Six-year monitoring of pesticide resistance in the Colorado potato beetle (Leptinotarsa decemlineata Say) during a neonicotinoid restriction period.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is an important potato pest with known resistance to pyrethroids and organophosphates in Czechia. Decreased efficacy of neonicotinoids has been observed in last decade. After the restriction of using chlorpyrifos, thiacloprid and thiamethoxam by EU regulation, growers seek for information about the resistance of CPB to used insecticides and recommended antiresistant strategies. The development of CPB resistance to selected insecticides was evaluated in bioassays in 69 local populations from Czechia in 2017-2022 and in 2007-2022 in small plot experiments in Zabcice in South Moravia. The mortality in each subpopulation in the bioassays was evaluated at the field-recommended rates of insecticides to estimate the 50% and 90% lethal concentrations (LC50 and LC90, respectively). High levels of CPB resistance to lambda-cyhalothrin and chlorpyrifos were demonstrated throughout Czechia, without significant changes between years and regions. The average mortality after application of the field-recommended rate of lambda-cyhalothrin was influenced by temperature before larvae were sampled for bioassays and decreased with increasing temperature in June. Downwards trends in the LC90 values of chlorpyrifos and the average mortality after application of the field-recommended rate of acetamiprid in the bioassay were recorded over a 6-year period. The baseline LC50 value (with 95% confidence limit) of 0.04 mg/L of chlorantraniliprole was established for Czech populations of CPBs for the purpose of resistance monitoring in the next years. Widespread resistance to pyrethroids, organophosphates and neonicotinoids was demonstrated, and changes in anti-resistant strategies to control CPBs were discussed.

Bias in sex ratios and polyandry rate in reproduction of Leptinotarsa decemlineata.

The Colorado potato beetle (CPB, Leptinotarsa decemlineata Slechtd.) is an invasive pest with economic importance worldwide. Sex ratios during egg-hatching and a frequency of polyandry in single-female families were analysed to clarify the reproduction strategy of CPB, which was still known only in fragments. 1296 just hatching 1st instar CPB larvae were collected from 19 single-female families, of which 13 were random families collected from potato fields and 6 were families produced by laboratory farming of naturally fertilised females. All larvae were analysed to detect a sex using a qPCR-based method and to detect polymorphisms in genotypes of 9 microsatellite (SSR) markers. The bias in sex ratio in favour of females was confirmed using linear mixed-effects model in both experimental groups of families: field collections (F = 36.39; P = 0.0001) and laboratory farming (F = 13.74; P = 0.0139). The analysis of diversity in microsatellites proved the polyandry in all progenies as 73% of analysed segregation patterns did not match with the patterns expected for full-sib progenies; on average per locus, 46% of allelic and 49.7% of genotype ratios showed irregular segregation. Both findings contribute toward understanding CPB success rate as an invasive species, as the preferential bearing of females with polyandry has a great potential to keep fitness of progenies, to maintain and operate population diversity, and to accelerate the reproduction of the pest.

Phenotypic, molecular and biochemical evaluation of somatic hybrids between Solanum tuberosum and S. bulbocastanum.

Somatic hybridization has been frequently used to overcome sexual incompatibility between potato and its secondary germplasm. The primary objective of this study was to produce and evaluate somatic hybrids of Solanum tuberosum (Stub) and S. bulbocastanum (Sblb) for breeding purposes. In 2007, 23 somatic hybrids were produced using an electrofusion of mesophyll protoplasts of diploid (2n = 2x = 24) potato line StubDH165 and S. bulbocastanum PI24351 (Sblb66). Phenotype of somatic hybrids in field conditions were evaluated, together with constitution and stability of 30 nuclear (ncSSR) and 27 cytoplasmic (cpSSR) microsatellite markers and content of main glycoalkaloids. All somatic hybrids had very high field resistance against late blight, but the plants were infertile: the viability of pollen grains insignificantly varied between 0.58 and 8.97%. A significant somaclonal variation was observed in terms of the morphology of plants, the date of emergence, the quantity of harvested tubers, the content of glycoalkaloids in foliage, and nuclear microsatellite markers (ncSSR). The analysis of ncSSR identified five distinct genotypes of hybrids partly associated with phenotype variations. The process of somatic hybridization with regeneration of shoots was identified as the most likely source of somaclonal variation because the ncSSR genotypes of hybrids, which were maintained in vitro, remained stable for more than 10 years. The infertile somatic hybrids have no practical breeding potential, but they are considered very suitable for advanced studies of the differential expression of genes in the pathways linked to dormancy of tubers and synthesis of glycoalkaloids.

