Characterizing the differential susceptibility and resistance to insecticides in populations of Chrysodeixis includens and Rachiplusia nu (Lepidoptera: Noctuidae) in Brazil.

BACKGROUND: Chrysodeixis includens (Walker) and Rachiplusia nu (Guenée) are major Plusiinae pests of soybean in the Southern Cone region of South America. In recent decades, C. includens was the main defoliator of soybean in Brazil, but from 2021 onwards, R. nu emerged as an important soybean pest in various regions of the country. Here, we characterize the differential susceptibility and resistance to insecticides in these Plusiinae pests from two soybean regions of Brazil. RESULTS: Except for spinetoram and chlorfenapyr (comparable lethality against both species) and a Bt-based biopesticide (more lethal for C. includens), the tested insecticides showed higher lethality against R. nu than against C. includens, but populations of the same species, even separated by long distances, presented similar resistance levels. For both species, the 90% lethal concentration (LC90) values of most insecticides were higher than the field-recommended dose. Nevertheless, the field-recommended doses of spinetoram, metaflumizone, emamectin benzoate, cyclaniliprole and chlorfenapyr showed comparable control efficacy against both species, whereas indoxacarb, chlorantraniliprole, flubendiamide, teflubenzuron and chlorfluazuron were more lethal for R. nu, and methoxyfenozide and the Bt-based insecticide were more lethal for C. includens. Thiodicarb, methomyl and lambda-cyhalothrin showed low lethality against both species. CONCLUSIONS: Large interspecific differences in the susceptibility to insecticides was found in major Plusiinae pests of soybean in Brazil. Furthermore, variations in susceptibility to insecticides occurred consistently among species and populations, regardless of the collection site and thus despite unequal temporal and spatial exposure to insecticides. These results demonstrate that accurate species identification is essential for effective control of Plusiinae in soybean. © 2024 Society of Chemical Industry.

CRISPR/Cas9-mediated knockout of scarlet gene produces eye color mutants in the soybean looper, Chrysodeixis includens.

The CRISPR/Cas9 technology has greatly progressed research on non-model organisms, demonstrating successful applications in genome editing for various insects. However, its utilization in the case of the soybean looper, Chrysodeixis includens, a notable pest affecting soybean crops, has not been explored due to constraints such as limited genomic information and the embryonic microinjection technique. This study presents successful outcomes in generating heritable knockout mutants for a pigment transporter gene, scarlet, in C. includens through CRISPR/Cas9-mediated mutagenesis. The scarlet locus identified in the genome assembly of C. includens consists of 14 exons, with a coding sequence extending for 1,986 bp. Two single guide RNAs (sgRNAs) were designed to target the first exon of scarlet. Microinjection of these two sgRNAs along with the Cas9 protein into fresh embryos resulted in the successful production of variable phenotypes, particularly mutant eyes. The observed mutation rate accounted for about 16%. Genotype analysis revealed diverse indel mutations at the target site, presumably originating from double-strand breaks followed by the nonhomologous end joining repair, leading to a premature stop codon due to frame shift. Single-pair mating of the mutant moths produced G1 offspring, and the establishment of a homozygous mutant strain occurred in G2. The mutant moths exhibited lightly greenish or yellowish compound eyes in both sexes, confirming the involvement of scarlet in pigmentation in C. includens. Notably, the CRISPR/Cas9-mediated genome editing technique serves as a visible phenotypic marker, demonstrating its proof-of-concept applicability in C. includens, as other pigment transporter genes have been utilized as visible markers to establish genetic control for various insects. These results provide the first successful case that the CRISPR/Cas9 method effectively induces mutations in C. includes, an economically important soybean insect pest.

Molecular characterization and expression patterns of a ryanodine receptor in soybean looper, Chrysodeixis includens.

