Accurate identification of Helicoverpa armigera-Helicoverpa zea hybrids using genome admixture analysis: implications for genomic surveillance.

Helicoverpa armigera, the cotton bollworm moth, is one of the world's most important crop pests, and is spreading throughout the New World from its original range in the Old World. In Brazil, invasive H. armigera has been reported to hybridize with local populations of Helicoverpa zea. The correct identification of H. armigera-H. zea hybrids is important in understanding the origin, spread and future outlook for New World regions that are affected by outbreaks, given that hybridization can potentially facilitate H. zea pesticide resistance and host plant range via introgression of H. armigera genes. Here, we present a genome admixture analysis of high quality genome sequences generated from two H. armigera-H. zea F1 hybrids generated in two different labs. Our admixture pipeline predicts 48.8% and 48.9% H. armigera for the two F1 hybrids, confirming its accuracy. Genome sequences from five H. zea and one H. armigera that were generated as part of the study show no evidence of hybridization. Interestingly, we show that four H. zea genomes generated from a previous study are predicted to possess a proportion of H. armigera genetic material. Using unsupervised clustering to identify non-hybridized H. armigera and H. zea genomes, 8511 ancestry informative markers (AIMs) were identified. Their relative frequencies are consistent with a minor H. armigera component in the four genomes, however its origin remains to be established. We show that the size and quality of genomic reference datasets are critical for accurate hybridization prediction. Consequently, we discuss potential pitfalls in genome admixture analysis of H. armigera-H. zea hybrids, and suggest measures that will improve such analyses.

Fungal identification in peanuts seeds through multispectral images: Technological advances to enhance sanitary quality.

The sanitary quality of seed is essential in agriculture. This is because pathogenic fungi compromise seed physiological quality and prevent the formation of plants in the field, which causes losses to farmers. Multispectral images technologies coupled with machine learning algorithms can optimize the identification of healthy peanut seeds, greatly improving the sanitary quality. The objective was to verify whether multispectral images technologies and artificial intelligence tools are effective for discriminating pathogenic fungi in tropical peanut seeds. For this purpose, dry peanut seeds infected by fungi (A. flavus, A. niger, Penicillium sp., and Rhizopus sp.) were used to acquire images at different wavelengths (365 to 970 nm). Multispectral markers of peanut seed health quality were found. The incubation period of 216 h was the one that most contributed to discriminating healthy seeds from those containing fungi through multispectral images. Texture (Percent Run), color (CIELab L*) and reflectance (490 nm) were highly effective in discriminating the sanitary quality of peanut seeds. Machine learning algorithms (LDA, MLP, RF, and SVM) demonstrated high accuracy in autonomous detection of seed health status (90 to 100%). Thus, multispectral images coupled with machine learning algorithms are effective for screening peanut seeds with superior sanitary quality.

Improvement of the Mass-Rearing Protocols for the South American Fruit Fly for Application of the Sterile Insect Technique.

The existing rearing protocols for Anastrepha fraterculus must be reviewed to make economically viable the production of sterile flies for their area-wide application. Additionally, evidence of sexual incompatibility between a long-term mass-reared Brazilian strain and wild populations has been found. To address these issues, this study aimed to refine rearing protocols and to assess the suitability of an A. fraterculus strain for the mass production of sterile flies. A series of bioassays were carried out to evaluate incubation times for eggs in a bubbling bath and to assess the temporal variation of egg production from ovipositing cages at different adult densities. A novel larval diet containing carrageenan was also evaluated. Egg incubation times higher than 48 h in water at 25 °C showed reduced larval and pupal yields. Based on egg production and hatchability, the density of 0.3 flies/cm2 can be recommended for adult cages. The diet with carrageenan was suitable for mass production at egg-seeding densities between 1.0 and 1.5 mL of eggs/kg of diet, providing higher insect yields than a corn-based diet from Embrapa. Even after two years of being reared under the new rearing protocols, no sexual isolation was found between the bisexual strain and wild flies.

Convolutional Neural Networks Using Enhanced Radiographs for Real-Time Detection of Sitophilus zeamais in Maize Grain.

