Lack of fitness costs in dsRNA-resistant Leptinotarsa decemlineata ([Coleoptera]: [Chrysomelidae]).

The Colorado potato beetle, Leptinotarsa decemlineata (Say) ([Coleoptera]: [Chrysomelidae]), is the most important defoliator of solanaceous plants worldwide. This insect displays a notorious ability in adapting to biological and synthetic insecticides, although in some cases this adaptation carries relevant fitness costs. Insecticidal gene silencing by RNA interference is a novel mode of action pesticide against L. decemlineata that is activated by ingestion of a double stranded RNA (dsRNA) targeting a vital L. decemlineata gene. We previously reported laboratory selection of a > 11,000-fold resistant strain of L. decemlineata to a dsRNA delivered topically to potato leaves. In this work, we tested the existence of fitness costs in this dsRNA-resistant colony by comparing biological parameters to the parental strain and an additional susceptible reference strain. Biological parameters included length of egg incubation period, number of eggs per clutch, egg viability, larval viability, length of larval and pupal periods, adult emergence, number of eggs laid per day, sex ratio, and adult longevity. Comparisons between the 3 beetle strains detected no fitness costs associated with resistance to dsRNA. This information is important to guide effective insect resistance management plans for dsRNA insecticides against L. decemlineata applied topically to potato leaves.

Evidence of a shared binding site for Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Cnaphalocrocis medinalis cadherin.

Insect midgut cadherins function as receptors and play critical roles as protein receptors of insecticidal Bacillus thuringiensis (Bt) toxins used as biopesticides and in Bt transgenic crops worldwide. Here, we cloned and characterized the full-length midgut cadherin (CmCad) cDNA from the rice leaffolder (Cnaphalocrocis medinalis), a destructive pest of rice in many Asian countries. Expression of recombinant proteins corresponding to the extracellular domain of CmCad allowed testing binding of Cry proteins. Results from in vitro ligand blotting and enzyme-linked immunosorbent assays supported that the extracellular domain of CmCad contains regions recognized by both Cry1Ac and Cry2Aa. Molecular modelling and docking simulations indicated that binding to both Cry1Ac and Cry2Aa is localized primarily within a CmCad motif corresponding to residues T1417-D1435. A recombinant CmCad protein produced without residues T1417-D1435 lacked binding to Cry1Ac and Cry2Aa, confirmed our modelling predictions that CmCad has a shared Cry1Ac and Cry2Aa binding site. The potential existence of a shared binding region in CmCad suggests that caution should be taken when using combinations of Cry1Ac and Cry2Aa in pyramided transgenic rice, as their combined use could speed the evolution of resistance to both toxins.

Insecticidal Gene Silencing by RNAi in the Neotropical Region.

Insecticidal gene silencing by RNA interference (RNAi) involves a post-transcriptional mechanism with great potential for insect control. Here, we aim to summarize the progress on RNAi research toward control of insect pests in the Neotropical region and discuss factors determining its efficacy and prospects for pest management. We include an overview of the available RNAi information for Neotropical pests in the Lepidoptera, Coleoptera, Diptera, and Hemiptera orders. Emphasis is put on significant findings in the use of RNAi against relevant Neotropical pests, including diamondback moth (Plutella xylostella L.), Asian citrus psyllid (Diaphorina citri Kuwayama), and the cotton boll weevil (Anthonomus grandis Boheman). We also examine the main factors involved in insecticidal RNAi efficiency and major advances to improve screening of lethal genes, formulation, and delivery. Few studies detail resistance mechanisms to RNAi, demonstrating a need for more research. Advances in formulation, delivery, and resistance management tools for insecticidal RNAi in the Neotropics can provide a basis for efficient field application.

Dynamics of transcriptomic response to infection by the nematode Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus temperata in Heliothis virescens larvae.

Entomopathogenic nematodes in the Heterorhabditis genus and their symbiotic Photorhabdus bacteria are important biocontrol agents of insect pests and models for the study of microbe-host interactions. In this work, we used larvae of the tobacco budworm (Heliothis virescens) as a model to study its defensive mechanisms against Heterorhabditis bacteriophora nematodes carrying symbiotic Photorhabdus temperata. We first determined time points of initial nematode entry and release of bacteria into the haemolymph to perform transcriptional analysis of insect gene expression during these steps in the infective process. RNA-Sequencing analyses were then performed to profile differential gene expression in the insect during nematode invasion, bacterial release and final steps of infection, relative to the untreated controls. Our results support the theory that insect immune response genes are induced upon nematode invasion, but the majority of these genes are suppressed upon the release of bacteria by the nematodes into the haemolymph. Overall, these findings provide information on the dynamics of the insect's response to a progressing infection by this entomopathogenic nematode-bacteria complex and facilitate development of Hel. virescens as a pest model for future functional studies of the key insect defence factors.

The association of low-molecular-weight hydrophobic compounds with native casein micelles in bovine milk.