Diamide insecticides, such as chlorantraniliprole, have been widely used to control insect pests by targeting the insect ryanodine receptor (RyR). Due to the efficacious insecticidal activity of diamides, as well as an increasing number of resistance cases, the molecular structure of RyR has been studied in many economically important insects. However, no research has been conducted on diamide resistance and RyR in the soybean looper, Chrysodeixis includens, a significant crop pest. In this study, we found moderate resistance to chlorantraniliprole in a field population from Puerto Rico and sequenced the full-length cDNA of the C. includens RyR gene, which encodes a 5124 amino acid-long protein. Genomic analysis revealed that the CincRyR gene consists of 113 exons, one of the largest exon numbers reported for RyR. Alternative splicing sites were detected in the cytosolic region. The protein sequence showed high similarity to other noctuid RyRs. Conserved structural features included the selectivity filter motif critical for ryanodine binding and ion conduction, as well as various domains involved in ion transport. Two mutation sites associated with diamide resistance in other insects were screened but not found in the Puerto Rico field populations or in the susceptible lab strain. Gene expression analysis indicated high expression of RyR in the third instar larval stage, particularly in muscle-containing tissues. Furthermore, exposure to a sublethal dose of chlorantraniliprole reduced RyR expression levels after 96 h. This study provides a molecular basis for understanding RyR structure and sheds light on potential mechanisms of diamide resistance in C. includens.

The impact of thiamethoxam on the feeding and behavior of 2 soybean herbivore feeding guilds.

Over the past few decades, inadvertent consequences have stemmed from the intensified use of neonicotinoids in agroecosystems. Neonicotinoid applications can result in both positive (e.g., reduced persistent virus transmission) and negative (e.g., increased host susceptibility) repercussions exhibiting ambiguity for their use in crop production. In soybean, aspects of neonicotinoid usage such as the impact on nonpersistent virus transmission and efficacy against nontarget herbivores have not been addressed. This study evaluated the interaction between the neonicotinoid thiamethoxam and soybean variety and the impact on different pest feeding guilds. Feeding and behavioral bioassays were conducted in the laboratory to assess the effect of thiamethoxam on the mortality and weight gain of the defoliator, Chrysodeixis includens (Walker). Bioassays evaluated impacts dependent and independent of soybean tissue, in addition to both localized and systemic efficacy within the soybean plant. Additionally, using the electrical penetration graph technique (EPG), the probing behavior of 2 piercing-sucking pests, Aphis gossypii Glover and Myzus persicae (Sulzer), was observed. Results from defoliator bioassays revealed thiamethoxam had insecticidal activity against C. includens. Distinctions in thiamethoxam-related mortality between bioassays dependent and independent of soybean tissue (~98% versus ~30% mortality) indicate a contribution of the plant towards defoliator-related toxicity. Observations of defoliator feeding behavior showed a preference for untreated soybean tissue relative to thiamethoxam-treated tissue, suggesting a deterrent effect of thiamethoxam. EPG monitoring of probing behavior exhibited a minimal effect of thiamethoxam on piercing-sucking herbivores. Findings from this study suggest neonicotinoids like thiamethoxam may provide some benefit via insecticidal activity against nontarget defoliators.

Investigating the Migratory Behavior of Soybean Looper, a Major Pest of Soybean, through Comparisons with the Corn Pest Fall Armyworm Using Mitochondrial Haplotypes and a Sex-Linked Marker.

The Noctuid moth soybean looper (SBL), Chrysodeixis includens (Walker), is an economically important pest of soybean (Glycine max (Linnaeus) Merrill). Because it is not known to survive freezing winters, permanent populations in the United States are believed to be limited to the southern regions of Texas and Florida, yet its geographical range of infestations annually extend to Canada. This indicates annual migrations of thousands of kilometers during the spring and summer growing season. This behavior is like that of the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), also a Noctuid that is a major global pest of corn. SBL and FAW are projected to have very similar distributions of permanent populations in North America based on climate suitability modeling and the overlap in the distribution of their preferred host plants (corn and soybean). It therefore seems likely that the two species will display similar migratory behavior in the United States. This was tested by identifying genetic markers in SBL analogous to those successfully used to delineate FAW migratory pathways and comparing the distribution patterns of the markers from the two species. Contrary to expectations, the results indicate substantial differences in migratory behavior that appear to be related to differences in the timing of corn and soybean plantings. These findings underscore the importance of agricultural practices in influencing pest migration patterns, in particular the timing of host availability relative to mean seasonal air transport patterns.

Evidence for Two Soybean Looper Strains in the United States with Limited Capacity for Cross-Hybridization.

