Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k_La of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5×10⁶ cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the μX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day⁻¹). The glucose consumption and lactate production were higher in the infected cultures (μGlucose and μLactate of, respectively, 0.0248 and 0.0089×10⁻⁸ g/cell×day in infected cultures and 0.0151 and 0.0046×10⁻⁸ g/cell×day in non infected ones). The glutamine consumption did not differ in both cultures (μGlutamine of 0.0034 and 0.0037×10⁻⁸ g/cell×day in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10⁶ to 2×10⁶ cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL₅₀. Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL₅₀. The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy (TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control.     

Accumulation of few-polyhedra mutants upon serial passage of Anticarsia gemmatalis multiple nucleopolyhedrovirus in cell culture

Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. To date, AgMNPV has been produced by larval infection and, due to in vivo production limitations and the continuing high demand for the biopesticide, attempts should be made to develop in vitro production of this virus. In order to investigate the effects caused by serial passage of AgMNPV in cell culture, we carried out a total of ten passages and analyzed the morphological and the genomic changes of the virus. After six passages, the many-polyhedra (MP) phenotype started to switch to the few-polyhedra (FP) phenotype which rapidly accumulated in the virus population. Ultrastructural analysis showed typical signs of FP mutant formation such as decrease in the number of polyhedra per cell, polyhedra aberrant morphology and low numbers of virions occluded in the protein matrix. Also enhanced BV production was observed from the fifth passage indicating that FP mutants were becoming predominant in comparison to the wild type virus. Restriction endonuclease analysis of the viral DNA revealed that lower and higher passages had similar profiles indicating that there were no large insertions or deletions or rearrangements in their genomes and indicating the generation of FP mutants instead of defective interfering viruses.     

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

Temperature and population density: interactional effects of environmental factors on phenotypic plasticity,immune defenses,and disease resistance in an insect pest

Temperature and crowding are key environmental factors mediating the transmission and epizooty of infectious disease in ectotherm animals. The host physiology may be altered in a temperature‐dependent manner and thus affects the pathogen development and course of diseases within an individual and host population, or the transmission rates (or infectivity) of pathogens shift linearly with the host population density. To our understanding, the knowledge of interactive and synergistic effects of temperature and population density on the host–pathogen system is limited. Here, we tested the interactional effects of these environmental factors on phenotypic plasticity, immune defenses, and disease resistance in the velvetbean caterpillar Anticarsia gemmatalis. Upon egg hatching, caterpillars were reared in thermostat‐controlled chambers in a 2 × 4 factorial design: density (1 or 8 caterpillars/pot) and temperature (20, 24, 28, or 32°C). Of the immune defenses assessed, encapsulation response was directly affected by none of the environmental factors; capsule melanization increased with temperature in both lone‐ and group‐reared caterpillars, although the lone‐reared ones presented the most evident response, and hemocyte numbers decreased with temperature regardless of the population density. Temperature, but not population density, affected considerably the time from inoculation to death of velvetbean caterpillar. Thus, velvetbean caterpillars succumbed to Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) more quickly at higher temperatures than at lower temperatures. As hypothesized, temperature likely affected caterpillars' movement rates, and thus the contact between conspecifics, which in turn affected the phenotypic expression of group‐reared caterpillars. Our results suggest that environmental factors, mainly temperature, strongly affect both the course of disease in velvetbean caterpillar population and its defenses against pathogens. As a soybean pest, velvetbean caterpillar may increase its damage on soybean fields under a scenario of global warming as caterpillars may reach the developmental resistance faster, and thus decrease their susceptibility to biological control by AgMNPV.     

Density‐dependent prophylaxis in primary anti‐parasite barriers in the velvetbean caterpillar