The application of artificial intelligence (AI) such as deep learning in the quality control of grains has the potential to assist analysts in decision making and improving procedures. Advanced technologies based on X-ray imaging provide markedly easier ways to control insect infestation of stored products, regardless of whether the quality features are visible on the surface of the grains. Here, we applied contrast enhancement algorithms based on peripheral equalization and calcification emphasis on X-ray images to improve the detection of Sitophilus zeamais in maize grains. In addition, we proposed an approach based on convolutional neural networks (CNNs) to identity non-infested and infested classes using three different architectures; (i) Inception-ResNet-v2, (ii) Xception and (iii) MobileNetV2. In general, the prediction models developed based on the MobileNetV2 and Xception architectures achieved higher accuracy (≥0.88) in identifying non-infested grains and grains infested by maize weevil, with a correct classification from 0.78 to 1.00 for validation and test sets. Hence, the proposed approach using enhanced radiographs has the potential to provide precise control of Sitophilus zeamais for safe human consumption of maize grains. The proposed method can automatically recognize food contaminated with hidden storage pests without manual features, which makes it more reliable for grain inspection.

Determining the Sterilization Doses under Hypoxia for the Novel Black Pupae Genetic Sexing Strain of Anastrepha fraterculus (Diptera, Tephritidae).

A common strategy used to maintain sterile fly quality without sacrificing sterility is to irradiate the insects under an oxygen-reduced atmosphere. So far, sterilizing doses for the South American fruit fly Anastrepha fraterculus have only been determined under normoxia. Our study reports for the first time the dose-sterility response under hypoxia for two different A. fraterculus strains. The pupae were derived from a bisexual strain (a Brazilian-1 population) and a recently developed genetic sexing strain (GSS-89). Two hours prior to irradiation, pupae were transferred to sealed glass bottles and irradiated when oxygen concentration was below 3%. Four types of crosses with nonirradiated flies of the bisexual strain were set to assess sterility for each radiation dose. For males from both strains, Weibull dose-response curves between radiation doses and the proportion of egg hatch, egg-to-pupa recovery, and recovery of adults were determined. The GSS males revealed high sterility/mortality levels compared to males from the bisexual strain at doses < 40 Gy, but a dose of 74 Gy reduced egg hatch by 99% regardless of the male strain and was considered the sterilizing dose. The fertility of irradiated females was severely affected even at low doses under hypoxia.

Cafeteria-Type Feeding of Chickens Indicates a Preference for Insect (Tenebrio molitor) Larvae Meal.

This study aimed to determine whether broiler chickens display a preference for Tenebrio molitor larvae (TM) meal by evaluating ingredient acceptability and birds' performance. Sixty 14-day-old male chickens were assigned into two treatment groups (5 birds/pen, n = 6) in a cafeteria-type study: the control (C) group, and the TM group. Each pen was equipped with one bell drinker and four through feeders allocated side by side; all feeders of the C group contained a complete standard diet whereas each feeder of the TM group contained one of the following ingredients: ground corn, extruded semi-whole soybean, vitamin-mineral supplement mixture, and TM meal. Feed intake was recorded daily and growth was monitored periodically up to day 32. Chickens which had access to individual feed components showed a delay to display preference for TM, but consumed, overall, up to 50% of the total intake as TM meal. Feed intake and growth performance were lower in all periods for TM group (p < 0.02), whereas feed conversion ratio was improved on days 22-28 and days 29-32 of age (p < 0.01). Data from bivariate and multidimensional analysis indicate that birds started to reach a balance of ingredient intake at 25 days of age, showing a high correlation between consumption of each ingredient and the day of the experiment. Chickens exhibited a preference for T. molitor meal, resulting in improved feed efficiency, which allows us to conclude that it can be a suitable feed alternative for poultry.

Invasion origin, rapid population expansion, and the lack of genetic structure of cotton bollworm (Helicoverpa armigera) in the Americas.

