Biologia deMeteorus rubens (Hym.: Braconidae), parasitoide primario deAgrotis ipsilon (Lep.: Noctuidae)

Resumen El desarrollo del bracónidoMeteorus rubens (Nees) se estudió en condiciones de laboratorio sobre las especies de noctuidosAgrotis ipsilon (Hufnagel),A. puta (Hübner),A. segetum (Denis & Schiffermüller),Peridroma saucia (Hübner),Spodoptera littoralis (Boisduval) yAutographa gamma (L.). Las larvas deA. ipsilon yA. puta fueron las únicas adecuadas para el desarrollo del parasitoide. EnA. ipsilon este bracónido parasitó y completó su desarrollo en el tercero, cuarto, quinto y sexto estadios larvarios, mostrando preferencia por los dos últimos estadios. El número de hembras que parasitan, el porcentaje de larvas parasitadas y el número de parasitoides emergidos por hospedante estuvo en relación directa con la edad del hospedante en el momento de la parasitación. En las larvas parasitadas en el tercer estadio el tiempo de desarrollo del parasitoide fue significativamente mayor que en los otros tres estadios. Sin embargo, el estadio en que fueron parasitadas las larvas no influyó en la longevidad de los adultos.      

Effect of carbohydrate on age-related feeding behaviors and longevity in adult black cutworm,Agrotis ipsilon (Lepidoptera: Noctuidae)

Age-related consumption and longevity were monitored in the laboratory for adultA. ipsilon fed either a 1M sucrose solution or water. An additional group was completely starved. Adults consumed sucrose solution and water just after eclosion; the percentage feeding daily and the mean daily consumption for females and males fed sucrose solution declined with time, whereas the percentage feeding daily and the mean daily consumption of those fed water increased with time. Total consumption was significantly higher for those fed sucrose solution (P<0.01) because they lived longer, but consumption per day averaged over the entire adult stage was not significantly different between those fed sucrose solution and those fed water (P>0.05). Mean longevity was significantly extended for females and males fed sucrose solution over those fed water or starved (P<0.01). Moreover, consumption of either fluid was significantly correlated with extended longevity in all groups (P<0.05). These data on fluid consumption by adultA. ipsilon are discussed relative to posteclosion migratory activities.     

The Masc gene product controls masculinization in the black cutworm,Agrotis ipsilon

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity AiMasc in BmN cells and found that/z'Masc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.     

Studies onBonnetia comta [Dipt.: Tachinidae] parasitizingAgrotis ipsilon [Lep.: Noctuidae] larvae

Laboratory studies showed that 1st-instarBonnetia comta (Fallén) maggots (planidia) had a significant impact (P<0.05) on mortality of all black cutworm (BCW),Agrotis ipsilon (Hufnagel), instars, either by killing 1st- and 2nd-instar BCWs 2.3 to 9.7 days after parasitization or by producing a puparium from older host instars. Diet consumption and utilization by BCW larvae parasitized byB. comta as 4th instars were similar to those of nonparasitized larvae until 1 to 2 days before the parasitoid emerged. In a 2-year host exposure study in Iowa, it was found thatB. comta primarily parasitized released BCWs in June through September and did not seem to play a role in controlling the damaging 1st generation of BCW larvae. Techniques were developed to produce and store large numbers ofB. comta planidia.Bonnetia comta deposited large numbers of planidia on filter paper treated with a fecal supernatant. These planidia could be stored on filter paper in a covered Petri dish at 4.4°C for 5 days with minimal mortality. Preliminary field data show that planidia placed around corn seedlings infested with 4th-instar BCW larvae do parasitize the pest and reduce the hosts cutting potential.     

Host range of an NPV and a GV isolated from the common cutworm, Agrotis segetum: pathogenicity within the cutworm complex

The term cutworm covers a range of species with a similar life history that can be very damaging pests on a wide range of crops. Attacks by cutworms are often made up of more than one species; thus, the most cost effective microbial control agent needs to be pathogenic for multiple species within this complex. In this study we investigate the host range of Agrotis segetum NPV and A. segetum GV for other cutworm species and closely related Noctuinae. Eight species, A. segetum, Agrotis ipsilon, Agrotis exclamationis, Agrotis puta, Noctua comes, Peridroma saucia, Xestia sexstrigata, and Xestia xanthographa, were clearly susceptible to AgseNPV, which was confirmed by DNA analysis. Aglais urticae, Diarsia rubi, Noctua pronuba, and Xestia c-nigrum were not susceptible to AgseNPV at the doses used. Noctua fimbriata, Noctua janthina, and Ochroplura plecta gave ambivalent results: larvae died of NPV infection when they were challenged with AgseNPV, but these individuals only produced weak positives in a squash blot analysis and there was insufficient DNA for confirmation by restriction endonuclease profiling. These ambivalent results could suggest either a weak infection by AgseNPV or partial homology between their own virus and AgseNPV. The untreated control insects of several species died of NPV infection, which indicates that these field-collected insects were probably carrying a vertically transmitted NPV. Fewer species were tested with AgseGV and only N. pronuba and N. comes were susceptible. N. fimbriata and Helicoverpa armigera were not susceptible to AgseGV.     

