On 25^th September 2018 Cupressus × leylandii trees were examined in the suburban region of Székesfehérvár from where a freshly emerged specimen of Cydia interscindana had been received from a horticulturist. Larvae of L. festiva (Fig. 2) and an unidentified Lepidoptera species (Fig. 3) were collected from under tree bark. The caterpillar soon died, but it was preserved for genetic analysis. In that year similar Lepidoptera larvae were found in three neighbouring villages (Velence, Sukoró, Pákozd).

Figures 1–6. 

Damage and developmental stages of pests in Cupressaceae. 1. Larva of Lamprodila festiva; 2. Damage caused by the larva of Cydia interscindana in the phloem; 3. Tunnel of the larva of Lamprodila festiva; 4. Exit hole of C. interscindana; 5. Fully grown larva of C. interscindana; 6. Adult of C. interscindana. Scale bars: 1 mm (Figs 1, 3, 5, 6); 10 mm (Fig. 2); 5 mm (Fig. 4). Photos were taken in Velence on 15.05. 2019 (5), 15.09.2019. (2) and 10.01.2021 (1, 3, 4) and in Budapest on 18.06.2016 (6) by Attila Takács.

In order to collect actual data on the distribution of C. interscindana, an investigation was launched in 2019, when CSALOMON-type C. pomonella pheromone traps were used at three sites: in Zalaegerszeg, Békéscsaba and Kápolnásnyék. In 2020 this survey was extended country-wide to a total of 18 localities: Békéscsaba, Berettyóújfalu, Bernecebaráti, Budapest, Bugac, Debrecen, Derecske, Gárdony, Horpács, Kápolnásnyék, Kaposvár, Nagyatád, Nyíregyháza, Pákozd, Sukoró, Székesfehérvár, Velence, Zalaegerszeg and with 21 sampling sites in total (Fig. 1 and Table 1). In 2020 the same type of pheromone trap was used. Two traps were used in each site on or close to potential host plants of the pest. During the studies, traps worked from mid-May to the end of September. Traps were generally checked once a week except in Budapest where they were checked yearly. Baits were changed after four weeks, whereas sticky sheets were changed if it was necessary.

Identification of sampled adults (Fig. 6) was made based on the description of Razowski (2003). Where traps caught enough C. interscindana, data were used to characterise population dynamics and life cycle of the species.

In 2019 and 2021 further damages were studied and larvae were collected in Budapest and Velence. Intact specimens and larval damage were documented with photographs made with a Canon 450 D camera, applied to a Carl Zeiss Stemi-2000 binocular stereomicroscope.

Three specimens were selected for molecular analyses (Table 2). Specimens were killed and stored in 70% alcohol. The genomic DNA was isolated from two legs of adult specimens or from a piece of the larval body. DNA was isolated with Quick-DNATM Tissue/Insect Miniprep Kit (Zymo Research) according to the recommended protocol of the manufacturer.

Amplification of the 658 bp DNA COI barcode region was performed with the primers LCO-1490 and HCO-2198 (Folmer et al. 1994). The PCR mixture contained 2 μl of template DNA, 7.5 μl of 2× HotStart Taq Plus Master Mix (HotStart Taq Plus Master Mix Kit, Qiagen), 0.5 μl of each primer (10 µM) and double distilled water to a final volume of 15 µl. The amplification profile consisted of a preheating step of 5 min at 95 °C followed by 5 cycles of 95 °C for 30 seconds (s), 45 °C for 30 s and 72 °C for 1 min, and additional 35 cycles at 95 °C for 30 s, 51 °C for 1 min and 72 °C for 1 min and a final extension at 72 °C for 10 min. Reaction was performed in a PTC-100 DNA thermal cycler (MJ Research). Products of amplification were analysed in 2% agarose gel with addition of ethidium bromide and visualised under UV light. After visualization of the PCR-products on agarose gel, they were purified and sequenced bidirectionally (BaseClear B.V.). Specimens used in this study are listed in Table 1 along with GenBank accession numbers.

The forward and reverse sequences were assembled with Staden Package 2.0.0b9. Sequences were inspected and translated using the translate tool of ExPASy Bioinformatics Resource Portal (Artimo et al. 2012) to verify that they were free of stop codons.

For further analysis, we used only available sequences of Austrian C. interscindana specimen with other gymnosperm-feeding Cydia species: Cydia cognatana (Barrett, 1874), C. colorana Kearfott, 1907, C. conicolana (Heylaerts, 1874), C. duplicana (Zetterstedt, 1839), C. indivisa (Danilevsky, 1963), C. illutana (Herrich-Schäffer, 1851), C. inopiosa (Heinrich, 1926), C. phyllisi Miller, 1986, C. strobilella (Linnaeus, 1758). Multiple sequence alignment was carried out by ClustalW (Larkin et al. 2007), with default parameters for gene sequences of examined Cydia species and with outgroup taxa (Ecdytolopha insiticiana Zeller, 1875). The distance analysis within the examined taxa was obtained based on uncorrected p-distances using MEGA 7 (Kumar et al. 2016).

