Béla Péter Molnár

Host finding behaviour and host acceptance for oviposition is a rather complex process amongst insects; it is a major decision that will have a direct effect on the performance of offspring. It is not uncommon that herbivorous insects oviposit on unsuitable hosts. The level of insect-host specialisation correlates with the likelihood of mis-oviposition incidence; polyphagous herbivore insect species have a higher chance to lay eggs on unsuitable hosts compared to specialised monophagous ones. Over several seasons, large numbers of walnut husk fly adults were observed on the canopy and fruit of European quince. Males were engaging in behaviours like fighting, courting, mating, and guarding oviposition sites, while females copulated, oviposited, or rested. Oviposition marks were found on quince fruits, with unhatched eggs, chorions, and newly hatched maggots inside. Molecular identification confirmed the species as Rhagoletis completa. To investigate this unusual host interaction, electrophysiological recordings were conducted on the antennae and maxillary palpi of WHF adults using quince and walnut fruit and foliage volatiles. Behavioural experiments also explored any preference for quince. The results showed that WHF adults, especially females, exhibited a positive attraction to quince fruit volatiles, indicating that their peripheral detection of volatiles is not limited to host-specific compounds. Based on these findings, we conclude that WHF adults treated quince as a suitable host for oviposition in a natural condition.

The selection of oviposition sites by female moths is crucial in shaping their progeny performance and survival, and consequently in determining insect fitness. Selecting suitable plants that promote the performance of the progeny is referred to as the Preference−Performance hypothesis (or ‘mother‐knows‐best’). While root infestation generally reduces the performance of leaf herbivores, little is known about its impact on female oviposition. We investigated whether maize root infestation by the Western corn rootworm (WCR) affects the oviposition preference and larval performance of the European corn borer (ECB). ECB females used leaf volatiles to select healthy plants over WCR‐infested plants. Undecane, a compound absent from the volatile bouquet of healthy plants, was the sole compound to be upregulated upon root infestation and acted as a repellent for first oviposition. ECB larvae yet performed better on plants infested below‐ground than on healthy plants, suggesting an example of ‘bad motherhood’. The increased ECB performance on WCR‐infested plants was mirrored by an increased leaf consumption, and no changes in the plant primary or secondary metabolism were detected. Understanding plant‐mediated interactions between above‐ and below‐ground herbivores may help to predict oviposition decisions, and ultimately, to manage pest outbreaks in the field.

Over two decades ago, an intercropping strategy was developed that received critical acclaim for synergizing food security with ecosystem resilience in smallholder farming. The push–pull strategy reportedly suppresses lepidopteran pests in maize through a combination of a repellent intercrop (push), commonly Desmodium spp., and an attractive, border crop (pull). Key in the system is the intercrop’s constitutive release of volatile terpenoids that repel herbivores. However, the earlier described volatile terpenoids were not detectable in the headspace of Desmodium, and only minimally upon herbivory. This was independent of soil type, microbiome composition, and whether collections were made in the laboratory or in the field. Furthermore, in oviposition choice tests in a wind tunnel, maize with or without an odor background of Desmodium was equally attractive for the invasive pest Spodoptera frugiperda. In search of an alternative mechanism, we found that neonate larvae strongly preferred Desmodium over maize. However, their development stagnated and no larva survived. In addition, older larvae were frequently seen impaled and immobilized by the dense network of silica-fortified, non-glandular trichomes. Thus, our data suggest that Desmodium may act through intercepting and decimating dispersing larval offspring rather than adult deterrence. As a hallmark of sustainable pest control, maize–Desmodium push–pull intercropping has inspired countless efforts to emulate stimulo-deterrent diversion in other cropping systems. However, detailed knowledge of the actual mechanisms is required to rationally improve the strategy, and translate the concept to other cropping systems.