In 2013, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was officially declared as present in Brazil and, after two years, the species was detected in the Caribbean and North America. Information on genetic features and accurate distribution of pests is the basis for agricultural protection policies. Furthermore, such knowledge is imperative to develop control strategies, understand the geographical range, and genetic patterns of this species in the Americas. Here, we carried out the widest sampling of H. armigera in the South American continent and Puerto Rico, after we estimated the diversity, demographic parameters, and genetic structure. The Internal Transcribed Spacer 1 (ITS1) nuclear marker was used to investigate the presence of putative hybrids between H. armigera and H. zea, and they were observed at a frequency of 1.5%. An ABC analysis, based in COI gene fragment, suggested Europe as the origin of South America specimens of H. armigeraand following a movement northward through the Caribbean. Three mtDNA genes and three nDNA markers revealed high genetic diversity distributed without the defined population structure of H. armigera in South America. Most of the genetic variation is within populations with a multidirectional expansion of H. armigera among morphoclimatic regions. High genetic diversity, rapid population expansion, and hybridization have implications for pest management since they suggest that adaptive alleles are spread through wide areas in South America that favor rapid local adaptation of H. armigera to new and disturbed environments (e.g., in agricultural areas).

Optimization of the sterilizing doses and overflooding ratios for the South American fruit fly.

The Sterile Insect Technique (SIT) is an autocidal control method that relies on inundative releases of sterilized mass-reared insects. This technology has been used in several area-wide programmes for the suppression/eradication of fruit fly populations. Choosing the optimum sterilizing dose and the sterile release density is an essential step of the SIT. Considering unsolved issues related to the application of this technique against Anastrepha fraterculus (Wiedemann), this study aimed to define accurately the central target dose for both sexes of this species and to verify the induction of sterility in fertile flies at different sterile:fertile ratios. The results from the regression analyses proved that the sterilization process for the A. fraterculus Brazilian-1 morphotype (the most common in southern Brazil and Argentina) could consist of irradiating pupae 72 h before adult emergence at 40 Gy, with no detrimental effects to standard quality control parameters. The ovarian development in irradiated females was characterized, demonstrating that doses equal to or higher than 25 Gy cause complete and irreversible ovarian atrophy. The laboratory and field cage tests showed that the sterility induction increased with the proportion of sterile flies, and a sterile:fertile ratio of 50:1 should be appropriate in SIT field trials. The sterile females apparently did not distract the sterile males, despite of the slightly higher reductions in pupal yield for all ratios in their absence. The data generated in this study have a great practical value and will help decision-makers in planning field trials to evaluate the efficacy of the SIT against A. fraterculus populations.

Can stable isotope markers be used to distinguish wild and mass-reared Anastrepha fraterculus flies?

The availability of accurate techniques to discriminate between marked laboratory-reared flies and unmarked wild flies captured in monitoring traps is essential for programs that integrate the Sterile Insect Technique (SIT) to manage fruit flies. In this study, the feasibility of using a stable isotope marking technique for the South American fruit fly, Anastrepha fraterculus (Wiedemann), was assessed. Wild flies were collected from apple orchards, which are a target of a SIT project in southern Brazil. To verify if adult flies could be labelled by the stable isotopes from larval diets, larvae were reared on two different C4-based diets and fruits in laboratory. To evaluate the influence of the two most common attractants applied to capture A. fraterculus (grape juice and CeraTrapTM) and the most common preservation method in fruit fly collections (ethanol), laboratory-reared flies were immersed in McPhail traps containing the respective treatments for two periods of time. Samples were analyzed in an elemental analyzer coupled to a Continuous Flow Isotope Ratio Mass Spectrometer (CF-IRMS) at CENA/USP. The δ13C signatures of flies reared on artificial diets differed significantly from the δ13C of flies whose larvae were reared on fruits and from wild flies. In contrast, the δ15N values were less conclusive and the technique could not rely solely on them. In all cases considered, the δ13C and δ15N signatures from males did not differ from females. Despite the alterations caused by the attractants tested and ethanol, laboratory-flies could be distinguished from the wild ones based on δ13C signatures. This is the first comprehensive study to demonstrate that it is possible to distinguish wild A. fraterculus from flies reared on larval diets containing C4 sugar. The first experimentally derived trophic discrimination factors were also obtained for this species. Thus, intrinsic isotope labelling can serve as a backup to conventional dye marking.