Agrotis segetum granulosis virus as a control agent against field populations ofAgrotis ipsilon andA. Segetum [Lep.: Noctuidae] on tobacco,okra, potato and sugar beet in northern pakistan

Agrotis segetum Schiff granulosis virus (AsGV) propagated in Denmark was supplied against naturally occurring cutworm populations (A. ipsilon and to a less extentA. segetum) in experimental field plots of tobacco, okra, potato and sugar beet in northern Pakistan. AsGV doses varied between 4 × 10⁷ and 4 × 10¹¹ capsules per m² plot, and no. of applications between 1 and 3. One treatment with AsGV did not reduce cutworm damage significantly to tobacco seedlings and potato plants. Two treatments with AsGV reduced cutworm damage significantly. In tobacco, reduction was 64–82%, in okra and potato 85% and 77% respectively. Damage in sugar beet was reduced 78%. Three treatments with AsGV dis not reduce damage significantly better than two treatments. AsGV and the chemical insecticides Tamaran and Dieldrin, andBacillus thuringiensis (Thuricide) were about equally effective, reducing damage by 85%, 79%, 87% and 69%, respectively. No difference was found between the efficiency of highly purified AsGV to which activated charcoal was added and partially purified AsGV without charcoal.      

Establishment and characterization of insect cell lines from 10 lepidopteran species

Summary Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer,Ostrinia nubilalis, a plutellid, the diamondback moth,Plutella xylostella, as well as eight noctuids: the black cutworm,Agrotis ipsilon, the celery looper,Anagrapha falcifera, the velvetbean caterpillar,Anticarsia gemmatalis, the corn earworm,Helicoverpa zea, the tobacco budworm,Heliothis virescens, the beet armyworm,Spodoptera exigua, the fall armyworm,Spodoptera frugiperda, and the cabbage looper,Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines fromA. ipsilon andH. virescens being adapted to suspension culture, using shaker flasks. The potential use for these cell lines in baculovirus production is discussed. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion sex, age, marital status, or handicap.     

Mycotoxin-producing potential of fungi isolated from red kidney beans

The predominant fungi present in samples of reject and retail red kidney beans were Aspergillus glaucus, Penicillium spp. and Alternaria spp. Together with A. ochraceus, A. flavus, Fusarium spp., and Trichoderma, these isolates from the reject beans were screened for numerous mycotoxins by TLC. The most consistently produced mycotoxins were penicillic acid (from A. ochraceus and Penicillium spp.) and Alternaria toxins (tenuazonic acid and alternariol). A. glaucus strains were tested for cytotoxicity in three tissue culture cell lines with positive results.     

Multiple factors promoting narrow host range in the sea hare,Aplysia californica

Summary Juvenile sea hares, Aplysia californica, utilize only the red algae Plocamium cartilagineum and Laurencia pacifica as host plants at Santa Catalina Island, CA. I tested three hypotheses which might account for this pattern of host choice: 1) A. californica specialize on the algae on which they grow best, 2) A. california specialize on algae from which they acquire secondary compounds that protect them from predators, and 3) A. californica specialize on certain algae in order to lower their encounter rates with predators. The results suggested that host range in the Aplysia californica system is affected by more than one factor. The first hypothesis was supported. A. californica of three size classes grew well on Plocamium, but could not grow at all on most other species of algae. Larger A. californica were able to grow on species of algae that smaller ones could not. The second hypothesis was also supported. Small A. californica grown on Plocamium were rich in terpenes. Small A. californica grown on Ulva sp. were terpene-free. Rock wrasses, Halichoeres semicinctus, were more likely to eat Ulva-fed A. californica than Plocamium-fed A. californica. Other fish and lobster, Panulirus interruptus, did not discriminate between the two groups. Kelp bass, Paralabrax clathratus, which were force-fed Ulva-fed A. californica regurgitated them less often, and after digesting them more completely, than did Paralabrax force-fed Plocamium-fed A. californica. The third hypothesis was rejected. A. californica located on Plocamium were not more cryptic to the opisthobranch Aglaja inermis (navanax), or to the pomacentrid fish, Hypsypops rubicundus, than were A. californica located on other algae. In addition, navanax, a specialist predator of opisthobranchs, was significantly more abundant on Plocamium than on other algae.     