The cypress bark beetle, Phloeosinus aubei, is of increasing concern in the European market of scale‐leafed conifers. Neither the reproductive behaviour nor the chemical ecology have been studied in detail so far, and no specific attractant traps are available to monitor the flight of adults. We established that stridulation by males is crucial for acceptance by calling females. To unravel the chemical communication, hindgut extracts from unmated females were analysed by GC‐EAD, using male antennae. Gut extracts and Thuja occidentalis leaf extracts were analysed by GC/MS. Two antennally active compounds from gut extracts were identified as (−)‐myrtenol and (−)‐α‐pinene by enantioselective GC/MS. (−)‐Myrtenol was shown to be female‐specific, while (−)‐α‐pinene was found as a 2:1 mixture with the (+)‐enantiomer also among the volatiles of the host tree, T. occidentalis. To prove that the production of the above compounds in the beetles is related to reproduction, we treated females with JHIII. The gut extracts from these sexually mature females showed elevated levels of (−)‐α‐pinene compared to untreated control groups. Synthetic enantiomers of myrtenol and α‐pinene were tested separately by electroantennography, and all were found to be electrophysiologically active. The attractiveness of different mixtures of these compounds was compared using 4‐arm olfactometer tests: 4:1 mixtures of racemic α‐pinene with either (−)‐ or (+)‐myrtenol were significantly attractive to males and females, respectively. In field tests, multifunnel traps baited with a 5:1 mixture of (−)‐α‐pinene and (−)‐myrtenol caught significantly more males and females than unbaited control traps. We concluded that (−)‐α‐pinene and (−)‐myrtenol are components of a female‐produced aggregation pheromone. EAD activity and behaviour assays indicated that α‐pinene from Thuja is used by the beetles as part of a host plant kairomone. The complex communication system of P. aubei is discussed in a chemoecological context.

The cypress bark beetle, Phloeosinus aubei (Coleoptera, Curculionidae, Scolytinae) prefers to build breeding galleries in decaying host trees. However, volatile cues specific to decaying or actively infested trees have not yet been reported. Any such information would be useful for the management of this invasive, wood-boring species that has become established across Europe in the temperate zone and is now a key pest of ornamental Cupressaceae in nurseries and urban landscapes. Our objective was to reveal key components in host volatiles specific to stressed trees, which could be potential signals for the pest of its suitability for colonisation. Volatile profiles of arborvitae, Thuja occidentalis ‘Smaragd’ suffering from either dehydration or fresh infestation of P. aubei was collected and compared to that samples from healthy control trees. Analyses of volatiles by gas chromatography coupled to an electroantennographic detector revealed substantial differences between healthy, dehydrated and infested trees. Structural elucidation of the major antennally active components revealed that both α-pinene and α-thujene were prevalent in the volatiles of infested trees, whilst α-thujone was the major component in volatiles of healthy trees. In volatiles collected from trunks housing fresh P. aubei female nuptial chambers, sharp increases of β-pinene, myrcene, limonene, and p-cymene were noticed, alongside trace amounts of camphene and fenchene. Volatiles collected from fresh frass showed a similar profile, but with a somewhat smaller amount of limonene. Fenchone was present in each type of volatiles, however, only in low amounts. Further studies should be directed to reveal the behavioural role of these components, which could be helpful in developing kairomone-based techniques for monitoring the flight of the pest in stands of scale-leafed trees.

