Grace's Medium Research Articles

Development of an animal-derived component-free medium for Spodoptera frugiperda (Sf9) cells using response surface methodology.

To develop an animal-derived component-free medium for Spodoptera frugiperda (Sf9) growth and green fluorescent protein (GFP) expression. OSF9-ADCFM contained optimum concentrations of CDLC, YE and ST at 0.5% (v/v), 11.0g/L, and 3.0g/L, respectively. A mean viable cell concentration of 1.71 ± 0.14 × 105 cells/mL was obtained from 5 passages (P1-P5). The use of both peptones after 10kDa ultrafiltration had a significant effect on Sf9 cell growth. Grace's insect medium with 10% FBS gave higher un-infected cell number than SF-900II and OSF9-ADCFM for 4.29 and 5.38 times, respectively. The average cell number of un-infected cells and GFP-fluorescent cells of SF-900II were higher than OSF9-ADCFM 1.25 and 7 times, respectively. In-house OSF9-ADCFM could support growth and GFP expression in Sf9 less than commercial SF-900II. However, it could lower the production cost at least 50% comparing to commercial SF-900II. The development of in- house OSF9-ADCFM would be continued to increase both cell numbers and protein expression in the next step.

Establishing and characterising a new cell line from Calliphora vicina (diptera: calliphoridae) fly embryonic tissues

Insect cell lines represent a promising and expanding field as they have several research applications including biotechnology, virology, immunity, toxicology, cell signalling mechanisms and evolution. They constitute a powerful tool having a direct impact on human and veterinary medicine and agriculture. Although more than 1000 cell lines have currently been established from various insect species, Calliphora vicina-derived fly cell lines are lacking. This study was aimed at establishing a new C. vicina embryonic tissue-derived cell line. Adult flies were collected and embryonated eggs were mechanically homogenised and seeded in four types of culture media (L15, Grace's insect medium, Grace's/L15 and DMEM). Cell growth and morphological characteristics were recorded and cytogenetic and molecular patterns were determined. The CV-062020-PPB cell line was established and was shown to have optimal growth in Grace's/L15 medium. CV-062020-PPB cell monolayers that had been sub-cultured over 16 times consisted of firmly adhering cells having different morphologies; a fibroblast-like shape dominated and the karyotype had a 12-chromosome diploid number. RAPD-PCR analysis of the CV-062020-PPB cell line revealed a high similarity index and strong intraspecific relationship with C. vicina adult flies and a weaker relationship with the Lutzomyia longipalpis-derived cell line (Lulo). The CV-062020-PPB cell line constitutes the first cell line obtained from C. vicina embryonic tissues and represents an important basic and applied research tool.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii (Xb-Sj), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath (xbpS1) or tail fibre (xbpH1) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii (Xsz-Sr), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj. Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr. To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj, suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta, while in competition with the xenorhabdicin-deficient strain (Xb-Sj:S1), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace’s insect cell culture medium (Grace’s), liver infusion tryptose (LIT), and Schneider’s insect medium (Schneider’s), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider’s with or without FCS when compared to that in Grace’s. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace’s promoted higher percentage of infected macrophages and parasite load compared with Schneider’s media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.Electronic supplementary materialThe online version of this article (10.1007/s00436-020-06893-z) contains supplementary material, which is available to authorized users.

Developing an Ephestia kuehniella Hemocyte Cell Line to Assess the Bio-Insecticidal Potential of Microencapsulated Helicoverpa armigera Nucleopolyhedrovirus Against Cotton Bollworm (Lepidoptera: Noctuidae) Larva.

Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) (genus: Alphabaculovirus, incertae sedis: Baculoviridae) has been used to control Helicoverpa armigera (Hübner). A reproducible and susceptible cell line was prepared from the hemocytes of Ephestia kuehniella in Grace and Ex-Cell 420 media. The population doubling time of these cloned cell cultures during the logarithmic phase were about 2.3 and 3.7 d for Ex-Cell 420 and Grace's media, respectively. When 60% confluence occurred, cells were infected by viral inoculums. All biochemical compounds were significantly changed relevant to cellular metabolism due to HearNPV infection. In order to improve its stability, two polymer formulations were used, i.e., formulation A (sodium alginate, gelatin, starch, and molasses) and formulation B (cottonseed kernel extract, Bran, glycerol, boric acid, egg white, and sugar). Formulant A provided high photostability by exhibiting 83.2 ± 3% efficacy and 88.66 ± 2.1% original activities remaining after 72 h UV exposure. Percentage original activity remaining of unformulated HearNPV and formulated mixture of B was 38.66 ± 2.6% and 9.33 ± 1.3%, respectively, after 72 h UV-irradiation. The virulence of the HearNPV proliferated from the Ex-Cell medium was similar to the virulence of wild-type HearNPV with LC50 of 7.7×105 OBs/ml. Formulant A, revealed only 20.0 ± 1% reduction in efficacy while the unformulated virus and formulant B faced a reduction of 90.0 ± 3% and 64.0 ± 2% after 72 h of UVA irradiation. Formulant A thus showed a high potential to protect HearNPVs microparticles against UV-inactivation suggesting a new platform for more efficient biological-management of cotton bollworm (specific name Helicoverpa armigera, genus: Helicoverpa, Lepidoptera: Noctuidae) in vivo.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25μg/ml gentamicin sulfate, pH5.5 at 35°C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25μg/ml gentamicin sulfate, pH6.8 at 26°C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

Establishment of an embryonic cell line from the American cockroach Periplaneta americana (Blattaria: Blattidae) and a preliminary study of telomerase activity changes during the culturing process.

