the growth-promoting effects of M. sexta larval hemolymph. However, the most effective fraction contained components whose molecular weights were ≥1000 kd. In diets that were devoid of insect materials, the best results were achieved when hen egg yolk, FreAmine, yeast extract, lactalbumin, trehalose, fetal bovine serum and bovine milk were included. This is the first report of an artificial diet for in vitro rearing an eulophid parasitoid from the egg through the pupal stage.

Effect of calcium ions on ecdysteroid secretion by testes of Heliothis virescens

Abstract 1. 1. Testes of Heliothis virescens synthesized ecdysteroid in media containing low titers of calcium; the optimum calcium titer for testis sheaths stimulated to synthesize ecdysteroid in vivo was ca 1 mM, while the optimum of testes stimulated in vitro with the peptide testis ecdysiotropin was ca 0.3 mM calcium. 2. 2. Verapamil at concentrations lower than 10−3 M induced increases in ecdysteroid synthesis, indicating more efficient synthesis when calcium influx was inhibited. 3. 3. Hemolymph of H. virescens was 7 mM in calcium, while whole testes were maintained at 1–2 μM calcium.

IN VITRO AND IN VIVO EFFECTS OF A FAT BODY EXTRACT ON SPODOPTERA LITTORALIS

Dear Editor: In insects, growth and metamorphosis are governed by ecdysteroids and juvenile hormones, growth factors, and cytokines. When these compounds bind to their receptor s, the resulting gene activation aiad transcription may induce a number of processes such as cell division, tissue repair, chemotaxis, differentiation, or even cell death. Oberlander and Fulco (1967) showed that ecdysteroids stimulated metamorphosis of cultured Galleria mellonella wing imaginal disks. More recently, 20-hydroxyecdysone (20E) has been shown to induce development of imaginal wing disks of various Lepidoptera in vitro (Smagghe et al., 1996, 1999). Imaginal wing disks are masses of cells that give rise to adult wings during metamorphosis in holometabolous insects, triggered by an increase in ecdysteroids. During this process, epithelial cells show numerous mitoses, Golgi bodies, and well-developed endoplasmic reticulum appear, and a new cuticular layer is deposited (Riddiford, 1985). A few yr later, Dutkowski and Oberlander (1974) reported on interactions between ecdysteroids and the fat body during wing disk development. A lowmolecular weight nonpeptidic factor from the fat body (Oberlander and Tomblin, 1972) or a low-molecular weight, heat-stable, peptidic factor isolated from the fat body tissue (Benson et al., 1974), potentiated the action of 20E in inducing tracheation and elongation of isolated wing disks. In continuation of these results, Loeb and Hakim (1991) and Loeb (1994) reported that an extract of fat body (FBX) from the abdomen of newly pupated Manduca sexta promoted the growth and development of the genital tract (Loeb and Hakim, 1991) and mitosis in cultured midgut cells (Sadrud-din et al., 1994). In the present study, we have tested the effects of FBX on imaginal disk development in vitro and toxicity against intact caterpillars of the cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), an economically important pest of cotton, vegetables, and ornamentals. Pesticide-resistant populations of this insect cause severe problems in numerous countries. Previously, we showed that 20E induces evagination in cultured wing disks after 3 d (Smagghe et al., 2000). All developmental stages of a continuous colony of S. littoralis were kept at standard conditions of 23 -+ 2 ~ C, 60 + 5% r.h. and a 16:8-h LD (light:dark) cycle. Larvae were raised on an artificial diet, and adults provided with 15% honey water as food, as described in Smagghe et al. (2000). FBX was obtained as an aqueous extract from the fat body of pupal abdomens of M. sexta as described in Loeb and Hakim (1991), and kept frozen at 2 0 ~ C until application. To evaluate the action of FBX, we used cultured imaginal wing disks that were incubated with 25 txl/ml FBX alone or in combination with different concentrations of 20E (from 10 p~M to 1 nM; Sigma Chemical Co., Bornem, Belgium), and compared with cultures with 20E alone. Individual wing disks were dissected from 3d-old (0-1 h) last-instar larvae in which the natural peak of ecdysteroids has not yet occurred. Before dissection, larvae were wateranesthetized and surface-sterilized in 70% ethanol for 15 min. The imaginal disks were cultured in modified Graces insect tissue culture medium with heat-inactivated fetal calf serum (9%, Sigma), 30% bovine serum albumin solution (3%, Sigma), and gentamycine sulfate (50 ~g/ml medium, Sigma) at 25 -+ 1 ~ C and 97 -+ 2% r.h., to prevent evaporation (Smagghe et al., 2000). Ten disks were kept in 1-ml culture medium in a 35 • 10-mm plastic tissue culture plate (Falcon 3001; Becton Dickinson and Co., Belgium). After 72 h, wing disks were inspected for treatment-induced evagination. Completion of the first phase of evagination in which epidermal folds are developed inside the epidermal sac was regarded as the minimum requirement for a positive result (Chihara et al., 1972; Mandaron, 1976). Culturing imaginal wing disks with 20E at 10 txM induced evagination and new cuticle deposition in the disks (Fig. 1). Imaginal disks cultured with 25 ~l/ml FBX alone showed no signs of evagination. Figure 2 shows that in all cases of combination with different concentrations of 20E, FBX at 25 M/ml clearly promoted the effect of 20E on the development in imaginal wing disks. With 20E at 1 ~M alone, 58% of the cultured disks showed evagination, while this percentage was 100% with the addition of FBX. About 50% of evagination was already scored in the case of 20E at 0.01 ~M, in combination with 25 txl/ml FBX. This represents an enhancing activity of FBX that reaches nearly 100-fold. Our observation that FBX potentiated the stimulation of disk evagination as well as new cuticle deposition by 20E in S. littoralis imaginal disks clearly indicates an interaction between some component of fat body and ecdysone for the development of the wings disks. Similarly, Oberlander and Tomblin (1972) reported extensive cuticle deposition in disks of Plodia when cultured in medium containing 20E and fat body. In addition, they observed inhibition of cuticle deposition in disks with juvenile hormone in the culture medium containing 2 ~g/ml 20E. As such, the FBX may supply an additional factor promoting disk development, and/or it may have modified the 20E molecules to a more active form, and/or it may potentiate the complex of 20E, ecdysteroid receptor (EcR), and ecdysteroid response element (EcRE). Howevel, further studies are necessary to draw firm conclusions. Nothing was known, until the present study, of the physiological response of intact lepidopteran larvae to FBX factors from pupae. We assayed FBX from M. sexta pupae on intact, newly molted (012 h), last-instar (6th) larvae from a laboratory colony ofS. littoralis. Larvae were treated orally with 75 ~l of aqueous FBX by covering the artificial diet in 5 • 5-well tissue culture plates (Castor, Belgium) uniformly, in a manner similar to Smagghe et al. (2000). Concentrations of FBX were prepared in distilled water, and at least 25 larvae were tested per concentration. Mortality counts were made 7

Isolation of crustacean erythrophore concentrating hormone from nerve tissue of Homarus americanus.

Crustacean erythrophore concentrating hormone (CECH) was isolated from lobster (Homarus americanus) eyestalks by reverse phase high-performance liquid chromatography (rp-hplc). Putative lobster CECH fractions co-chromatographed with commercially available pure CECH on two independent rp-hplc systems, and were positive for activity by bioassay. CECH was detected in eyestalk, but not brain homogenates.