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.     

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.     

Established cell lines from the beetleDiabrotica undecimpunctata (Coleoptera: Chrysomelidae)

Summary Two new cell lines, designated IPLB-DU182A and IPLB-DU182E, were developed from embryos of the southern corn rootworm,Diabrotica undecimpunctata. Cells were grown in the lepidopteran cell culture media IPL-52B and IPL-76 in a 3∶1 ratiowith 9% fetal bovine serum. The IPL-52B was modified by deleting CaCl₂·2H₂O and NaHCO₃ out of the formulation. The osmotic pressure was adjusted to the optimal osmolarity of 400 mOsm/kg by the addition of2 g mannitol/100 ml medium. The cells were primarily epithelial-like, but some spindle-shaped cells were also present. The lines were 65% diploid and were characterized with respect to 10 isozymes. Cellscurrently grow with a 5-d doubling time and are subcultured by trypsinization at 1-wk intervals and at a 1∶2 to 1∶5 split ratio.     

In vitro rearing of Edovum puttleri,an egg parasitoid of the Colorado potato beetle,on artificial diets: Effects of insect cell line‐conditioned medium

Effects of conditioned media prepared from cell lines derived from 11 insect species (six families, three orders) on the in vitro growth and development of the egg parasitoid Edovum puttleri were investigated. The parasitoid exhibited significantly different responses to the various insect cell line‐conditioned media that were incorporated into the artificial diets. When cell lines were derived from embryos, higher percentages of 3rd instars and prepupae were observed than when cell lines were derived from fat body or ovaries. Medium conditioned with cell line IPLB‐CPB2 derived from the embryos of the Colorado potato beetle produced the best result. Preconditioning time was important. In general, 5 days of preconditioning appeared to be optimal. The growth‐ and development‐promoting effect may have resulted from growth factors or growth‐supporting factors produced/ released by the insect cell lines into the culture medium. Upon storage at 0–4°C for 7–14 days, the ability of cell line‐conditioned medium to promote development beyond the second instar was greatly reduced (approximately 10–55%). Our studies demonstrated that to support the in vitro growth and development of E. puttleri, insect hemolymph could be successfully replaced with insect cell line‐conditioned medium. These findings should facilitate the development of a cost‐effective mass‐rearing system for E. puttleri and/or other parasitoids. Arch. Insect Biochem. Physiol. 40:173–182, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Establishment and characterization of the Bombyx mandarina cell line

A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2 years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 °C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei’s genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

In vitro development ofCampoletis sonorensis (Hym.: Ichneumonidae), a larval endoparasitoid ofheliothis virescens (Lep.: Noctuidae) in an artificial medium with insect sources from egg to third larval instar

Effects of host hemolymph, fat body, and epidermal cell extracts on growth and development ofCampoletis sonorensis in vitro were studied. A simple cell culture medium preconditioned with intact fat body for several days improved growth of the parasitoid larvae while the addition of macerated fat body had a negative effect. Addition of co-cultured host epidermal cell mixture, without any preconditioning with the artificial medium, promoted growth and development ofC. sonorensis. The beneficial chemicals in the epidermal cell mixture were larger than 10 kd but were not cold or heat stable. Addition of unparasitized host larval hemolymph improved the hatching and development of the parasitoid larvae in the artificial medium but hemolymph from parasitized hosts did not. Host pupal hemolymph was also found to be beneficial to growth and development ofC. sonorensis. The effective components of pupal hemolymph were also neither cold nor heat stable and had a molecular weight range between 1 kd and 300 kd. While these host-derived growth factors increased molting and growth, they failed to stimulateC. sonorensis to develop to stages beyond the third instar.     

Initiation and characterization of five embryonic cell lines from the cotton boll weevil anthonomus grandis in a commercial serum- free medium

Summary Five continuous cell lines were initiated from embryonic tissue of the cotton boll weevilAnthonomus grandis Boheman in a commercially available, serum-free medium (Excell 401) and have undergone in excess of 60 passages. Isoenzyme analysis confirmed that the lines originated from boll weevil tissue. Four of the lines grew as single attached cells of either epithelioid or fibroblastoid morphology. The fifth line, BRL-AG-2, grew primarily as cell aggregates and was found to release ecdysteroids (primarily ecdysone) into the culture medium. Evidence was also obtained suggesting that line BRL-AG-2 synthesizes chitin. Three lines, BRL-AG-1, BRL-AG-3A, and BRL-AG-3C, could be induced to produce an antibacterial factor(s) which was released into the culture medium.     