The noctuid moth soybean looper (SBL), Chrysodeixis includens (Walker) is an economically important pest of soybeans (Glycine max (L.) Merr.) in the southeastern United States. It has characteristics that are of particular concern for pest mitigation that include a broad host range, the capacity for annual long-distance flight, and resistance in some populations to important pesticides such as pyrethroids and chitin synthesis inhibitor. The biology of SBL in the United States resembles that of the fellow noctuid fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), a major pest of corn and several other crops. FAW exhibits a population structure in that it can be divided into two groups (host strains) that differ in their host preferences but are broadly sympatric and exhibit incomplete reproductive isolation. In this paper, strategies used to characterize the FAW strains were applied to SBL to assess the likelihood of population structure in the United States. Evidence is presented for two SBL strains that were defined phylogenetically and display differences in the proportions of a small set of genetic markers. The populations exhibit evidence of reproductive barriers sufficient to allow persistent asymmetry in the distribution of mitochondrial haplotypes. The identified molecular markers will facilitate studies characterizing the behaviors of these two populations, with relevance to pest mitigation and efforts to prevent further dispersal of the resistance traits.

Development and biological evaluation of nanoencapsulated-based pyrethroids with synergists for resistance management of two soybean pests: insights for new insecticide formulations.

BACKGROUND: Chemical control is commonly used against Euschistus heros (F.) and Chrysodeixis includens (Walker) in soybean fields in South America. However, previous studies reported that these pests have reduced susceptibility to pyrethroids in Brazil. On this basis, we developed and evaluated nanoencapsulated-based bifenthrin (BFT) and λ-cyhalothrin (LAM) with the synergists piperonyl butoxide (PBO) and diethyl maleate (DEM) for insect resistance management (IRM). RESULTS: Nanoformulations of BFT and LAM with PBO and DEM presented good physical-chemical characteristics and were stable. The spherical morphology of all systems and the encapsulation efficiency in nanostructured lipid carriers did not change when synergists were added. Nanoencapsulated BFT with DEM applied topically increased the susceptibility of E. heros to BFT by 3.50-fold. Similarly, nanoencapsulated BFT and LAM with PBO in diet-overlay bioassays increased the susceptibility of C. includens to both chemicals by up to 2.16-fold. Nanoencapsulated BFT and LAM with synergists also improve control efficacy of both species, causing higher mortality than commercial products containing these chemistries. CONCLUSIONS: It is possible to develop nanoencapsulated-based formulations of BFT and LAM with PBO or DEM, and these nanoformulations have the potential to improve control of E. heros and C. includens with recognized low susceptibility to pyrethroids. This study provides updates for designing new insecticide formulations for IRM. © 2022 Society of Chemical Industry.

Bioactivity of essential oils from Croton grewioides and its major compounds: toxicity to soybean looper Chrysodeixis includens and selectivity to the predatory stink bug Podisus nigrispinus.

Natural biological control is a key factor that ensures the regulation of insect pest populations in agroecosystems. However, the indiscriminate use of pesticides has compromised this environmental service. Thus, the search for environmentally safe pesticides is an increasing requirement for sustainable food production. In this study, we analyzed the toxicity of essential oils from two accessions (CGR112 and CGR126) of Croton grewioides and its major compounds, methyl eugenol and eugenol, on the soybean pest Chrysodeixis includens. In addition, we investigated the sublethal effects of these compounds on the predatory bug Podisus nigrispinus, analyzing its developmental, reproduction and life table parameters. Essential oils and their major compounds were toxic to C. includens and P. nigrispinus. In general, the presence of eugenol made the essential oil more toxic to the pest and selective to the natural enemy. Eugenol was the most toxic compound for 2nd instar larvae of C. includens at LD50, followed by CGR126 essential oil from C. grewioides which was equally toxic at higher doses. The estimated lethal times for essential oils to cause mortality in 50% of the population of C. includens were less than 15 h. There was selectivity of the essential oil of CGR126 accession of C. grewioides at lethal doses above 90%. Although the treatments showed little effect on the development of P. nigrispinus, body mass and reproductive parameters were negatively affected, with the exception of the essential oil of CGR126 accession of C. grewioides. The essential oil of C. grewioides may be a promising active ingredient for the synthesis of new insecticides, which are efficient against C. includens and at the same time are safer for the natural enemy P. nigrispinus.

The role of glyphosate-resistant weeds and starvation on biological, reproductive, and preference parameters of Chrysodeixis includens (Lepidoptera: Noctuidae).