Despite the pest status and medicinal value of the American cockroach Periplaneta americana, few attempts have been made to establish cell lines from this insect owing to the difficulty of culturing Blattarian cells. Here, we describe the establishment of the RIRI-PA1 line from P. americana embryo tissue following primary culture in modified Grace's medium containing 20% fetal bovine serum. RIRI-PA1 was found to primarily consist of attached spindle-shaped and giant cells, which attach themselves to their container. The population-doubling time of 40th-passage cells was approximately 84.8h. The average chromosome number at the 30th passage was 42, with 40% of cells demonstrating substantial variations, with the highest number of variations of 78 and lowest of 24. The identity of RIRI-PA1 was confirmed by comparing the COI gene of these cells to that of P. americana embryo tissue. Telomerase activity decreased in primary cells after 7d of culture and 5th-passage cells in comparison to embryo tissues; however, compared to the other cultured cells tested, the telomerase activity significantly increased at the 20th passage. We propose that the stagnation periods and cessation of proliferation observed relate to cellular telomerase activity, but the relationship between insect cell proliferation and telomerase as well as the regulatory mechanism involved remains to be elucidated.

Cryopreservation of Luciola praeusta Kiesenwetter (Coleoptera: Lampyridae) embryos by vitrification

There is an urgent need to preserve the ever-decreasing number of different species of fireflies all over the world. We sought to develop a vitrification procedure to cryopreserve the firefly embryos. The late stages of Luciola praeusta Kiesenwetter embryos were collected. Several impermeable and permeable protectants with various concentrations in different mediums (TNM-FH insect medium, Grace's medium, Dulbecco's Modification of Eagle's Medium (DMEM) and Dulbecco's Phosphate-Buffered Saline (DPBS)) were used. Embryos culturing in TNM-FH medium yielded the highest survival rate of 75.3 ± 3.6%. One-step, two-step and three-step methods were used in equilibrium procedure respectively. The highest survival rate (73.7% ±3.2%) occurred in embryos treated by three-step method ((1.5 M ethylene glycol (EG) + 2 M EG plus 8% polyvinylpyrrolidone (PVP) + 3 M EG, 8% PVP and 15% trehalose). Additionally, embryos exposed to 0.5 M trehalose presented a significantly higher survival rate (71.8 ± 2.7%) than embryos preserved in 0.5 M sucrose.

Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor.

Background Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.Methodology/Principal FindingsCultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R)—an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite—is important for the transformation process.Conclusion/SignificanceOur findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.

Establishment and characterization of a novel cell line from midgut tissue of Helicoverpa armigera (Lepidoptera: Noctuidae).

The midgut of lepidopteran larvae serves as a target for many pathogens such as Bacillus thuringiensis (Bt). Cell lines originating from midgut tissues will be very helpful tools in many research fields. However, to date, no Bt-susceptible midgut-derived cell lines are available. Here, we reported that a novel cell line, designated as HNU-Ha-MG1, was established from midgut tissue of the fourth instar larvae of Helicoverpa armigera. This cell line grew well in Grace's insect cell culture medium supplemented with 10-15% fetal bovine serum. The shape of the most cells was round or polygonal, and some tended to aggregate to form multiple cell masses. The size of the cells was 13.8 ± 1.8μm in diameter, and the maximum density reached (2.40 ± 0.15) × 10(6) cells/ml. The population doubling time during logarithmic growth phase was 58.6 ± 7.0h at 28°C. The number of chromosomes was about 90-130, which exhibited typical chromosome characteristics of lepidopteran cell lines. The patterns of random amplified polymorphic DNA of the cell line were different from those of Sl-HP and Hi5 cell lines which were frequently used in our laboratory. 20-Hydroxyecdysterone induced apoptosis in a very small part of cells at 2μg/ml but did not affect expression of autophagy-related protein 8 (Atg8) and its lipidation at 36h post-treatment. The cell line was permissive to Autographa californica nuclear polyhedrosis virus (AcMNPV) and H. armigera nuclear polyhedrosis virus (HaSNPV). This cell line was found to be susceptible to activated Cry1C at the final concentration of 0.5-1.0μg/ml but not to the activated Cry1Ac.

Role of secondary metabolites in establishment of the mutualistic partnership between Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (ΔxcnKL strain), xenocoumacin and the newly discovered antibiotic F (ΔxcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace's insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ΔxcnKL strain or ΔxcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ΔxcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction.

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph.