Characterization of cell lines developed from the colorado potato beetle, Leptinotarsa decemlineata say (coleoptera: Chrysomelidae)

In order to isolate new pathogens (viruses, microsporidia, etc.) or to evaluate the efficiency of some pathogens (serovarieties and mutants of Bacillus thuringiensis, fungi, etc.) in the control of Colorado potato beetle, an economically important pest, we established four cell lines from tissues of this insect. One was initiated from embryonated egg fragments in the M3 medium supplemented with 20% fetal bovine serum (FBS) and then transferred after several passages to the Ex-Cell 400 medium with 20% FBS. Another was initiated from larval hemocytes in Ex-Cell 400 with 5% FBS. Finally, two other cell lines were initiated from adult hemocytes: one in the Ex-Cell 400 with 20% FBS and 1% of lipid mixture and the other in the Ex-Cell 400 with 5% FBS only. These cell lines have been characterized by their morphology with light and electron microscopy, their karyotypes, cell growth, and isozyme analysis. Each cell line differed in morphologic, karyologic, growth, and isozyme patterns. The cell line initiated from embryonated eggs was growing slower than the three initiated from hemocytes. The cytotoxicity of solubilized crystal delta-endotoxins from different B. thuringiensis formulations (M-One, Trident, MYX-1806, Teknar-HPD, and Thuricide) and of destruxins, mycotoxins from Metarhizium anisopliae, was tested on these cell lines. They are sensitive to the solubilized toxins of some strains of B. thuringiensis (serovar. San Diego and serovar. tenebrionis) and to destruxins, and they can be used for the bioassay and detection of toxins and for the study of the mechanism of their action on coleopteran cells.     

Growth and mitogenic effects of arylphorin in vivo and in vitro

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.     

Nonreciprocal gonadal dysgenesis in hybrids of the chironomid midgeChironomus thummi. IV. Behavior of a female-specific protein,probably a vitellogenin

Using denaturing SDS-polyacrylamide gel electrophoresis, a protein with a subunit MW of about 148,000 daltons could be detected in the fat body of females of the reciprocal hybrids of Chironomus thummi thummi and Chironomus thummi piger, which is not present in males. This protein is presumably a vitellogenin and can be found in both hybrids during the late fourth-instar larval stage until eclosion of the adults, i.e., in early vitellogenesis. After eclosion, the reciprocal hybrids behave differently concerning the 148-kd protein. In females of the piger female x thummi male cross, which are fertile and produce yolky eggs, the 148-kd protein disappears from the fat body immediately after eclosion. In females of the reciprocal cross (thummi female x piger male) which are affected by gonadal dysgenesis and in which the oocytes only rarely contain yolk, the 148-kd protein is still present in the fat body of the adult up to 50 hr after eclosion until the fat body degrades. It is concluded that the inability of the sterile thummi female x piger male females to produce yolky eggs is caused by an impaired uptake of the presumed 148-kd vitellogenin into oocytes and not by a defective vitellogenesis. The impaired vitellogenin deposition into oocytes is taken as another aberrant trait of gonadal dysgenesis of the thummi female x piger male hybrids.     

Synthesis of rotavirus-like particles in insect cells: comparative and quantitative analysis

When the three major structural proteins, VP2, VP6, and VP7, of rotavirus are co-expressed in insect cells infected with recombinant baculoviruses, they self-assemble into triple-layered virus-like particles (VLPs) that are similar in morphology to native rotavirus. In order to establish the most favorable conditions for the synthesis of rotavirus VLPs, we have compared the kinetics of 2/6/7-VLP synthesis in two different insect cell lines: High Five cells propagated in Excell 405 medium and Spodoptera frugiperda 9 cells in Excell 400 medium. The majority of VLPs produced in both cell lines were released into the culture medium, and these released VLPs were predominantly triple-layered and were found to be stable for the period of six or seven days examined. The optimal synthesis of VLPs depended upon the cell line and the culture medium used as well as the time of harvesting infected cell cultures. The highest yield of VLPs was obtained from High Five cultures in the late phase of infection when the yield was at least 5-fold higher than that from S. frugiperda 9 cultures on a per cell basis. Our results demonstrate the usefulness of High Five cells for the production of VLPs as potential rotavirus subunit vaccines.     

Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 10⁵/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.     

Description of a New Species of Microsporidia from Muscidifurax raptor (Hymenoptera: Pteromalidae), a Pupal Parasitoid of Muscoid Flies

ABSTRACT. A microsporidian parasite, Nosema muscidifuracis n. sp., has been found in Muscidifurax raptor , a parasitoid of muscoid flies. Stages of the parasite developed in direct contact with the host cell cytoplasm and were detected in midgut epithelium, Malpighian tubules, ovaries (including oocytes) and fat body of larvae and adults. Spores were also detected within eggs deposited on the host. Light and electron microscopy revealed a developmental cycle with diplokaryotic stages dividing by binary fission and disporous sporulation sequences producing diplokaryotic spores of three morphological classes, differing significantly only in length of the polar filament. Two of the classes were found in larvae, pupae and adults. One of these, with about five turns in the coiled polar filament, is presumed to be responsible for transmission from cell to cell within the host (autoinfection) and the other, with about 10 turns, responsible for transmission from host to host. A third class, with about 15 turns in the polar filament, was found in eggs of M. raptor . It is, presumably, either involved in initiation and spread of the infection at eclosion or is responsible for horizontal transmission to a new host individual when eggs are cannibalized.     