The most appropriate model of DNA sequence analysis was determined with MEGA7 under the Bayesian Information Criterion (BIC). The General Time Reversible model with discrete Gamma distribution (GTR+G) (Tavaré 1986) was selected for our phylogenetic reconstruction. Phylogenetic trees were calculated with Maximum Likelihood (ML) method as implemented in MEGA 7, starting from a random Neighbour Joining tree, with the default initial rearrangement settings. To obtain an estimate of the support for each node, a bootstrap analysis using 5000 replicates was performed. Bootstrap support is given on appropriate clades in the ML tree.

Amongst preimaginal stages, ovum and morphology of larvae were not studied in detail, but damage and signs of occurrence that are important in the recognition of damage are described hereunder.

The mines of C. interscindana larvae can be found on the branches and trunk of the host plant. The mines are full of frass (Fig. 2) that differs conspicuously from that of the co-occurring L. festiva (Fig. 3). At the entrance of the tunnel, the beetle’s reddish frass can be also found (Fig. 4). The mine reaches the phloem. The plant tissue around the mine becomes loose, slightly swollen (Fig. 8), somewhat sponge-like; this tissue is consumed by the larva. Perhaps it represents a case of gall-induction.

Figures 7–10. 

Pupa of C. interscindana. 7. Exuviae, close view; 8. Two exuviae on a Chamaecyparis lawsoniana tree; 9. Pupa in wood, ventral view; 10. Caudal end of pupa, dorsal view. Scale bars: 1 mm (Figs 7, 9); 10 mm (Fig. 8); 0.1 mm (Fig. 10). Photos were taken in Velence on 10.01.2021 (7, 8) and 10.05.2020 (9, 10) by Attila Takács.

The pupa is light brown (Fig. 9). The caudal edge of each abdominal segment has a row of tiny, but strong teeth on the dorsal side, accompanied by an additional transverse row in the central third of the segment. The caudal end of the pupa is slightly rounded, nearly truncated, with three pairs of narrow and hooked cremastal setae (Fig. 10). The pupal stage lasts for 10–12 days.

The exuviae are brown, extruding by 2/3 from the wood (Figs 7, 8). They were found in Velence and Székesfehérvár.

In Hungary, C. interscindana was detected together with of L. festiva, on Cupressus × leylandii, Platycladus orientalis and Chamaecyparis lawsoniana (Cupressaceae). Pheromone traps were also used near one or two or even all of these host plants, and where it was possible, traps were placed on these trees.

Larvae consume the phloem (Fig. 2), which has the very important physiological role of transport and storage of nutrients. Due to the attacks the plants turn yellow, begin to fade and dry out and in cases of heavy infestation plants may even die.

All examined Cupressus × leylandii, Platycladus orientalis, and Chamaecyparis lawsoniana plants (n=52) were infected by C. interscindana, but L. festiva attacked only 30 plants. The number of C. interscindana larvae was highest in Cupressus × leylandii, while the least preferred host plant was Platycladus orientalis (Table 3).

The present study shows that Cydia interscindana develops two generations per year in Hungary and that these do not overlap. Adults of 1^st generation are on the wing from mid-May to mid- or late June; those of the 2^nd generation from mid-August until mid- or late September, depending on weather conditions (Fig. 11). Specimens overwinter as mature larvae and pupate in early spring.

Figure 11. 

Temporal changes of 3-day moving average of abundance of Cydia interscindana based on Cydia pomonella sex pheromone trap catches in Budapest, 2020.

The distribution of Cydia interscindana has been studied in 11 localities in Hungary between 2018 and 2019. In 2018 it was found in Székesfehérvár and damage that may have been caused by this pest was also detected in the three neighbouring localities of Velence, Sukoró and Pákozd. In 2019, traps collected C. interscindana in Békéscsaba and Kápolnásnyék, but not in Zalaegerszeg. In 2020 C. interscindana specimens were caught by traps in Velence, Sukoró, Pákozd, Gárdony, Kápolnásnyék, Székesfehérvár, Békéscsaba, Debrecen and Budapest (Fig. 12).

Figure 12. 

Known distribution of Cydia interscindana in Hungary. Black dots: the species is present; empty dots: the species was searched for but not found between 2018 and 2020. (Sampling sites are detailed in Table 1).

The recovered length of the COI region of the mitochondrial DNA numbered 547 nucleotide positions in the final dataset. Based on the results of our analysis, the three Hungarian specimens had the same nucleotide sequence in this examined region. This was the reason for characterising the three Hungarian populations with a single sequence in our analysis. Relationships among examined species inferred from the analysed mitochondrial region are shown in Fig. 13.

Figure 13. 

Relationships of examined Cydia species based on a ML analysis was inferred using COI-5P sequences (547 aligned nucleotides) under the GTR+G model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) is shown next to the branches. Species names and GenBank Accession or BOLD Sample/Process ID numbers are listed for each taxon. The scale bar represents the number of substitutions per site.

Within the “gymnosperm” group the three Hungarian Cydia interscindana populations shared an identical sequence with the Austrian specimen. These populations formed a common clade with the most closely related Cydia species (C. duplicana). The genetic distance between them was 3% (17 nucleotides). Since this value is very similar to typical species-level genetic differences (e.g. Huemer et al. 2014), we also assume that these two closely related species are not conspecific (bootstrap value was 98%).