Glyphosate-resistant weeds are difficult to manage and can serve as hosts for pests that threaten cultivated crops. Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) is one of the main polyphagous pests of soybean in Brazil that can benefit from weeds' presence during season and off-season. Despite its pest status, little is known about C. includens survival and development on alternative hosts, including those resistant to glyphosate. Therefore, we assessed the biology, reproduction, preference, and survival at different feeding periods of C. includens on seven glyphosate-resistant weeds (Sumatran fleabane, Italian ryegrass, sourgrass, goosegrass, smooth pigweed, wild poinsettia, hairy beggarticks) commonly found in Brazilian agroecosystems, under laboratory conditions. Our results showed that C. includens survival and reproduction were similar on soybean and wild poinsettia. Survival and reproduction were lower on smooth pigweed and hairy beggarticks. Also, these plants prolonged the larval stage. Larvae did not pupate when fed on sourgrass, goosegrass, Italian ryegrass, and Sumatran fleabane. However, on Sumatran fleabane their biomass was higher. The mean generation time was lower on wild poinsettia. This weed was preferred to soybean. An antifeeding factor was observed on Sumatran fleabane. Larvae fed for 11 days on soybean, wild poinsettia and smooth pigweed developed into pupae. In agricultural systems, farmers must pay attention to the management of these weeds, especially wild poinsettia, smooth pigweed, and hairy beggarticks, to interrupt the cycle of this pest, since these plants can serve as main sources of infestation for the soybean crop.

Evaluation of Hemp (Cannabis sativa) (Rosales: Cannabaceae) as an Alternative Host Plant for Polyphagous Noctuid Pests.

Hemp (Cannabis sativa L.) is a reemerging crop in the United States with increasing outdoor acreage in many states. This crop offers a potential host for polyphagous, defoliating lepidopteran pests currently present in Louisiana. The ability of soybean looper [Chrysodeixis includens (Walker)] (Lepidoptera: Noctuidae), fall armyworm [Spodoptera frugiperda (J.E. Smith)] (Lepidoptera: Noctuidae), and beet armyworm [Spodoptera exigua (Hübner)] to develop and reproduce on hemp was investigated in this study. Insects were reared on two hemp varieties, Maverick and Pipeline, as well as documented host plants soybean [Glycine max (L.) Merr.] (Fabales: Fabaceae) variety UA5414RR and cowpea [Vigna unguiculata (L.)] (Fabales: Fabaceae) variety Quickpick Pinkeye. Larvae of all three species reared on 'Maverick' had significantly faster preadult developmental times compared to the other hosts. Chrysodeixis includens larvae fed excised leaves of 'Maverick' and 'Pipeline' experienced higher intrinsic and finite rates of increase, higher net reproductive rates, and faster mean generation and doubling times. Spodoptera frugiperda larvae reared on 'Maverick', 'Pipeline', and 'UA5414RR' had higher intrinsic and finite rates of increase, higher net reproductive rates, and faster mean generation and doubling times compared to 'Quickpick Pinkeye'. Spodoptera exigua larvae had the highest survivorship on 'Maverick' and similar, positive lifetable statistics when reared on 'Maverick' and 'Pipeline'. The results of this study indicate hemp is an alternative host plant that has the potential to influence the population dynamics of C. includens, S. frugiperda, and S. exigua in Louisiana agroecosystems they co-occur in.

The Effect of Synergistic Compounds on the Susceptibility of Euschistus heros (Hemiptera: Pentatomidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) to Pyrethroids.

The Neotropical brown stink bug, Euschistus heros (F.), and the soybean looper, Chrysodeixis includens (Walker), are key pests of soybean in South America. Low susceptibility to pyrethroids has been reported for both species in Brazil. Here, we evaluate the addition of synergistic compounds piperonyl butoxide (PBO) and diethyl maleate (DEM) to manage E. heros and C. includens with resistance to λ-cyhalothrin and bifenthrin. The LD50 of technical grade and commercial products containing λ-cyhalothrin and bifenthrin decreased against field-collected E. heros exposed to PBO and DEM relative to unexposed insects; synergistic ratios up to 4.75-fold. The mortality also increased when E. heros were exposed to commercial formulations containing λ-cyhalothrin (from 4 to 44%) and bifenthrin (from 44 to 88%) in the presence of synergists. There was also a higher susceptibility of field-collected C. includens to technical grade λ-cyhalothrin when PBO was used; synergistic ratio of 5.50-fold. High lethally of technical grade λ-cyhalothrin was also verified in the presence of PBO, with mortality increasing from 6 to 57%. Our findings indicate the potential utility of synergists in reversing the resistance to λ-cyhalothrin and bifenthrin in E. heros and C. includens and suggest a significant role of metabolic mechanisms underlying the detoxification of both pyrethroids.