Pre-B cell generation potentiated by soluble factors from a bone marrow stromal cell line

Two bone marrow stromal cell lines isolated from the adherent layer of a Dexter-type long term bone marrow culture differ markedly in their hemopoietic support capacity. S17 supports myelopoiesis and the differentiation of early B cell precursors into B lymphocytes while S10 supports myeloid cell differentiation and not B lymphopoiesis. The identification of a stromal cell line with B cell support capacity prompted an investigation of whether the effects of S17 were mediated via soluble factors. Results presented herein indicate that medium conditioned by S17 but not S10 contains an activity that can induce the expression of the 220,000 m.w. 14.8 antigen and cytoplasmic mu H chain of Ig in B lymphocyte progenitors that have not yet expressed these markers. Bone marrow cells were depleted of 14.8+, cytoplasmic mu+ pre-B cells on antibody-coated petri dishes. After 24-h liquid culture newly generated pre-B cells were enumerated as cells that expressed cytoplasmic mu H chain of Ig but not Ig L chains by immunofluorescence. Expression of Ly5(220) was monitored by 14.8 antibody binding. This pre-B cell differentiation activity was abrogated by digestion with pronase, aminopeptidase, or carboxypeptidase. Isoelectric focusing data revealed the activity to have isoelectric point of 5.9 to 6.2. S17-conditioned medium was fractionated using HPLC and each fraction tested for pre-B cell-generating activity. Fractions collected from a Superose 12 gel filtration column were found to have two peaks of activity associated with molecules of apparent m.w. of approximately 60,000 and 10,000. Virtually identical peaks of activity were observed when medium conditioned by heterogeneous stromal cell cultures was fractionated. Separation of S10-conditioned medium revealed no cryptic activity. S17-conditioned medium was further characterized by anion exchange chromatography and the majority of the pre-B cell generating activity shown to be associated with the void volume that eluted from a MonoQ column. These fractions were rechromatographed on Superose and the activity again found to be associated with two fractions corresponding to apparent m.w. of 60,000 and 10,000. The S17 pre-B cell differentiation activity appears to result from the presence of a novel molecule because other well characterized mediators had no activity in this short-term liquid culture system. No pre-B cell-generating activity was observed when IL-1 or conditioned medium containing IL-2, IL-3, or IL-4 (B cell stimulatory factor 1) were added to cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

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).     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Expression of parathyroid hormone-related protein in a human T cell lymphotrophic virus type I-infected T cell line

We analyzed human T cell lymphotrophic virus type I (HTLV-I)-infected T cells for the presence of mRNA coding for parathyroid hormone-related protein (PTHrP) by Northern blotting using synthetic DNA probes. We report here that PTHrP mRNAs were detected in a HTLV-I-infected T cell line, MT-2, but not in uninfected T cell or B cell lines, and that PTH-like bioactivity was detected only in the conditioned medium of MT-2 cells. Our study suggests that the pathophysiology of hypercalcemia in patients with adult T cell leukemia/lymphoma may resemble that which occurs with solid tumors.     

舞毒蛾卵寄生蜂大蛾卵跳小蜂发育与温度的关系及利用替代寄主柞蚕卵繁育的子代品质评价

舞毒蛾Lymantria dispar (L.)是国际性检疫害虫。大蛾卵跳小蜂Ooencyrtus kuwanae (Howard)是舞毒蛾卵的重要寄生蜂, 对舞毒蛾有一定的控制作用。为了在规模化繁育大蛾卵跳小蜂时控制小蜂的发育进度, 设置不同的温度梯度研究了该蜂发育与温度的关系; 同时, 为了对替代寄主繁育出的天敌质量进行评价, 对利用其自然寄主舞毒蛾卵和替代寄主柞蚕Antheraea pernyi卵繁育出的子代成蜂的寿命、 胸宽、 雌雄性比进行了比较。结果显示： 大蛾卵跳小蜂的发育起点温度和有效积温分别为10.50±1.41℃和260.74±25.09日·度, 温度与发育速率的关系为T=10.50+260.74V。当采用30%的蜂蜜水饲喂成蜂时, 柞蚕卵繁育出的大蛾卵跳小蜂雌、 雄蜂的平均寿命分别为15.01和10.38 d, 采用原寄主舞毒蛾卵繁育出的雌、 雄蜂平均寿命分别为20.94和15.95 d, 两者差异显著; 采用柞蚕卵繁育出的雌蜂个体显著大于用舞毒蛾卵繁育出的雌蜂个体; 柞蚕卵和舞毒蛾卵繁育出的大蛾卵跳小蜂雌雄性比没有显著差异, 分别为2.42∶1和2.57∶1。结果表明, 在野外开展舞毒蛾的生物防治时, 释放利用柞蚕卵繁育出的大蛾卵跳小蜂具有可行性。     

Establishment of a germ-line competent C57BL/6 embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.