Detection of sex in adults and larvae of Leptinotarsa decemlineata on principle of copy number variation.

The identification of sex in larvae of insects is usually challenging or even impossible, while in adults the sexual dimorphism is usually evident. Here, we used copy number analysis to develop a method of sex detection in Colorado potato beetle (Leptinotarsa decemlineata), which has an X0 sex determination system. The X linked gene LdVssc and autosomal gene LdUBE3B were identified as appropriate target and reference loci, respectively. The copy numbers (CNV) of LdVssc in males and females were estimated using standard droplet digital PCR (ddPCR) and real-time PCR (qPCR). With both methods, CNVs were bimodally distributed (BAddPCR = 0.709 and BAqPCR = 0.683) with 100% ability to distinguish females from males. The use of qPCR-based sex detection in a broad collection of 448 random CPB adults showed a perfect association (Phi = 1.0, p < 0.05) with the true sexes of adults, with mean CNV in females of 2.032 (SD = 0.227) and 0.989 in males (SD = 0.147). In the collection of 50 random 4th instar larvae, 27 females and 23 males were identified, consistent with the expected 1:1 sex ratio (p = 0.689). The method is suitable for sexing in all stages of ontogenesis. The optimal cost-effective application of the method in large populations requires the DNA extraction using CTAB, the qPCR assay in one biological replicate and three technical replicates of each marker, and the use of one randomly chosen male per run to calibrate calculation of CNV.

Reliable molecular differentiation of Trichuris ovis and Trichuris discolor from sheep (Ovis orientalis aries) and roe deer (Capreolus capreolus) and morphological characterisation of their females: morphology does not work sufficiently.

The main aim of the study was to evaluate associations between morphological variability of Trichuris females from sheep and roe deer and their rDNA polymorphism in whipworm populations from the Czech Republic. The results introduced the use of new molecular markers based on the internal transcribed spacer (ITS)1-5.8S RNA-ITS2 region polymorphisms, as useful tools for the unambiguous differentiation of congeners Trichuris ovis and Trichuris discolor. These markers revealed both parasites in roe deer and in sheep; however, T. ovis females predominated in sheep while T. discolor females occurred mostly in roe deer. Additional analysis of ITS1-5.8 rRNA-ITS2 discovered the genetic uniformity of the analysed T. discolor but high haplotype variation of T. ovis. Simultaneously, molecularly designated female individuals of both species were categorised into four morphotypes (MT) on the basis of morphology of genital pore area. MT1 and MT4 (vulvar opening on everted vaginal appendage/on visible cuticular bulge) occurred only in T. ovis, MT2 (uneverted vagina-vulvar opening without any elevation) was identified only in T. discolor and MT3 (transient type of vulvar opening on a small swelling) was observed in both species. Statistical analysis of biometric data confirmed that morphology of vulva is not a reliable marker for the species determination. On the basis of the ITS1-5.8S RNA-ITS2 region variability, we carried out a phylogenetic analysis (maximum likelihood method, Hasegawa-Kishino-Yano model) which showed that T. ovis haplotypes from the Czech Republic and Ireland and T. discolor haplotypes from the Czech Republic, Spain, Iran and Japan are sister OTUs.