1. Organisms rely on a set of primary barriers to prevent invasion by parasites, and secondary defences to fight parasites that breach the primary barriers. However, maintaining these defences to be active and effective is costly. Thus, hosts increase investment in anti‐parasite defences under situations of high risk of infection and reduce defences when the risk is reduced (the ‘Density‐Dependent Prophylaxis’ hypothesis). 2. In the present study, it was tested whether the midgut primary defences of the velvetbean caterpillar Anticarsia gemmatalis Hübner present density‐dependent plasticity, and also whether these defences could be induced by a viral pathogenic challenge. The aim was to examine whether morphometry and the structure of the midgut and peritrophic matrix (PM) change in accordance with colour transition in caterpillars, and whether such changes may provide the caterpillars a more protective barrier against invasion by Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). 3. It was found that PM and the midgut epithelium of the velvetbean caterpillar change plastically according to phenotype, itself a response to changes in population density. Caterpillars reared at high densities (black phenotype) had a considerably thicker midgut epithelia and peritrophic matrices than those reared individually (green phenotype), and there was also more chitin in the PM of the former. 4. This was interpreted as the first demonstration of increased investment in primary, barrier, defences against parasites, in response to increased conspecific density and an increased risk of infection. The possibility that this arises as a positive result of pleiotropy is discussed further, wherein the biochemical pathways responsible for the up‐regulation of the immune system are also involved in midgut properties.     

Predatory behaviour of Podisus nigrispinus (Heteroptera: Pentatomidae) on different densities of Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae

The functional response of the predatory bug Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) feeding on its prey, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was studied in a greenhouse compartment. Each cage enclosed three soybean plants plus two, four, six, eight, 10, 12 or 14 prey larvae. One adult predator was released and kept inside the cages for 24h. The predation rate of adult male P. nigrispinus was highest at densities of eight or more A. gemmatalis larvae with a handling time of 5.76h and an attack rate of 0.68h^?1. Adult females had higher predation rates on plants with 10 or more A. gemmatalis larvae, with a handling time of 3.84h and an attack rate of 0.65h^?1. The maximum number of larvae consumed by males and females of this predator were 4.1 and 6.0 per day, respectively, in groups of three plants. The results suggest that P. nigrispinus may be used in biological control programmes against A. gemmatalis in soybean fields.     

In vitro and in vivo host range of Anticarsia Gemmatalis multiple nuclear polyhedrosis virus

Summary A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID₅₀ values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7×10⁶ TCID₅₀/ml) followed by BCIRL-HA-AM1 cells (8.3×10⁵ TCID₅₀/ml) and BCIRL-AG-AM1 cells (3.2×10⁵ TCID₅₀/ml). In addition, a low ECV titer of 1.37×10³ TCID₅₀/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID₅₀ results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC₅₀ values were 96.9, 564.6, 733.3, and 1.1×10⁴ occlusion bodies/cm² of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively. This article presents the results of research only. Mention of proprietary products in this article does not indicate endorsement or a recommendation for use by USDA-ARS. All programs and services of the USDA are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, marital status or handicap.     

Feasibility of using an alternative larval host and host plants to establish Cotesia flavipes (Hymenoptera: Braconidae) in the temperate Louisiana sugarcane ecosystem

Attempts to establish Cotesia flavipes Cameron (Hymenoptera: Braconidae) in Louisiana sugarcane fields to control the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae) have been unsuccessful. Experiments were conducted to investigate the feasibility of using an alternative larval host and host plants to overcome barriers preventing establishment. In addition, we evaluated C. flavipes' ability to search for D. saccharalis in sugarcane without above-ground internodes. Diatraea evanescens Dyar (Lepidoptera: Crambidae) was investigated as an alternative host for C. flavipes. Cotesia flavipes was reared for five generations on D. evanescens without any indication of diminishing fitness as measured by days to parasite pupation and average cocoon mass weight. However, there was a significant reduction in percent parasitism, cocoon mass weight, and percent emergence when C. flavipes parasitized D. evanescens as compared with D. saccharalis, resulting in a 75% reduction in the gross reproductive rate (R(0)). Greenhouse studies indicated little difference in parasitism of D. saccharalis on the weed hosts johnsongrass, Sorghum halepense (L.), and vaseygrass, Paspalum urvillei Steud. However, when planted as refuge plots, we found it difficult to establish infestations of D. saccharalis in either of these hosts, or in two energy sugarcanes. After 3 yr of infesting host plants and releasing parasitoids only one parasitized D. saccharalis larvae was recovered within the johnsongrass refuge. Diatraea evanescens readily established in vaseygrass; however, these larvae appear inaccessible to C. flavipes. In contrast, parasitism of D. saccharalis by C. flavipes infesting young sugarcane was 30%.     

Ultrastructural studies on the fungus,Nomuraea rileyi,infecting the velvetbean caterpillar,Anticarsia gemmatalis

Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.     