Interspecific Variation in Susceptibility to Insecticides by Lepidopteran Pests of Soybean, Cotton, and Maize Crops From Brazil.

The interspecific variation in susceptibility to insecticides by lepidopteran species of soybean [Glycine max L. (Merr.)], cotton (Gossypium hirsutum L.), and maize (Zea mays L.) crops from Brazil were evaluated. Populations of Anticarsia gemmatalis (Hübner) (Lepidoptera: Erebidae), Chrysodeixis includens (Walker), Helicoverpa armigera (Hübner), Spodoptera frugiperda (Smith), Spodoptera eridania (Stoll), Spodoptera cosmioides (Walker), and Spodoptera albula (Walker) (Lepidoptera: Noctuidae) were collected from 2019 to 2021. Early L3 larvae (F2 generation) were exposed to the formulated insecticides methoxyfenozide, indoxacarb, spinetoram, flubendiamide, and chlorfenapyr in diet-overlay bioassays. The median lethal concentrations (LC50) were used to calculate tolerance ratios (TR) of each species in relation to the most susceptible species to each insecticide. The lowest LC50 values were verified for A. gemmatalis to all insecticides tested. Chrysodeixis includens and most of the Spodoptera species were moderately tolerant to methoxyfenozide (TR < 8.0-fold) and indoxacarb (TR < 39.4-fold), whereas H. armigera was the most tolerant species to methoxyfenozide (TR = 21.5-fold), and indoxacarb (TR = 106.4-fold). Spodoptera cosmioides, S. eridania, and S. albula showed highest tolerance to spinetoram (TR > 1270-fold), S. eridania, S. frugiperda, and S. albula to flubendiamide (TR from 38- to 547-fold), and S. albula to indoxacarb (TR = 138.6-fold). A small variation in susceptibility to chlorfenapyr (TR < 4.4-fold) was found among the lepidopteran evaluated. Our findings indicate a large variation in susceptibility to indoxacarb, spinetoram, and flubendiamide and a relatively low variation in susceptibility to methoxyfenozide and chlorfenapyr by lepidopteran species of soybean, cotton, and maize from Brazil.

Susceptibility of Alabama argillacea and Chrysodeixis includens (Lepidoptera: Noctuidae) larvae to Beauveria bassiana associated with kaolin / Susceptibilidade de larvas de Alabama argillacea e Chrysodeixis includens (Lepidoptera: Noctuidae) a Beauveria bassiana associada com caulim

Abstract The mortality of the Alabama argillacea and Chrysodeixis includens (Lepidoptera: Noctuidae) larvae caused by the kaolin inert powder and the entomopathogenic fungus Beauveria bassiana were determined under laboratory conditions. Using the caterpillar submersion method, the CG 138 B. bassiana isolate was more pathogenic to A. argillacea than the CG 70, GC 82, ESALQ 634, and ESALQ 645. All five tested isolates caused similar mortality of C. includens. The mortality of first-instar larvae of A. argillacea and C. includens by feeding on leaf-disc impregnated with B. bassiana (CG 138) and kaolin was also determined. Higher A. argillacea mortalities were observed in the B. bassiana (CG 138) treatments, regardless of the presence of kaolin. However, the activity of kaolin + B. bassiana (CG 138) against C. includens was higher than each ingredient alone, indicating an additive action against C. includes larvae. The mortality of A. argillacea and C. includens larvae treated with kaolin + B. bassiana (CG 138) was similar, and the A. argillacea mortality was higher than that of C. includens with kaolin and B. bassiana (GC 138) separated. The treatment kaolin + B. bassiana (CG 138) is promising for the simultaneous management of these two defoliator pests, mainly A. includes. In addition, the monophagous A. argillacea is more susceptible to both kaolin and B. bassiana (GC 138) than the polyphagous C. includens, suggesting that the nutritional ecology plays an important role in the susceptibility of these defoliator species to alternative insecticides.

