Embryonic development of an endoparasitoid,Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media

Summary Embryos of the parasitoidMicropolitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical hostLymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace’s, and ExCell 400. The developmental response ofM. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace’s media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ hand stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace’s media had a significant effect on eggs attaining germ band stage compared with the Grace’s control medium. However, Grace’s medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace’s control medium. Although the BCIRL-HZ-AMI cell line, which is derived from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for promoting development ofM. croceipes in vitro.     

Stimulation of embryonic development in Microplitis croceipes (Braconidae) in cell culture media preconditioned with a fat body cell line derived from a nonpermissive host, gypsy moth, Lymantria dispar

A cell culture medium, IPL-52B, was preconditioned with host fat body and two insect cell lines to determine if they would support embryonic development of Microplitis croceipes in vitro. The medium was preconditioned with the cell line IPL-LdFB, derived from fat body of the gypsy moth, Lymantria dispar, cell line IAL-TND1, derived from imaginal discs of the cabbage looper, Trichoplusia ni, and whole fat body tissue from host Helicoverpa zea. A second cell culture medium, Excell 400, was preconditioned with only the cell line, IPL-LdFB. Pregerm band eggs were dissected from third instar host larvae and incubated in the conditioned medium for 20 h. Newly laid parasitoid eggs did not develop in unconditioned IPL-52B, but did develop to germ band stage in unconditioned Excell 400. The IPL-52B medium conditioned with both cell lines induced germ band formation, but only the L. dispar cell line (IPL-LdFB) promoted significant development to eclosion comparable to host far body tissue. Excell 400 medium preconditioned with the cell line, IPL-LdFB also supported development to eclosion.     

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l⁻¹ 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.     

Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However,in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.     

Gypsy moth cell lines divergent in viral susceptibility

Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus. This research was funded in part through a reimbursable agreement with the U.S. Forest Service.     

Development of IVM-IVF produced 8-cell bovine embryos in simple,serum-free media after conditioning or Co-culture with buffalo rat liver cells

Development of 8-cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum-free media (CZB and SOM) with buffalo rat liver cells co-culture (BRLC) or after conditioning compared to a commonly used, serum-supplemented complex medium TCM-199. In a 3 x 4 factorial design, 578 eight-cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co-culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co-culture and 48 hr conditioning within media type resulted in better development when compared to the 24-hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co-culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co-culture and 48-hr conditioned groups. Hatching occurred only with co-culture and 48-hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM-199 with serum. © 1994 Wiley-Liss, Inc.     

Rearing of ectoparasitoid Diapetimorpha introita on an artificial diet: supplementation with insect cell line-derived factors

We investigated the use of two insect cell lines to improve an artificial diet (DI) for the pupal ectoparasitoid Diapetimorpha introita. DI was supplemented with Grace's culture medium conditioned with IPL-LdFB, a cell line derived from fat body of the gypsy moth, Lymantria dispar (FBCell diet), and with Grace's medium conditioned with Sf9, a cell line derived from ovaries of the fall armyworm, Spodoptera frugiperda (Sf9Cell diet). The diets were also chemically analyzed for nutrients and any deficiencies were filled by the addition of nutrients. One-half ml aliquots of each diet were encapsulated in paraffin domes and newly hatched larvae of D. introita were placed on each diet (one larva/dome) and allowed to develop to the adult stage. Providing fresh diet on day four when the larvae were in the third instar did not improve parasitoid production. Compared with DI, only Sf9Cell had a positive effect on the parasitoid's growth, increasing the size of male parasitoids. The parasitoids, however, took longer to develop to the adult stage than those reared on the natural host. Neither cell line significantly enhanced the average weight of female parasitoids, shortened developmental time, nor increased % cocoons produced and % adult emergence. Providing additional nutrients (amino acids, vitamins, cations and anions, fatty acids and milk/egg protein) to both diets (based on chemical analyses of the cell line-supplemented diets) enhanced the average weight of the females on Sf9Cell and males and females on FBCell. The nutritional additions, however, did not improve the developmental time, pupation and adult emergence.     

Selection and breeding for salinity tolerancein vitro

Summary Selection for tolerance to NaCl inCitrus sinensis andC. aurantium has been carried out in agar and suspension cultures. Callus was subjected to culture media containing up to 0.17M NaCl for ten passages. Selected cell lines were grown for three passages on media without salt before further tests on saline media. Four stable tolerant cell lines, differing in degree of tolerance, have been selected fromC. sinensis. Four lines of similar tolerance have been selected fromC. aurantium. The stability of most lines was very satisfactory. MostC. sinensis lines grew well in media containing up to 0.2M NaCl, andC. aurantium lines in media of up to 0.15M NaCl.Embryos were regenerated in most selected cell lines fromC. sinensis and, more sporadically, fromC. aurantium. Addition of 0.5–0.6% NaCl to the media often enhanced embryogenesis. Embryos from a selected line ofC. sinensis showed higher tolerance to NaCl in the medium than comparable embryos from an unselected line.Single embryos derived from both selected and unselected cell lines ofC. sinensis were successfully cloned. A limited comparison of plantlets from one tolerant line (R14) with plantlets from unselected control lines showed better adaptation of the former to salt (0.085 to 0.12M NaCl in the medium), and a lesser degree of leaf burn symptoms.Contribution No. 1045-E, 1984 series.     