Susceptibility/resistance of Anticarsia gemmatalis larvae to its nucleopolyhedrovirus (AgMNPV): Structural study of the peritrophic membrane

This investigation compares the peritrophic membrane (PM) morphology along the midgut of susceptible (SL) and resistant (RL) Anticarsia gemmatalis larvae to the AgMNPV. The PM increased the thickness from the anterior to the posterior midgut region in both insects strain; however, the intensity of FITC-WGA reaction of the PM in the RL were greater than in SL. The PM in RL was ultrastructurally constituted by several layers of fibrous/vesicular materials in comparison with the few ones in SL. Our results showed that the structure of PM in the RL could be one of the resistance barriers to AgMNPV.     

Comparison of Hemolymph and Holotissues of Different Species of Insects as Diet Components forin VitroRearing ofTrichogramma galloiZucchi andT. pretiosumRiley

The aim of this work was to compare pupal holotissues (Diatraea saccharalis, Anticarsia gemmatalis,andBombyx mori) and larval hemolymph of several moth species (Helicoverpa zeaandBombyx mori) commonly produced in Brazilian laboratories as components of artificial diets forin vitrorearing ofTrichogramma galloiandTrichogramma pretiosum.Diets based on larval hemolymph ofH. zeaproduced the best development of both natural enemies, although a diet with pupal holotissues ofB. moriallowed full development ofT. pretiosum.Amino acid analysis showed qualitative differences among the insect derivates and eggs of the natural and alternative hosts, but with a quantitative similarity. Electrophoretic analysis resulting in a diverse protein-stained bands evidenced the qualitative differences among these components.     

Simultaneous monitoring of flight and oviposition of individual velvetbean caterpillar moths

ABSTRACT. A 'pivot' flight actograph was combined with a rolling oviposition surface to characterize the flight and oviposition behaviour of velvetbean caterpillar moths, Anticarsia gemmatalis Hubner. Tethered, caged control females laid significantly more eggs that those flown on the actograph. Mated females laid more eggs than unmated ones. However, mating did not affect longevity nor fight frequency and duration. Laboratory-reared and wild-type females also did not differ signficantly in longevity and flight. Both laboratory and wild mated females laid most of their eggs during the first 7 days whereas unmated ones delayed their oviposition. None of the female types produced a definite hourly, daily or lifetime pattern in flight frequency or duration. Of the 7672 recorded flights, about 3% were greater than 0.5 h. 'Long' flights (>0.5h) were made by some females before oviposition, as in colonization migration, but most interspersed flight with oviposition, as in extended search migration.     

Fluorescent Brighteners Improve Anticarsia gemmatalis (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (AgMNPV) Activity on AgMNPV-Susceptible and Resistant Strains of the Insect

Fluorescent (optical) brighteners are known for their characteristics of protecting baculoviruses against deactivation by ultraviolet (UV) light and enhancing the activity of these agents as microbial insecticides on hosts and semipermissive hosts. These substances were evaluated in combination with the velvetbean caterpillar, Anticarsia gemmatalis Hübner, multiple-embedded nucleopolyhedrovirus (AgMNPV). The first trial involved 4 fluorescent brighteners (Blankophor BBH, Blankophor HRS, Blankophor RKH, and Tinopal LPW) obtained from the United States. The second trial was conducted with 11 fluorescent brighteners (Tinopal UNPA-GX, Tinopal DMS, Tinopal CBS, Leukophor DUB, Leukophor BSBB, Hostalux KS-N, Hostalux ETBN, BRY 10 D2 100, BRY 10 D2 150, Uvitex BHT, and Uvitex NFW) available in Brazil in combination with the AgMNPV to determine the degree of enhancement of viral activity. These brighteners were also evaluated with regard to AgMNPV protection against deactivation by UV light. Combinations of the virus with selected fluorescent brighteners were tested against both AgMNPV-susceptible and resistant strains of A. gemmatalis. In the first trial, brighteners obtained from the United States promoted increases in AgMNPV activity from 5.2-fold (Blankophor HRS) to 76.6-fold (Blankophor RKH) and reduced the mean time to death by 2.8 to 3.5 days. In the second trial, the most effective brightener (Tinopal UNPA-GX) reduced the LC₅₀ in A. gemmatalis larvae from 7083 occlusion bodies (OBs)/ml (virus alone) to 77.8 OBs/ml (≈90-fold). When 4 selected brighteners were tested in combination with the AgMNPV in resistant insects, the LC₅₀ was reduced by ca. 10,000-fold (Leukophor DUB) to ca. 62,000-fold (Tinopal UNPA-GX), in comparison to the LC₅₀ of 3.7 × 10⁷ OBs/ml observed for the virus alone. Therefore, mortality of highly resistant A. gemmatalis larvae to the AgMNPV increased dramatically when the virus was combined with some fluorescent brighteners. UV protection measured by original activity remaining (OAR) varied from <30% OAR (Uvitex NFW) to >90% OAR (Tinopal UNPA-GX and BRY 10 D2 100). All efficacious brighteners were stilbene disulfonic acid derivatives and, when used alone, none showed negative effects against A. gemmatalis larvae.     