The agreed biological function of the casein micelles in milk is to carry minerals (calcium, magnesium, and phosphorus) from mother to young along with amino acids for growth and development. Recently, native and modified casein micelles were used as encapsulating and delivery agents for various hydrophobic low-molecular-weight probes. The ability of modified casein micelles to bind certain probes may derive from the binding affinity of native casein micelles. Hence, a study with milk from single cows was conducted to further elucidate the association of hydrophobic molecules into native casein micelles and further understand their biological function. Hydrophobic and hydrophilic extraction followed by ultraperformance liquid chromatography-high resolution mass spectrometry analysis were performed over protein fractions obtained from size exclusion fractionation of raw skim milk. Hydrophobic compounds, including phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, showed strong association exclusively to casein micelles as compared with whey proteins, whereas hydrophilic compounds did not display any preference for their association among milk proteins. Further analysis using liquid chromatography-tandem mass spectrometry detected 42 compounds associated solely with the casein-micelles fraction. Mass fragments in tandem mass spectrometry identified 4 of these compounds as phosphatidylcholine with fatty acid composition of 16:0/18:1, 14:0/16:0, 16:0/16:0, and 18:1/18:0. These results support that transporting low-molecular-weight hydrophobic molecules is also a biological function of the casein micelles in milk.

Spodoptera frugiperda (J.E. Smith) with field-evolved resistance to Bt maize are susceptible to Bt pesticides.

Field-evolved resistance to maize event TC1507 expressing the Cry1Fa toxin from Bacillus thuringiensis (Bt) was detected in populations of Spodoptera frugiperda from Puerto Rico. We tested for cross-resistance to purified Cry1A toxins and commercial Bt pesticides in susceptible (Benzon) and TC1507-resistant (456) strains of S. frugiperda. Larvae from the 456 strain exhibited cross-resistance to Cry1Ab and Cry1Ac toxins, while no differences in susceptibility to XenTari WG and DiPel ES pesticides were detected. These data support cross-resistance to toxins that share binding sites with Cry1Fa and no cross-resistance to Bt pesticides in S. frugiperda with field-evolved resistance to Bt maize.

Fitness costs associated with field-evolved resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Increasing adoption of transgenic crops expressing cry toxin genes from Bacillus thuringiensis (Bt crops) represents an augmented risk for development of insect resistance. While fitness costs can greatly influence the rate of resistance evolution, most available data related to Bt resistance have been obtained from laboratory-selected insect strains. In this article, we test the existence of fitness costs associated with high levels of field-evolved resistance to Bt maize event TC1507 in a strain of Spodoptera frugiperda (JE Smith) originated from maize fields in Puerto Rico. Fitness costs in resistant S. frugiperda were evaluated by comparing biological performance to susceptible insects when reared on meridic diet, maize or soybean leaf tissue, or cotton reproductive tissues. Parameters monitored included larval survival, larval and pupal weights, developmental time (larval and pupal), adult longevity, reproductive traits (fecundity and fertility), and sex ratio. We found that all monitored parameters were influenced to a similar extent by the host, independently of susceptibility to Bt maize. The only parameter that significantly differed between strains for all hosts was a longer larval developmental period in resistant S. frugiperda, which resulted in emergence asynchrony between susceptible and resistant adults. To test the relevance of fitness costs in resistant S. frugiperda, we performed a selection experiment to monitor the stability of resistance in a heterogeneous strain through 12 generations of rearing on meridic diet. Our data demonstrate lack of fitness costs relevant to stability of field-evolved resistance to Bt maize and help explain reported stability of field-evolved resistance in Puerto Rican populations of S. frugiperda.

Binding of vitamin A by casein micelles in commercial skim milk.

Recent studies have shown that reassembled micelles formed by caseinates and purified casein fractions (α(s)- and ß-casein) bind to hydrophobic compounds, including curcumin, docosahexaenoic acid, and vitamin D. However, limited research has been done on the binding of hydrophobic compounds by unmodified casein micelles in skim milk. In the present study, we investigated the ability of casein micelles in commercial skim milk to associate with vitamin A (retinyl palmitate), a fat-soluble vitamin commonly used to fortify milk. Milk protein fractions from different commercially available skim milk samples subjected to different processing treatments, including pasteurized, ultrapasteurized, organic pasteurized, and organic ultrapasteurized milks, were separated by fast protein liquid chromatography. The fractions within each peak were combined and freeze-dried. Sodium dodecyl sulfate-PAGE with silver staining was used to identify the proteins present in each of the fractions. The skim milk samples and fractions were extracted for retinyl palmitate and quantified against a standard using normal phase-HPLC. Retinyl palmitate was found to associate with the fraction of skim milk containing caseins, whereas the other proteins (BSA, ß-lactoglobulin, α-lactalbumin) did not show any binding. The retinyl palmitate content in the various samples ranged from 1.59 to 2.48 µg of retinyl palmitate per mL of milk. The casein fractions contained between 14 and 40% of total retinyl palmitate in the various milks tested. The variation in the retention of vitamin A by caseins was probably explained by differences in the processing of different milk samples, including thermal treatment, the form of vitamin A emulsion used for fortification, and the point of fortification during processing. Unmodified casein micelles have a strong intrinsic affinity toward the binding of vitamin A used to fortify commercially available skim milks.

Cryo-transmission electron tomography of native casein micelles from bovine milk.

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (∼20 to 30 nm in diameter), channels (diameter greater than ∼5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed.

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin.

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry.

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.

Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses.

Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as Cry1Ac binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind Cry1Ac were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a Cry1Ac binding protein. V-ATP synthase subunit A and actin were identified as novel Cry1Ac binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional Cry1Ac binding proteins.

Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.

Binding analyses of Bacillus thuringiensis Cry delta-endotoxins using brush border membrane vesicles of Ostrinia nubilalis.

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit (125)I-Cry1Ab binding to BBMV. Cry1F inhibited (125)I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.).

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K(com)] = 1.1 nM) for (125)I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM. Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.