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Selection and Validation of Reference Genes for Gene Expression Analysis in Tuta absoluta Meyrick (Lepidoptera: Gelechiidae).

ABSTRACT: The tomato leaf miner, Tuta absoluta is a destructive pest of tomato. The leaf-mining activities of its larvae can cause significant yield losses. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used to measure gene expression, and the selection of stable reference genes for calibration and standardization is critical for accurate use of RT-qPCR. We studied the stable expression of nine common housekeeping genes in T. absoluta. These were examined at different developmental stages, in larval tissues, as well as those induced by exposure to 20E and insecticides. Four dedicated algorithms (geNorm, BestKeeper, NormFinder, and ΔCt method) and online tool (RefFinder) were used to analyze and rank the tested reference genes. Based on the standardized gene expression data of target gene ecdysone receptor (EcR), the applicability of specific reference genes was verified. The results clarify that the optimal internal reference genes vary greatly under different experimental conditions. GAPDH and RPS11 were the best reference genes for developmental stages; RPL28 and RPL10 for different tissues; EF1α and RPL28 for 20E treatment; EF1α and RPL7A for insecticide treatments. The most suitable reference genes in all experimental conditions are EF1α and RPL28.

SUBMITTER: Yan X

PROVIDER: S-EPMC8305163 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tomato (Solanum esculentum) is one of the vegetable crops grown by both smallholder and commercial farmers in the Kingdom of Eswatini. Tomato leaf miner, Tuta absoluta M. is a major insect pest of tomatoes resulting in reduced tomato yields throughout the country. The study investigated the virulence of two sub-tropical EPN species on T. absoluta larvae. Steinernema yirgalemense and S. jeffreyense at different concentrations (0, 20, 40, 60 IJs/insect) were screened for efficacy (i.e. mortality rate) against larvae of T. absoluta in laboratory bioassays. Results obtained showed that S. yirgalemense and S. jeffreyense were able to kill T. absoluta larvae without significant differences between the two EPN species in 24-well bioassay trays. Significantly higher (p?<?0.05) mortality was observed when 60 IJs/insect was used. The combination of S. yirgalemense at 60 IJs/insect (100%) and S. jeffreyense at 60 IJs/insect (98.3%) resulted in significantly higher (p?<?0.05) mortality, compared with the other four combinations of EPN and concentration tested. In the leaf bioassays, S. yirgalemense (58.8%) resulted in significantly higher (p?<?0.05) mean mortality compared to S. jeffreyense (46.4%). Steinernema yirgalemense at 60 IJs/insect resulted in significantly higher mean mortality compared to the other EPN and concentration combinations in the leaf bioassay. The results indicated that both EPNs tested were effective against T. absoluta larvae. Steinernema yirgalemense at 60 IJs/insect can effectively find T. absoluta larvae inside leaf mines, but large-scale field trials are recommended to demonstrate the potential use of the biocontrol agent within integrated pest management programs.Tomato (Solanum esculentum) is one of the vegetable crops grown by both smallholder and commercial farmers in the Kingdom of Eswatini. Tomato leaf miner, Tuta absoluta M. is a major insect pest of tomatoes resulting in reduced tomato yields throughout the country. The study investigated the virulence of two sub-tropical EPN species on T. absoluta larvae. Steinernema yirgalemense and S. jeffreyense at different concentrations (0, 20, 40, 60 IJs/insect) were screened for efficacy (i.e. mortality rate) against larvae of T. absoluta in laboratory bioassays. Results obtained showed that S. yirgalemense and S. jeffreyense were able to kill T. absoluta larvae without significant differences between the two EPN species in 24-well bioassay trays. Significantly higher (p?<?0.05) mortality was observed when 60 IJs/insect was used. The combination of S. yirgalemense at 60 IJs/insect (100%) and S. jeffreyense at 60 IJs/insect (98.3%) resulted in significantly higher (p?<?0.05) mortality, compared with the other four combinations of EPN and concentration tested. In the leaf bioassays, S. yirgalemense (58.8%) resulted in significantly higher (p?<?0.05) mean mortality compared to S. jeffreyense (46.4%). Steinernema yirgalemense at 60 IJs/insect resulted in significantly higher mean mortality compared to the other EPN and concentration combinations in the leaf bioassay. The results indicated that both EPNs tested were effective against T. absoluta larvae. Steinernema yirgalemense at 60 IJs/insect can effectively find T. absoluta larvae inside leaf mines, but large-scale field trials are recommended to demonstrate the potential use of the biocontrol agent within integrated pest management programs.