Resumo A mortalidade de larvas de Alabama argillacea e Chrysodeixis includens (Lepidoptera: Noctuidae), causada pelo pó inerte do caulim e pelo fungo entomopatogênico Beauveria bassiana, foi determinada em condições de laboratório. Usando o método de submersão da lagarta, o isolado CG 138 de B. bassiana foi mais patogênico para A. argillacea que os CG 70, GC 82, ESALQ 634 e ESALQ 645 desse fungo. Os cinco isolados testados causaram mortalidade semelhante de C. includens. A mortalidade de larvas de primeiro ínstar de A. argillacea e C. includens pelo método de alimentação em discos foliares impregnados com B. bassiana (CG 138) e caulim foi, também, determinada. A mortalidade de A. argillacea foi maior nos tratamentos com B. bassiana (CG 138), independentemente da presença do caulim. A atividade do caulim + B. bassiana (CG 138) contra C. includens foi maior que cada ingrediente isolado, indicando ação aditiva contra larvas desse Lepidoptera. A mortalidade de larvas de A. argillacea e C. includens, com caulim + B. bassiana (CG 138), foi semelhante e as de A. argillacea maiores que as de C. includens com caulim e B. bassiana (GC 138) isoladamente. O tratamento caulin + B. bassiana (CG 138) é promissor para o manejo simultâneo dessas duas pragas desfolhadoras, principalmente A. includens. Além disso, a monófaga, A. argilacea, é mais suscetível ao caulim e à B. bassiana (GC 138) que a polífaga, C. includens, sugerindo que a ecologia nutricional desempenha papel importante na suscetibilidade dessas espécies desfolhadoras a inseticidas alternativos.

Seasonal Flight Patterns of Chrysodeixis includens (Lepidoptera: Noctuidae) in the Florida Panhandle and Inventory of Plusiine Species Cross-Attracted to Synthetic Pheromone.

Chysodeixis includens (Walker) is a polyphagous economic pest in agricultural landscapes. To detect the occurrence of this pest in the field, trapping using sex pheromone lures is often implemented. However, other plusiine species are cross-attracted to these lures and may be misidentified as C. includens due to their morphological similarities. The objectives of this study were to provide region-specific information on the abundance of C. includens throughout the year as well as document the occurrence of related plusiines cross-attracted to C. includens sex pheromone traps in the Florida Panhandle. Twelve commercial fields of peanut and twelve commercial fields of cotton located across Escambia, Santa Rosa, and Jackson counties were monitored with Trécé delta traps baited with C. includens sex pheromone lures (Alpha Scents, Inc.; West Linn, OR) from June 2017 to June 2019. There was no difference in C. includens flight across dryland or irrigated fields. Identifications revealed that in addition to C. includens, the following species of the subfamily Plusiinae were crossed-attracted: Argyrogramma verruca (Fabricius) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), Ctenoplusia oxygramma (Geyer) (Lepidoptera: Noctuidae), and Rachiplusia ou (Guenée) (Lepidoptera: Noctuidae). The occurrence of each species in the region and their flight phenology are documented. Chysodeixis includens abundance was greatest in September and decreased through December. Due to the high abundance of C. oxygramma and similar flight phenology to C. includens, this is the likeliest species to skew estimations and influence management decisions of C. includens, especially early in the crop season, when C. includens abundance is low.

Evaluation of Reference Genes and Expression Level of Genes Potentially Involved in the Mode of Action of Cry1Ac and Cry1F in a Susceptible Reference Strain of Chrysodeixis includens.