Applying spatial analysis of genetic and environmental data to predict connection corridors to the New World screwworm populations in South America.

The myiasis causing New World screwworm (NWS) fly is responsible for substantial losses to livestock breeders in the Americas. Due to the negative impact of the NWS fly in animal health, expansion of successful NWS fly eradication programmes is under discussion. However, the effects of geography and environmental diversity on NWS population structure and migration patterns need to be assessed before any political decision is made to implement such a programme. We present a GIS tool to construct potential connection corridors among sampling localities based on genetic and environmental data. We integrate, through a home-made python script, a friction raster based on a Maxent niche model and the pairwise ΦST statistic. Among 38 NWS fly sampling localities from South America, we find a high population connectivity among the sampling localities from the south of the Amazon region. The region along the Atlantic Ocean was identified as the most probable migration corridor between the north (NAG) and the south (SAG) of the Amazon region. The approach highlighted previously undetected population structure within NAG showing low to medium connectivity through the Andes, correlating with current understanding of NWS fly migration in South America. Also, the approach is flexible, allowing future research to incorporate other niche simulations and genetic differentiation metrics. With this flexibility, the tool could become part of any AW-IPM by helping to target regions for control.

Genetic diversity and population structure of the New World screwworm fly from the Amazon region of Brazil.

Cochliomyia hominivorax (Coquerel) is a myiasis fly that causes economic losses to livestock farmers in warmer American regions. Previous studies of this pest had found population structure at north and south of the Amazon Basin, which was considered to be a barrier to dispersal. The present study analyzed three mitochondrial DNA (mtDNA) markers and eight nuclear microsatellite loci to investigate for the first time the genetic diversity and population structure across the Brazilian Amazon region (Amazonia). Both mtDNA and microsatellite data supported the existence of much diversity and significant population structure among nine regional populations of C. hominivorax, which was found to be surprisingly common in Amazonia. Forty-six mtDNA haplotypes were identified, of which 39 were novel and seven had previously been found only at south of Amazonia. Seventy microsatellite alleles were identified by size, moderate to high values of heterozygosity were discovered in all regions, and a Bayesian clustering analysis identified four genetic groups that were not geographically distributed. Reproductive compatibility was also investigated by laboratory crossing, but no evidence of hybrid dysgenesis was found between an Amazonian colony and one each of from Northeast and Southeast Brazil. The results have important implications for area-wide control by the Sterile Insect Technique.

Dietas larvais alternativas para criação massal da mosca da bicheira, Cochliomyia hominivorax / Alternative larval diets for mass rearing of screwworm, Cochliomyia hominivorax

A criação massal da mosca da bicheira, Cochliomyia hominivorax (Diptera: Calliphoridae), para programas que utilizam a Técnica do Inseto Estéril nas Américas faz parte de um dos maiores programas do mundo de criação de insetos em larga escala. A maior parte do custo e do trabalho necessários para a produção massal está relacionada com a dieta larval, o que torna importante o seu aprimoramento levando-se em conta sua eficiência, custo e praticidade. Neste trabalho, foram realizados dois bioensaios com dietas alternativas feitas a partir de produtos disponíveis no mercado brasileiro com o objetivo de avaliar a viabilidade e eficiência dessas dietas para criações de mosca da bicheira no Brasil. Foram testadas duas dietas líquidas (tendo como bulking agents bagaço de cana ou fibra de coco), uma de carne, e outra de gel. Os parâmetros biológicos (peso das larvas, viabilidade larval, peso de pupas, porcentagem de emergência de adultos e razão sexual) estiveram, em todos os casos, dentro de parâmetros satisfatórios para a criação laboratorial de C. hominivorax e as dietas não diferiram estatisticamente entre si. Devido a vantagens importantes, como alta viabilidade larval e menor custo, recomenda-se a dieta de gel para as criações de larvas da mosca da bicheira no Brasil.