Project description:(1) Background: The tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae) is a key tomato insect pest. At present, it is considered to be a serious threat in various countries in Europe, North Africa, and Middle East. The extensive use and the developed resistance of T. absoluta to spinosad causes some concern, which leads to the need for alternative products. (2) Materials and Methods: Several laboratory experiments were conducted to investigate the ovicidal properties of a zeolite particle film on T. absoluta. The toxicity of three different zeolites and six zeolite formulations to T. absoluta eggs and larvae was determined using different exposure methods. (3) Results: In general, the formulated zeolites yielded higher egg and larvae mortality values, especially when the zeolite particle film was residually applied. Notable differences in mortality rates from exposure to zeolites compared to other products, such as kaolin, its formulated product Surround, and the insecticide spinosad, were observed. Kaolin and Surround exhibited little or no effect for both application methods, while the hatch rate was reduced by 95% when spinosad was applied topically. Spinosad yielded egg and larvae mortality rates of 100% for both application methods. Additionally, increased oviposition activity was observed in adults exposed to the wettable powder (WP) formulations. These WP formulations increased egg deposition, while Surround and spinosad elicited a negative oviposition response. (4) Conclusions: It can be derived that the tested products, zeolites BEA (Beta polymorph A), FAU (Faujasite), LTA (Linde type A), and their formulations, had no real insecticidal activity against the eggs of T. absoluta. Nevertheless, egg exposure to zeolites seemed to affect the development process by weakening the first instar larvae and increasing their mortality. Subsequently, based on the choice test, no significant difference was observed between the number of eggs laid on the treated leaves and control leaves.

Project description:Entomopathogenic nematodes (EPN) are excellent biocontrol agents against various insect pests. Novel biotechnological approaches can enhance their utility against insects above-ground, opening a new venue for selecting superior EPN against certain insects. We hypothesize that different populations of the same species but from different origins (habitat, ecoregion) will differ in their virulence. This study aimed to evaluate the virulence of various EPN populations against two pests of worldwide incidence and damage to high value crops: Frankliniella occidentalis (Thysanoptera: Thripidae) and Tuta absoluta (Lepidoptera: Gelechiidae). We tested 10 EPN populations belonging to three EPN species: Heterorhabditis bacteriophora (Koppert, MG-618b, AM-203, RM-102), Steinernema feltiae (Koppert, RS-5, AM-25, RM-107), and Steinernema carpocapsae (Koppert, MG-596a). Each EPN population was tested at two concentrations. Frankliniella occidentalis was tested at 160 and 80 IJs/cm2 and T. absoluta at 21 and 4 IJs/cm2. Control treatments followed the same experimental procedure but only adding distilled water. Overall, whenever different, higher IJs concentration resulted in lower adult emergence, higher larval mortality, and shorter time to kill the insects. Considering the low concentration, S. feltiae provided the best results for both insects and instars investigated, while H. bacteriophora and S. carpocapsae required a high concentration to reach similar or slightly better results. Differences among populations of each of the species were detected, but only the native populations of H. bacteriophora populations showed consistently higher control values against both insects/instar compared with the commercial one. Differences among S. feltiae and S. carpocapsae populations depended on the IJs concentration, insect, and instar. We consider S. feltiae a very promising species for their application against F. occidentalis and T. absoluta, with the Koppert population as the most consistent among the populations tested. Specific EPN-populations of S. carpocapsae and H. bacteriophora were good candidates against certain instar/insects at high concentrations. This study emphasized the importance of intraspecific variability for EPN virulence.

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Selection and Validation of Reference Genes for Gene Expression Analysis in Tuta absoluta Meyrick (Lepidoptera: Gelechiidae).

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