Soybean looper (SBL), Chrysodeixis includens (Walker), is one of the major lepidopteran pests of soybean in the American continent. SBL control relies mostly on the use of insecticides and genetically modified crops expressing Bacillus thuringiensis (Bt) insecticidal Cry proteins. Due to the high selection pressure exerted by these control measures, resistance has developed to different insecticides and Bt proteins. Nevertheless, studies on the mechanistic background are still scarce. Here, the susceptibility of the laboratory SBL-Benzon strain to the Bt proteins Cry1Ac and Cry1F was determined in diet overlay assays and revealed a greater activity of Cry1Ac than Cry1F, thus confirming results obtained for other sensitive SBL strains. A reference gene study across larval stages with four candidate genes revealed that RPL10 and EF1 were the most stable genes for normalization of gene expression data obtained by RT-qPCR. Finally, the basal expression levels of eight potential Bt protein receptor genes in six larval instars were analyzed, including ATP-binding cassette (ABC) transporters, alkaline phosphatase, aminopeptidases, and cadherin. The results presented here provide fundamental knowledge to support future SBL resistance studies.

Optimization of Large-Scale Production of Chrysodeixis includens nucleopolyhedrovirus for Its Use as a Biopesticide.

The baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) is pathogenic to Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) larvae, known as soybean looper, which is an important pest of soybean and bean. In this study, some parameters were tested to overcome the difficulties in the in vivo production of ChinNPV aiming to increase its use as a biopesticide. First, different combinations of larval instars (3rd and 4th instars), larval incubation temperatures (23 °C and 26 °C), and rearing densities (individually and 10 larvae/cup) were compared for larval weight and the production of occlusion bodies (OBs). A positive correlation (p< 0.001) was observed for OB production and larval weight. Fourth instar larvae produced more OBs than third instar larvae (p<0.05); however, no significant differences in OBs/larva (p>0.05) were observed for larvae kept in groups or individually. Therefore, a second assay was performed using fourth instar larvae incubated at 26 °C and two larval densities (10 larvae/cup and 40 larvae/cup). The losses of insects and OB production were evaluated as well as the influence of storage temperatures post-mortem (-20 °C, 4 °C, and 15 °C) in the OB yield. As expected, insect losses due to cannibalism or microbial contamination were greater (p<0.05) with the increase in larval density, although no difference was observed in OBs/larva (p>0.05). In addition, the storage temperature post-mortem did not influence the OB yield (p>0.05). The average production of ChinNPV OBs was 3×1010 OBs/40 larvae cup. The results demonstrate the viability of rearing C. includens in groups to enhance the mass production and reduce virus production costs.

Distribution and invasion risk assessments of Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae) using CLIMEX.

Chrysodeixis includens is a polyphagous pest restricted to the American continent. The occurrence of C. includens is allied, among other factors, by favorable conditions such as temperature, humidity, presence of hosts, and migratory behavior. In this work, we built spatiotemporal species distribution models at continental and global levels for the distribution of C. includens using CLIMEX to determine times and regions favorable for year-round survival and migration of this species and in case of invasion on other continents to apply timely and right phytosanitary measures. Our models estimated high climate suitability for C. includens in Central and large proportions of South America throughout the year. Moreover, there is suitability for C. includens growth in all months of the year in Central and northern part of South America. In the northern hemisphere, these conditions range from April to October, while in mid-southern parts of South America, favorable periods comprise October through June. The countries with the highest suitability for C. includens outside the American continent are located on the African and Asian continents. Our results show variable climate suitability for C. includens during the year that help to understand likely migration pattern in North America. This information would direct efforts for appropriate C. includens management during warm and moist periods of the year. Furthermore, our models notify the need for the development of strategies for the inspection and interception of C. includens especially in central Africa, India, South and Southeast Asia, and Northeast Australia.

Transcriptome Analysis of Pyrethroid-Resistant Chrysodeixis includens (Lepidoptera: Noctuidae) Reveals Overexpression of Metabolic Detoxification Genes.