The mass rearing of the screwworm fly, Cochliomyia hominivorax (Diptera: Calliphoridae), for programs that integrate the Sterile Insect Technique in the Americas is part of one of the largest insect mass rearing programs in the world. Most of the cost and labor required in mass rearing systems is related to the larval diet, what enhances the relevance of the constant development of novel diets taking into account their effectiveness, cost and practicability. In this research, two bioassays with alternative diets made using local products were performed in order to evaluate the feasibility and efficiency of these diets for screwworm rearing in Brazil. Two liquid diets (having sugarcane bagasse or coconut fiber as bulking agents), a meat diet, and a gelled diet were tested. The biological parameters evaluated (larval weight, larval viability, pupal weight, adult emergence, and sex ratio) were satisfactory according to mass rearing standards and the diets did not differ statistically (P > 0.05). Due to its higher number of advantages, as high larval viability and lower cost, the gelled diet developed is recommended for screwworm larvae rearing in Brazil.

Determinação da temperatura base para o desenvolvimento embrionário da mosca-da-bicheira / Assessment of the threshold temperature for the embryonic development of the screwworm fly

Após sucessivas campanhas de erradicação da mosca-da-bicheira, Cochliomyia hominivorax (Coquerel), através da utilização de insetos estéreis, sua distribuição geográfica atual abrange apenas áreas do Caribe e dos países da América do Sul, com exceção do Chile. Para o planejamento de novos programas de controle nos países do MERCOSUL, deve-se contar com o máximo de informações possíveis relacionadas à ecologia desse parasita, como suas exigências térmicas. O objetivo deste estudo foi determinar a constante térmica (K), as temperaturas base (Tb), ótima (To) e a influência de temperaturas entre 15 e 35°C sobre a duração do período embrionário de C. hominivorax. Depois de separados das massas, os ovos foram inoculados sobre papel de filtro umedecido em placas de Petri, as quais foram colocadas em câmaras climatizadas do tipo B.O.D. Considerando o menor período de desenvolvimento e máxima viabilidade de ovos, a temperatura ótima foi estimada em 35°C. A temperatura base foi estimada em 13,1°C, com uma constante térmica de 9,17GD.

After successful eradication campaigns against the screwworm fly, Cochliomyia hominivorax (Coquerel), through the release of sterile insects, its current geographical distribution comprehends only some Caribbean islands and South American countries, except Chile. Before planning new control programs in MERCOSUR countries, information related to the ecology of this parasite, as its thermal requirements, are necessary. This research aimed to assess the thermal constant (K), the threshold (Tb) and optimal (To) temperatures, as the influence of temperature between 15 and 35°C over the length of the embryonic period of C. hominivorax. Eggs were separated from the masses and inoculated on moistened filter paper in Petri dishes, which were held in acclimatized chambers. Considering the shortest development period and the highest egg hatch values, the optimal temperature was 35°C. The threshold temperature was 13.1°C and the estimated thermal constant was 9.17 degrees-days.

The host marking pheromone application on the management of fruit flies - a review

The aim of this work was to review the role of the host marking pheromone (HMP) and its application in integrated management programs for the fruit flies. Initially the oviposition behavior of tephritids has been analyzed with emphasis on Ceratitis capitata. The deposition of HMP, which consists in the last stage of the oviposition behavior has been characterized and discussed about evolutive aspects and the biological meaning of the tephritidae communication through the HMP. Finally, the perspectives on the use of HMP in the integrated management of fruit flies have been discussed.

An Overview of the Components of AW-IPM Campaigns against the New World Screwworm.

The New World Screwworm, Cochliomyia hominivorax (Coquerel), is one of the most damaging parasites of livestock, causing millions of dollars in annual losses to producers. The fly is an obligate parasite of warm-blooded animals, including humans. After a successful 50-year eradication campaign, C. hominivorax has been eradicated from the USA, Mexico and Central America by an area-wide integrated pest management approach. Recently, Caribbean and South American countries have expressed an interest in this approach. Aiming to support forthcoming projects in these countries, this review describes the main technical components of past and ongoing AW-IPM campaigns against C. hominivorax.