Toward the physiological basis for increased Agrotis ipsilon multiple nucleopolyhedrovirus infection following feeding of Agrotis ipsilon larvae on transgenic corn expressing Cry1Fa2

Larvae of the black cutworm, Agrotis ipsilon Hufnagel, were more susceptible to infection by A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV: Baculoviridae) after feeding on Herculex^® I, a transgenic corn hybrid expressing the Bacillus thuringiensis (Bt)-derived toxin Cry1Fa2 compared to larvae fed on isoline corn. We investigated the physiological basis for increased susceptibility to virus infection following exposure to Herculex^® I by analyzing the midgut pH, gut protease activity and peritrophic matrix structure which are important factors for both Bt toxin action and baculovirus infection. No significant treatment differences were found in the pH of anterior midgut, central midgut or posterior midgut in larvae fed Herculex^® I or isoline diets. Analysis of soluble and membrane-associated gut proteinase activities from larvae fed Herculex^® I or isoline diets indicated that membrane-associated aminopeptidase activity and soluble chymotrypsin-like proteinase activity were significantly lower in Herculex^® I -fed larvae compared to isoline-fed larvae. The number and relative molecular masses of soluble chymotrypsin-like proteinases did not differ. Baculoviruses were not susceptible to in vitro degradation by bovine chymotrypsin, suggesting that chymotrypsin degradation of baculovirus occlusion-derived virus did not result in reduced infection of larvae fed on isoline diet. Scanning electron micrographs of the peritrophic matrices of Herculex^® I -fed larvae and isoline-fed larvae indicated that Herculex^® I did not result in damage to the peritrophic matrix that could facilitate subsequent baculovirus infection. Additional research is required to further delineate the physiological basis for enhanced baculovirus infection following exposure to sublethal doses of Bt toxins.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

Distribution of some virulence related-properties of Vibrioalginolyticus strains isolated from Mediterranean seawater (Bay of Khenis, Tunisia): investigation of eight Vibriocholerae virulence genes

This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips. Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and Caco-2) were also investigated. Eight Vibrio cholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines. The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells and fish mucus.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

Identification of Zygowilliopsis californicaStrains of Different Origin by Means of Polymerase Chain Reaction with Universal Primers

After reevaluation of the taxonomic position of 27 yeast collection strains of different origin by UP-PCR followed by dot-hybridization, only 22 strains were assigned to the biological species Zygowilliopsis californica(Lodder) Kudriavzev. Four strains were identified as Williopsis suaveolens(Klöcker) Naumov et al. Universal primers L45 and N21 are recommended for identification of the Z. californicayeasts.     

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.     

Cell lines used for the selection of recombinant baculovirus

Summary Four insect cell lines were used to isolate two recombinant baculoviruses which had theβ-galactosidase (β-gal) gene for colorimetric assay purposes. Plaque assays were performed using twoTrichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and twoSpodoptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blueβ-gal⁺ recombinants) formed in theTrichoplusia cells was higher than in theSpodoptera cells. The appearance ofAutographa californica NPV polyhedra was also faster in theT. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.     

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.     

Characterization of theHelicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene region

A local strain ofHelicoverpa assulta nucleopolyhedrovirus (HasNPV) was isolated from infectedH. assulta larvae in Korea. Restriction endonuclease fragment analysis, using 4 restriction enzymes, estimated that the total genome size of HasNPV is about 138 kb. A degenerate polymerase chain reaction (PCR) primer set for the polyhedrin gene successfully amplified the partial polyhedrin gene of HasNPV. The sequencing results showed that the about 430 bp PCR product was a fragment of the corresponding polyhedrin gene. Using HasNPV partial predicted polyhedrin to probe the Southern blots, we identified the location of the polyhedrin gene within the 6 kbEcoRI, 15 kbNcoI, 20 kbXhoI, 17 kbBgl II and 3 kbClaI fragments, respectively. The 3 kbClaI fragment was cloned and the nucleotide sequences of the polyhedrin coding region and its flaking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame of 735 nucleotides which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of HasNPV polyhedrin shared 73.7&#x0025; identity with the polyhedrin gene fromAutographa californica NPV but were most closely related toHelicoverpa andHeliothis species NPVs with over 99% sequence identity.     

不同饲喂下棉铃虫肠道内生菌的分离鉴定及脱毒效果研究

[目的]本研究对比饲喂正常饲料和棉粕饲料下棉铃虫(Helicoverpa armigera)肠道中可培养微生物的异同,并对筛选菌株进行棉酚耐受及降解作用的研究,为棉铃虫肠道微生物在棉源饲料中的脱毒作用提供理论和实验支持.[方法]通过饲喂不同饲料、醋酸棉酚单一碳源微生物选择性培养,分离饲喂正常饲料和棉粕饲料下棉铃虫肠道可...     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.