An application of the synthetic sex attractants from the series “EFETOV-2” for studying Procridinae in Italy (Lepidoptera: Zygaenidae)

The enantiomers of 2-butyl 2-dodecenoate synthesised in the Crimean Federal University are known as the sex attractants for Procridinae species (Lepidoptera: Zygaenidae). These compounds, alone and in combination, were applied for studying the Zygaenidae fauna in Italy for four years from 2015 to 2018. We evaluated the effectiveness of the three following variants: EFETOV-2 (the racemic mixture of (2 R )-butyl 2-dodecenoate and (2 S )-butyl 2-dodecenoate), EFETOV-S-2 (the R -enantiomer alone) and EFETOV-S-S-2 (the S -enantiomer alone). The males of three genera of Procridinae were attracted to the lures from the series “EFETOV-2”: Rhagades Wallengren, 1863 (one species), Adscita Retzius, 1783 (six species), and Jordanita Verity, 1946 (four species). The sex attractants for species Adscita ( Adscita ) italica (Alberti, 1937), A. ( A. ) alpina (Alberti, 1937), A. ( A. ) dujardini Efetov & Tarmann, 2014, and one endemic species Jordanita ( Jordanita ) tenuicornis (Zeller, 1847) were found for the first time. Moreover, a hybridisation zone between A. ( A. ) italica and A. ( A. ) alpina was discovered by the application of the sex attractants. Rhagades ( Rhagades ) pruni ([Denis & Schiffermüller], 1775) was recorded for the first time for more than 100 years in Piemonte and for the first time ever in the Ligurian Alps.


Introduction
In recent years, there has been an increasing interest in studies of sex pheromones and sex attractants of Insecta.A considerable amount of research has been published on the application of these molecules for pest management and monitoring of rare and endangered species (WITZGALL et al., 2010;EFETOV et al., 2014b;OLEANDER et al., 2015;LARSSON, 2016;CIZEJ & TREMATERRA, 2017;WILSON et al., 2017;GREGG et al., 2018).Sex pheromones and sex attractants are also used for faunal and evolutionary studies of Lepidoptera (ROELOFS & ROONEY, 2003;SUBCHEV et al., 2016;RAZOV et al., 2017;CAN et al., 2019).Field experiments with sex attractants and/or sex pheromones, such as for Zygaenidae (Lepidoptera), have allowed researchers to discover new species (KEIL, 2016;EFETOV et al., 2018a), detect low-density populations of harmful or rare moths (EFETOV et al., 2019b;TARMANN et al., 2019;VRENOZI et al., 2019) and discover new data about the distribution of Zygaenids (CAN CENGIZ et al., 2018).
2-butyl 2-dodecenoate were synthesised in the Crimean Federal University as described in EFETOV et al. (2014c).We tested the attractiveness of (2R)-butyl 2-dodecenoate alone (EFETOV-S-2), (2S)-butyl 2dodecenoate alone (EFETOV-S-S-2) and their racemic mixture (EFETOV-2).For preparing baits, the different sex attractants were applied onto grey rubber vial caps fixed on cardboard rectangles and labelled.The lures were attached to twigs on bushes or on the clothes of the researcher (when he slowly crossed the biotope).The attracted specimens were collected by netting them near the lure.In some cases, transparent Delta traps were also used.The removable sticky layers were covered with Tanglefoot ® insect glue.Control traps (without attractants) were necessarily placed in these localities.All captured and attracted moths were recorded and determined by the genitalia examination.

Results and discussion
It was shown that (2R)-butyl 2-dodecenoate and (2S)-butyl 2-dodecenoate, alone and in combination, were attractive for the males of eleven Procridinae species and two hybrid populations (Table 2).The total numbers of the attracted specimens to the sex attractants from the series "EFETOV-2" are listed in Table 3.
AN APPLICATION OF THE SYNTHETIC SEX ATTRACTANTS FROM THE SERIES "EFETOV-2" SHILAP Revta.lepid., 48 (192) diciembre 2020 735   Table 3.-The total numbers of the males of Procridinae species attracted to the sex attractants from the series "EFETOV-2" in different provinces of Italy.Specimens collected by baited traps are marked with an asterisk.The two-letter code is an abbreviation of the province name.Some Procridinae species demonstrated a strong preference for only one type of the attractant.For example, the males of Rhagades (Rhagades) pruni were attracted only to the baits with EFETOV-S-S-2.It should be noted that Rh. (Rh.) pruni was found for the first time since its last record of more than 100 years ago in Piemonte near Torino in 1906 and for the first time ever in the Ligurian Alps.Three new populations (Fig. 1) of this presumed extinct species were found only with the help of the sex attractant.It has recently been shown that EFETOV-S-S-2 contributed to the rediscovery of Rh. (Rh.) pruni in Spain on the southern side of the Pyrenees where this species is also a very rare (EFETOV et al., 2019b).Together, these studies suggest that the sex attractant EFETOV-S-S-2 is a sensitive and efficient tool for monitoring populations of Rh. (Rh.) pruni.It is interesting that this species is mentioned as a pest on apple (Malus toringoides Hughes) and Prunus spp. in Japan (TARMANN, 2003).