Chrysodeixis includens (Walker, [1858]) is one of the most important defoliator of soybean in Brazil because of its extensive geographical distribution and high tolerance to insecticides compared with other species of caterpillars. Because of this, we conducted bioassays to evaluate the efficacy of pyrethroid λ-cyhalothrin on a C. includens resistant strain (MS) and a susceptible (LAB) laboratory strain. High throughput RNA sequencing (RNA-seq) of larval head and body tissues were performed to identify potential molecular mechanisms underlying pyrethroid resistance. Insecticide bioassays showed that MS larvae exhibit 28.9-fold resistance to pyrethroid λ-cyhalothrin relative to LAB larvae. RNA-seq identified evidence of metabolic resistance in the head and body tissues: 15 cytochrome P450 transcripts of Cyp6, Cyp9, Cyp4, Cyp304, Cyp307, Cyp337, Cyp321 families, 7 glutathione-S-transferase (Gst) genes, 7 α-esterase genes from intracellular and secreted catalytic classes, and 8 UDP-glucuronosyltransferase (Ugt) were overexpressed in MS as compared with LAB larvae. We also identified overexpression of GPCR genes (CiGPCR64-like and CiGPCRMth2) in the head tissue. To validate RNA-seq results, we performed RT-qPCR to assay selected metabolic genes and confirmed their expression profiles. Specifically, CiCYP9a101v1, CiCYP6ae149, CiCYP6ae106v2, CiGSTe13, CiCOE47, and CiUGT33F21 exhibited significant overexpression in resistant MS larvae. In summary, our findings detailed potential mechanisms of metabolic detoxification underlying pyrethroid resistance in C. includens.

Performance of Genetically Modified Soybean Expressing the Cry1A.105, Cry2Ab2, and Cry1Ac Proteins Against Key Lepidopteran Pests in Brazil.

The pyramided genetically modified (GM) soybean [Glycine max L. (Merr.)] MON87751 × MON87708 × MON87701 × MON89788, expressing Cry1A.105, Cry2Ab2, and Cry1Ac from Bacillus thuringiensis Berliner, was approved for commercial use in Brazil. We conducted laboratory, greenhouse, and field studies to assess the efficacy of this Bt soybean against key soybean lepidopteran pests. Neonates of Anticarsia gemmatalis (Hübner) (Lepidoptera: Erebidae), Chrysodeixis includens (Walker), and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) were exposed to Bt proteins in diet-overlay bioassays. MON87751 × MON87708 × MON87701 × MON89788 soybean and individual components were evaluated in laboratory (leaf disc), greenhouse (high artificial infestations), and in field conditions (natural infestations). Neonates of A. gemmatalis, C. includens, and H. armigera were highly susceptible to Cry1A.105 (LC50 from 0.79 to 48.22 ng/cm2), Cry2Ab2 (LC50 from 1.24 to 8.36 ng/cm2), and Cry1Ac (LC50 from 0.15 to 5.07 ng/cm2) in diet-overlay bioassays. In laboratory leaf disc bioassays and greenhouse trials, MON87751 × MON87708 × MON87701 × MON89788 soybean as well as the individual components were highly effective in controlling A. gemmatalis, C. includens, and H. armigera. Similarly, under field conditions, the pyramided genotypes expressing Cry1A.105, Cry2Ab2, and Cry1Ac were highly effective at protecting soybean against C. includens. We concluded that the individual Bt proteins expressed by GM soybean MON87751 × MON87708 × MON87701 × MON89788 killed all or nearly all the susceptible A. gemmatalis, C. includens, and H. armigera, fulfilling one important criterion for successfully delaying resistance to pyramided Bt crops.

A Novel Alphabaculovirus from the Soybean Looper, Chrysodeixis includens, that Produces Tetrahedral Occlusion Bodies and Encodes Two Copies of he65.

Isolates of the alphabaculovirus species, Chrysodeixis includens nucleopolyhedrovirus, have been identified that produce polyhedral occlusion bodies and infect larvae of the soybean looper, Chrysodeixis includens. In this study, we report the discovery and characterization of a novel C. includens-infecting alphabaculovirus, Chrysodeixis includens nucleopolyhedrovirus #1 (ChinNPV#1), that produces tetrahedral occlusion bodies. In bioassays against C. includens larvae, ChinNPV #1 exhibited a degree of pathogenicity that was similar to that of other ChinNPV isolates, but killed larvae more slowly. The host range of ChinNPV#1 was found to be very narrow, with no indication of infection occurring in larvae of Trichoplusia ni and six other noctuid species. The ChinNPV#1 genome sequence was determined to be 130,540 bp, with 126 open reading frames (ORFs) annotated but containing no homologous repeat (hr) regions. Phylogenetic analysis placed ChinNPV#1 in a clade with other Group II alphabaculoviruses from hosts of lepidopteran subfamily Plusiinae, including Chrysodeixis chalcites nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus. A unique feature of the ChinNPV#1 genome was the presence of two full-length copies of the he65 ORF. The results indicate that ChinNPV#1 is related to, but distinct from, other ChinNPV isolates.