The main function of the single-carbon metabolic cycle is maintaining nucleotide pool and DNA methylation which is highly influenced by the chemotherapy protocols. This study aimed to evaluate the ultrashort effect of reduced folate combined anticancer treatment on peripheral blood parameters of colorectal cancer (CRC) patients. Post-operative CRC patients were treated with chemotherapy using the combination of oxaliplatin, 5-FU, leucovorin, and capecitabine. Blood samples were taken until the beginning of the treatment and immediately after the therapy. Plasma fractions were separated and the levels of S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH) and the simultaneous quantification of nucleotides (adenine, cytosine, thymine, guanine and uracil) were detected by HPLC-MS/MS. Homocysteine (HCY) was also determined from plasma specimens. Cell-free DNA (cfDNA) was isolated from plasma and DNA methylation was analyzed by LINE-1 bisulfite pyrosequencing. Moreover, LINE-1 methylation statuses were also examined from PBMC cells that were separated from whole blood by a density gradient centrifugation. In each patient, the HCY level of plasma was decreased by an average of 17% after oxaliplatin treatment with the combination of capecitabine (XELOX) and also with 5-FU + leucovorin (FOLFOX) agents. Our study also confirmed that cancer patients have an elevated cfDNA level (avg. 20ng/plasma mL); however, its concentration decreased significantly (p=0.02) on average by 50% after treatment compared to the baseline. The same tendency was observed in the case of SAM after XELOX and FOLFOX adjuvant therapy in plasma samples (p=0.03). Moreover, mostly elevated SAM/SAH ratios were observed in parallel with reduced SAH level after the therapy. Significant (p=0.0001) mean LINE-1 hypomethylation was detected in PBMC (81.84%) compared to cfDNA of plasma (73.76%), furthermore, slightly DNA hypermethylation was noticed in CpG1 position of LINE-1 after chemotherapy in both mononuclear cells and circulating DNA. Lower level of plasma nucleotides was identified in case of adenine, cytosine and uracil after treatment, while thymine and guanine were below the detection limit. The present study demonstrates that the DNA-damaging chemotherapy results in a detectable decrease in cfDNA immediately after the treatment, that was supported by the decrease in the level of the nucleotides measured in the plasma fraction. The methionine cycle is responsible for the DNA methylation maintenance, and though the amount of members of this pathway was reduced, only a moderately increased DNA methylation could be detected even in such a short period time. Co-administration of high dose leucovorin as a folate derivative and a methyl donor with further chemotherapeutic agents may actively contribute to genome alterations. Citation Format: Zsófia Brigitta Nagy, Barbara Kinga Barták, Tamás Fodor, Gellért Balázs Karvaly, Sára Zsigrai, Krisztina Andrea Szigeti, Alexandra Kalmár, Magdolna Dank, István Takács, Béla Molnár. Folic acid combined chemotherapy has an immediate effect on C1 methionine cycle and consequential DNA methylation in liquid biopsy samples of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3741.

Background: Single cell sequencing is a powerful tool for the evaluation of intratumoral heterogeneity and the investigation of cancer evolution. Aims: By combining laser microdissection and single cell sequencing, we aimed to link tissue morphology and spatial information with sequencing data of the isolated cells. Materials & methods: In our preliminary study, we used fresh frozen tissue specimen of surgically resected material from a colorectal cancer (CRC) patient containing both cancerous and normal adjacent tissue (NAT). From part of the normal and cancerous tissue exome sequencing was performed in bulk (to assess somatic variants), while the other part was subjected to single cell sequencing. Fresh frozen tissues from both CRC and NAT were cryosectioned at -20°C with section thickness ranging from 16 to 25 µm to ensure that a layer of whole cells are present in the slides. Tissue slides were then scanned using a PANNORAMIC 1000 scanner (3DHISTECH Ltd.). After morphologic evaluation single normal colonocytes and cancerous cells were laser microdissected from the NAT and multiple CRC areas (invasive front, differentiated, non-differentiated cells) by using a CellCut Laser Microdissection system (MMI). The isolated cells were subjected to Repli-G Single Cell WGA Kit (Qiagen) and library preparation followed by whole exome sequencing (WES) on NextSeq 550 (Illumina). Blood sample was also collected before surgical treatment, cell-free DNA was isolated and exome sequencing was completed. Bioinformatic analysis was conducted using BaseSpace and GATK4 best practices. Common and unique variants were identified between cells and also compared with the bulk exom and cell free DNA sequencing results of the patient. Identified variants were further investigated using the oncoKB annotator. Results: Both healthy (1) and cancerous epithelial cells (3) were dissected and sequenced successfully. A median depth of coverage of 192 was achieved with a median of 43.3% of coverage of 50x or above in the target region compared to 73.9 and 52.8% 208 and 97.6% in tissue and plasma samples, respectively. Overall, we identified 105, likely oncogenic” and 2, predicted oncogenic” unique variants in the single cells using the oncoKB annotator. Among the cancerous single cells, we identified 10, likely oncogenic” and 1, predicted oncogenic” common variants (such as ARID4B, DNMT3B, MSH6 and HNF1A), while 15, 25 and 7, likely oncogenic” (such as CTLA4, MLH1, MSH2, CDK12, CDKN1B) variants were identified uniquely in the 3 cancerous cells. Conclusion: We were able to dissect, isolate and sequence single cells from CRC and NAT thus combining valuable morphologic information with sequencing data on a single cellular level with maintained spatial information. The distribution of variants among the single cells shows that it is a viable approach to investigate tumor heterogeneity and to link the morphologic phenotypes and genotypes of cancerous cells. Citation Format: William Jayasekara Kothalawala, Alexandra Kalmár, Gitta Szabó, Barbara Kinga Barták, Sára Zsigrai, Zsófia Brigitta Nagy, Ildikó Felletár, Krisztina Andrea Szigeti, István Takács, Béla Molnár. A histology based approach to spatial single cell analysis of colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1690.

Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.

Synthetic vitamin B9, also known as folic acid (FA), is an extensively used nutritional supplement as well as an adjunctive medication in cancer therapy. However, there is increasing evidence that FA can promote the progression of established colorectal cancers (CRC); therefore, great care is required in the case of its application. In order to gain knowledge about the underlying mechanisms, we analyzed the genomic and the epigenomic effect of different FA supplies on two CRC cell lines with distinct molecular backgrounds. HT-29 and SW480 cells were kept in FA-free media (0 ng/mL) or treated with 100 ng/mL and 10000 ng/mL FA for 72 hours. Firstly, cell proliferation and cell viability alterations were determined with Supforhodamine B and AlamarBlue assays; then, cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Global DNA methylation level was investigated with the pyrosequencing of long interspersed nuclear element 1 (LINE-1) retrotransposons. Micronucleus scoring performed on DAPI- and anti-γ-H2AX-stained slides, as well as Comet assay, were used to detect the impact of FA on genome integrity. Finally, we analyzed gene expression alterations with Human Transcriptome Array (HTA) 2.0. Our data revealed that 100 ng/mL FA induced significant (p≤0.05) elevation (HT-29100: 128.4±24.9%) of HT-29 cell proliferation compared to the other two circumstances (HT-290: 101.3±13.5%; HT-2910000: 86.1±20.8%). The tendency of cell viability was analogous to the results detected during cell proliferation analyses (HT-290: 91.6±13.3%; HT-29100: 115.8±30.9%; HT-2910000: 64.1±20.2%). The genomic stability of FA-supplemented HT-29 samples was improved in a significant manner (p≤0.05), based on the results of micronucleus scoring (HT-290: 0.56±0.05%; HT-29100: 0.17±0.05%; HT-2910000: 0.25±0.09%) and Comet assay (HT-290: 37.4±3.5%; HT-29100: 31.2±3.4%; HT-2910000: 20.1±3.6%). However, in SW480 cells, remarkable alterations were not detected concerning these parameters. Fundamental differences were observed between the two cell lines in the case of cell cycle (HT-29: G0/1 phase dominance; SW480: S phase dominance) and global DNA methylation analysis (HT-29: 59.1±1.0; SW480: 49.0±0.2), but exposing them to different FA doses did not lead to significant changes. Gene expression alterations were more diverse, as genes involved in carcinogenesis were either up- (HES1, SLC7A11) or downregulated (CCL2) for FA supplementation. We concluded that the effect of FA was considerably influenced by the cell type and the applied FA concentration. Thereby, translating our in vitro results to patient care, we would emphasize the importance of genetic and epigenetic investigations coupled with the choice of proper FA dose upon CRC diagnosis to achieve the best disease outcome. Citation Format: Sára Zsigrai, Alexandra Kalmár, Barbara Kinga Barták, Zsófia Brigitta Nagy, Krisztina Andrea Szigeti, Gábor Valcz, Titanilla Dankó, Anna Sebestyén, Gábor Barna, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3730.

Black plum sawfly (Hoplocampa minuta) is an important pest species of plum (Prunus domestica). In organic plum orchards, the yield loss caused by H. minuta larval damage can reach almost 100% occasionally. Adults feed on pollen and nectar of the plum flower; therefore, we hypothesize that, besides visual cues, also olfaction plays an important role in habitat and host finding. To understand the chemical communication mediated by flower volatiles to black sawflies, we investigated the chemical signals released from plum flowers, which can trigger the peripheral physiological responses of adult sawflies. First, using gas chromatography coupled with electroantennography (GC-EAD), we selected 18 physiologically active compounds from the headspace volatile collection of plum flowers, which triggered the H. minuta male and female antennae. Subsequently, we determine the volatilome of plum flower and identified those compounds, which elicited physiological responses, using gas chromatography coupled with mass spectrometry (GC-MS). These antennally active components in flower volatiles could be candidates for potential kairomone, which could later be used for attracting males and females of H. minuta and could contribute to developing pesticide-free, effective monitoring and lure and kill strategy against this pest.