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 1% (v/v) hemolymph extract, or 3% (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID50/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10% (v/v) FBS and 1% (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10% (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.

Establishing midgut cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against ten different insecticides

Midgut epithelial cell culture was successfully developed from red palm weevil (Rhynchophorus ferrugineus) during this study and named as RPW-1. Optimum conditions for four different commercial media were also worked out to successfully maintain the culture. Grace's medium was found to be the most effective for RPW-1 culturing which resulted in the highest cell density of 7.5 × 10(6) cells/ml after 72 h of cell seeding with 96% cell viability. It was followed by Schneider's medium and TNM-FH medium where cell densities reached up to 7.4 × 10(6) and 5.9 × 10(6) cells/ml, respectively, after 72 h having 91 and 89% cell viability. Comparatively, Media-199 was least effective for RPW-1 cell culturing. As a whole, temperature at 27°C and pH 6.3 were the best for RPW-1 culturing where the highest cell density and maximum cell viability were noted. Individually, Grace's medium, Schneider's medium, TNM-FH medium, and Media-199 produced better results at 27°C, 27°C, 24°C, and 21°C and pH 6.3, 6.4, 5.3, and 7.1, respectively. The toxicity assay and MTT cell proliferation assay revealed that, out of the ten insecticides used in this study, emamectin benzoate was the most toxic insecticide to RPW-1 cells resulting in 92% cell mortality and 74% cell growth inhibition. Dieldrin was the least potent, causing only 19% cell mortality and 18% cell growth inhibition.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

Establishment and characterization of a cell line developed from the neonate larvae of Papilio demoleus Linnaeus (Lepidoptera: Papilionidae)

A new cell line named RIRI-PaDe, developed from the neonate larvae of Papilio demoleus Linnaeus, was established in modified Grace's medium supplemented with 20% fetal bovine serum. The cell line was incubated at 28°C and consisted of attached round and short spindle-like cells. The population doubling time was 55 h. The chromosome numbers varied widely from 24 to 136 with a mode of 59 at the 71st passage. Comparison of mitochondrial cytochrome c oxidase subunit I gene of the cell line and neonate larvae confirmed that the cell line was of P. demoleus origin. This cell line was susceptible to the Autographa californica multicapsid nucleopolyhedrovirus and Apocheima cinerarius nucleopolyhedrovirus.

The potential of trehalose to replace insect hemolymph in artificial media for Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Insect additives have been shown to improve the value of artificial media for Trichogramma species, but at the same time maintain dependence on parallel cultures of host insects. In the present study, Trichogramma dendrolimi Matsumura was reared in vitro from egg to adult on artificial media with different contents of pupal hemolymph of Chinese oak silkmoth Antheraea pernyi (Guérin-Méneville) and with supplements of distilled water, or of trehalose dissolved in water or in Grace's insect medium. The results indicated insect hemolymph was the key component of artificial medium. Developmental parameters, including rates of parasitism, pupation, adult emergence and normal adults, and numbers of produced adults, were increased on media supplemented with trehalose even when the proportion of pupal hemolymph was reduced. Two artificial media, the first containing 30% hemolymph and 10% trehalose in water with 98.9% parasitism rate, 77.7.0% pupation rate, 77.2% emergence rate, 80.0% normal adult rate and 333 produced adults, and the second containing 25% hemolymph and 15% trehalose in Grace's insect medium with 97.8% parasitism rate, 91.0% pupation rate, 85.2% emergence rate, 76.1% normal adult rate and 757 produced adults, were believed to hold potential to mass produce T. dendrolimi. The use of trehalose to partially replace pupal hemolymph in artificial medium of this and other Trichogramma species may contribute to a significant reduction in their production cost and may as such help to evade problems related to short supplies of lepidopteran eggs, which currently constitute the main factitious host for the mass rearing of the parasitoids.

IgG Autoantibody Response against Keratinocyte Cadherins in Endemic Pemphigus Foliaceus (Fogo Selvagem)

It is well established that autoantibodies against desmoglein 3 and desmoglein 1 are relevant in the pathogenesis of pemphigus vulgaris and pemphigus foliaceus, including its endemic form, Fogo Selvagem (FS). Isolated reports have shown that in certain patients with these diseases, autoantibodies against other desmosomal cadherins and E-cadherin may also be present. The goal of this investigation was to determine if FS patients and normal individuals living in endemic areas possess autoantibodies against other desmosomal cadherins and E-cadherin. Testing a large number of FS and endemic control sera by ELISA we find a consistent and specific autoantibody response against desmoglein 1 and other keratinocyte cadherins in these individuals, which is quite different from US controls. Overall, the highest correlations among the autoantibody responses tested are in the endemic controls, followed by FS patients, and lowest in the US controls. These findings suggest that multiple, perhaps cross reactive, keratinocyte cadherins are recognized by FS patients and endemic controls.

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8-7.2. Osmolality was adjusted to 720±10mOsmkg(-1) and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace's insect medium, and found better performance in SCCM especially for lymphoid cells with 107% increase in activity and 85±9days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation "cocktail".