Effects of Fetal Bovine Serum deprivation in cell cultures on the production of <Emphasis Type="Italic">Anticarsia gemmatalis</Emphasis> Multinucleopolyhedrovirus

Background  

Anticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum.     

Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from theobroma seeds on midgut protease activity of lepidopteran pest insects

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.     

Dosage-mortality and time-mortality responses of the armyworm Spodoptera exempta to a nuclear polyhedrosis virus

Third-instar Spodoptera exempta larvae were fed on young maize leaves treated with 20 μl of polyhedral inclusion body (PIB) suspension of concentrations that varied from 1.6 × 10² to 1.6 × 10⁹ PIBs/ml. Daily observations were kept on mortality rates. A probit analysis on the results gave an LD₅₀ value of 48.4 PIBs/larva (lower and upper fiducial limits 39.2 and 59.4 PIBs/larva, respectively), and an LT₅₀ that varied from 146.2 to 221.3 hr, depending on the dosage. LD and LT values obtained show the high pathogenicity of S. exempta nuclear polyhedrosis virus to its host.     

Susceptibility of larvae of Trichoplusia ni and Anticarsia gemmatalis to intrahemocoelic injections of conidia and blastospores of Nomuraea rileyi

The LD₅₀ for larvae of Trichoplusia ni injected with blastospores of Nomuraea rileyi was 4.30 ± 1.16 hyphal bodies/larva; the LD₅₀ for injected conidia was ca. 25,000 conidia/larva. The dose-mortality regression line for blastospores was Y = 4.6504 + 0.5487 X. Larval mortalities of Anticarsia gemmatalis and T. ni at 100 blastospores/larva were 0.4 ± 0.5% and 96.7 ± 1.9%, respectively. At a dosage of 25,000 conidia/larva, larval mortalities for A. gemmatalis and T. ni were 0.4 ± 0.5% and 43.1 ± 8.7%, respectively. Thus, larvae of A. gemmatalis were > 100 times and >200 times more resistant to injected conidia and blastospores, respectively, than were larvae of T. ni. Resistance of A. gemmatalis to N. rileyi may not be solely at the integumental barrier, as is often believed, but may also be a function of an internal physiological response.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts,Phthorimaea operculella and Tecia solanivora

A granulovirus isolate collected from diseased Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) larvae in Costa Rican potato [Solanum tuberosum L. (Solanaceae)] crops was characterized at the molecular and biological level. Restriction endonuclease analysis identified this isolate as a novel P. operculella granulovirus (PhopGV) (Baculoviridae: Betabaculovirus) strain and was designated as PhopGV‐CR1. In addition, PCR amplification of four specific variable genomic regions yielded multiple amplicons for two open reading frames, revealing the presence of different genotypic variants within the virus population. Biologically, PhopGV‐CR1 was highly pathogenic for its two indigenous hosts, although significant differences of up to four‐fold were detected against P. operculella [LD₅₀ = 17.9 occlusion bodies (OBs) mm^?2] and Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) (LD₅₀ = 69.1 OBs mm^?2). The two P. operculella colonies, from Costa Rica and France, were equally susceptible to PhopGV‐CR1. Serial passage of PhopGV‐CR1 over four generations in T. solanivora increased its pathogenicity by five‐fold in three generations, suggesting an ongoing adaptation to its alternate host.