Cell culture techniques for studying insect cuticle

Evidence that biosynthetic pathways critical to the formation of insect cuticle are retained in continuous insect cell lines opens new possibilities for research on the cuticle system. Recent findings indicate that chitin, molting hormone, and catecholamines are all produced by a vesicle cell line derived from embryos of the cockroach Blattella germanica. The chitin that is formed by this cell line is particulate and does not show the characteristic featherlike crystalline structure found in mature cuticle. The molting hormone is produced as ecdysone and is released into the culture medium. The addition of 20-hydroxyecdysone to the cultures increases the production of chitin fourfold. These responses are similar to those found in insect organ cultures.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

REPRODUCTIVE ISOLATION BY SEX PHEROMONES IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

The Closterium peracerosum–strigosum–littorale (C. psl.) complex consists of unicellular algae and is known to be composed of several reproductively isolated mating groups of heterothallic strains. Group I‐E is completely isolated from mating groups II‐A and II‐B, groups II‐A and II‐B are partially isolated from each other, and only mating‐type plus (mt⁺) cells of group II‐A and mating‐type minus (mt^?) cells of group II‐B form zygotes. Based on the alignment of 1506 group I introns, significant phylogenetic relationships were observed among mating groups II‐A and II‐B, while mating group I‐E was distant from groups II‐A and II‐B. Sexual cell division in both mating‐type cells of group II‐A was stimulated in conditioned media in which cells of group II‐B had been cultured. When mt^? cells of group II‐B were stimulated in conditioned medium derived from group II‐A, mt⁺ cells of group II‐B did not respond to the conditioned medium. Conditioned media derived from group I‐E did not exhibit sexual cell division (SCD)–inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous protoplast‐release‐inducing protein (PR‐IP) Inducer genes, we detected a significant similarity among groups II‐A and II‐B, and mating group I‐E had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.     

Characterization of cell lines developed from the glassy-winged sharpshooter, Homalodisca coagulata (hemiptera: Cicadellidae)

Summary Four continuous cell lines were established from the embryos of the glassy-winged sharpshooter, Homalodisca coagulata (Say), an economically important insect vector of bacterial pathogens of grape, almond citrus, oleander, and other agricultural and ornamental plantings. The cell lines were designated GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH. The GWSS-Z10, GWSS-Z15, and GWSS-G3 lines were cultured in Ex-Cell 401 medium supplemented with 10% fetal bovine serum (FBS), whereas the GWSS-LH line was cultured in LH medium supplemented with 20% FBS. The cell lines were characterized in terms of their morphology, growth, protein composition, and polymerase chain reactionamplification patterns of their chromosomal deoxyribonucleic acid. The population doubling times of GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH were 46.2, 90.9, 100.3 and 60.2 h, respectively. These lines should be useful for the study of insect-pathogenic viruses of leafhoppers, aphids, treehoppers, and other related insects as well as plant-pathogenic viruses that are transmitted by these insects.     

Establishment of two cell lines from embryonic tissue of the tobacco hornworm, Manduca sexta (L).

Two cell lines were derived from primary cultures of embryonic tissue of the tobacco hornworm, Manduca sexta (L.). The cell lines were maintained on hemolymph-free synthetic insect medium. Cytogenetic and immunoligical identification of the lines were carried out. Techniques for obtaining the line and medium, subculturieng and freezing procedures for long-term storage, and the morphological and growth characteristics are described.     

Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.     

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos.

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.     

Abnormal ovarian morphogenesis in Drosophila melanogaster after injection of embryos with conditioned media from the Daudi cell line

Summary Drosophila melanogaster embryos were injected before the blastoderm stage with conditioned media from several male Burkitt's lymphoma human cell lines and the Daudi cell line. Such injections do not have any effect on the male genital apparatus or on the female tract. The Daudi conditioned medium modifies the ovarian morphogenesis of the flies and the rudimentary ovaries obtained look like nymphal gonads. Moreover, they have a drastically reduced number of germ cells. The ovaries that looked functional contain numerous necrotic germ cells and the mean number of ovarioles per fly is significantly smaller than that of the controls. The abnormalities observed resemble the results of experimental and genetic lack of germ cells. They disappear at very high dilution (1×10^–6).     

Enhanced somatic embryo production by conditioned media in cell suspension cultures ofDaucus carota

Summary The addition of conditioned media extracted from 8 day old embryo culture accelerated growth and production of torpedo embryos and plantlets in cell suspension cultures ofDaucus carota. The production of late-stage embryos was increased a maximum threefold (up to 1500 embryos/ml) compared with that of control culture, when spent medium was added during inoculation.     

Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co‐culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non‐macromastic epithelial cells when co‐cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia‐derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co‐culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co‐cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy.     

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor‐derived gametes

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES‐like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma‐irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor‐derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells. Mol. Reprod. Dev. 53:394–397, 1999. © 1999 Wiley‐Liss, Inc.