Attracted species EFETOV-2 EFETOV-S-2 EFETOV-S-S-2
In contrast to Rh. (Rh.) pruni the species Jordanita (Solaniterna) subsolana (Staudinger, 1862) (with more than 400 attracted specimens) reacted only to EFETOV-S-2.For instance, during 30 minutes enormous masses of this species (more than 100 specimens) were attracted to the lure with EFETOV-S-2 attached to the hat of the researcher (Fig. 2).The males showed active sexual behaviour: they trembled by wings, moved antennas, opened valves, touched the rubber cap by the end of abdomen and tried to copulate with it.This means that our type of the sex attractant was very effective for detection of J. (S.) subsolana in biotopes.New localities could be discovered in three provinces, viz.Torino, Imperia and Parma (Fig. 3).
All three variants of the sex attractants from the series "EFETOV-2" were active for Adscita (Tarmannita) mannii (Lederer, 1853).They are new synthetic sex attractants for this species.Besides this, the attractiveness of other esters of 2-butanol, viz.(2S)-butyl (9Z)-tetradecenoate, (2S)-butyl (7Z)dodecenoate and (2R)-butyl (7Z)-dodecenoate, were proved for the males of A. (T.) mannii some years ago (SUBCHEV et al., 2010;EFETOV et al., 2015b;RAZOV et al., 2017).It is interesting to note that for this species the S-enantiomers of these esters are more active than the corresponding R-enantiomers.EFETOV et al. (2015b) demonstrated that when three variants of the attractants ((2S)-butyl (7Z)-dodecenoate, (2R)butyl (7Z)-dodecenoate and their mixture) were present in the habitat, the males of A. (T.) mannii came mainly to the S-enantiomer alone.When only (2R)-butyl (7Z)-dodecenoate and the mixture were present, some specimens could be found in traps baited with the mixture.The males of A. (T.) mannii were captured by the traps baited with (2R)-butyl (7Z)-dodecenoate only if other types of the lures were absent in biotopes.Our results coincide with those observed in earlier studies.The males of A. (T.) mannii were attracted to the lures EFETOV-S-2 (R-enantiomer) if we used only this one attractant during observations.In case when all three types of the lures were applied, A. (T.) mannii came to EFETOV-S-S-2 (Senantiomer) or EFETOV-2 (the mixture).It should be noted that the differences between our sex attractants and other attractive esters for A. (T.) mannii are in the position of the double bond (at the 2 nd carbon atom instead of the 7 th or 9 th ) and for (2S)-butyl (9Z)-tetradecenoate in the carbon chain length of fatty acids (12 C instead of 14 C).There are two important implications from these findings.Firstly, the presence of the R-enantiomers of biologically active esters does not influence the attractiveness of the corresponding Senantiomers for A. (T.) mannii.Secondly, stereochemistry of molecules is important for the identification of the sex attractants by olfactory receptors of moths.
Three closely related species, viz.Adscita (Adscita) statices (Linnaeus, 1758), A. (A.) alpina (Alberti, 1937), and A. (A.) italica (Alberti, 1937), reacted actively to the investigated sex attractants.The males of A. (A.) statices were attracted only to the lures baited with EFETOV-S-2.The males of A. (A.) alpina were mainly attracted to EVETOV-S-2 and to EFETOV-2.The males of A. (A.) italica were caught by the sticky trap with EFETOV-2 (Fig. 4); in addition, they also came to the lures with EFETOV-S-S-2 and EFETOV-S-2.The sex attractants for A. (A.) alpina and A. (A.) italica have been found for the first time while the attractiveness of EFETOV-2 and EFETOV-S-2 for A. (A.) statices was demonstrated in our previous studies (EFETOV & GORBUNOV, 2016;CAN CENGIZ et al., 2018).In the Ligurian Alps these three species of the subgenus Adscita have their distributional borders.In recent years it has been observed that there is a hybrid belt between A. (A.) statices and A. (A.) alpina in the western Ligurian Alps (GUENIN & TARMANN, 2016).Hybrid zones between these two species have been known throughout the Alps for a long time (white squares on Fig. 5) (TARMANN, 1979;GUENIN, 2013GUENIN, , 2016)).However, there was still a distributional gap of about 70 km (as the crow flies) between A. (A.) alpina in the West and A. (A.) italica italica (Alberti, 1937) in the East in the Ligurian Alps and the Ligurian Apennine.A. (A.) italica italica is an endemic subspecies of the Apennine Peninsula and had its western known distribution around Genova.In summer 2017 the field work to study this so-called distributional gap in the Ligurian Alps and the Ligurian Apennines was carried out with the help of the sex attractants from the series "EFETOV-2".As soon as the males were detected, the females had to be found because A. (A.) alpina and A. (A.) italica can only be distinguished clearly by different structures in the female genitalia (Figs 6,7).The distributional limits of A. (A.) alpina and A. (A.) italica were determined and hybrids between A. (A.) alpina and A. (A.) italica were discovered for the first time.The contact area between these two species is farther to the East than was expected and A. (A.) alpina is recorded for the first time from the Apennines (province Savona: Montenotte inferiore) (Fig. 5).Hybrids Adscita alpina × italica with intermediate female genitalic structures (Fig. 8) were found at five localities, four in the Ligurian Apennines (province Genova: Valle Gorgasso, Madonna della Guardia; province Savona: Piampaludo, Case del Oca) and one in the eastern Ligurian Alps (province Savona: Bardinetto) (black squares on Fig. 5).
Adscita (Adscita) dujardini Efetov & Tarmann, 2014, was found in large numbers in 2015 at its typelocality (more than 50 specimens on flowers of Geranium sanguineum L.).In spite of this fact, there were only two males (one in EFETOV-2 trap and one in EFETOV-S-2 trap) glued from 16-VI-2015 to 18-VI-2015.It means that the attractants have no strong attraction for the males of this species.However, the empty control trap (without attractants) indicates that it was not accidently that the specimens were attracted into traps with the attractants EFETOV-2 and EFETOV-S-2.The males of the endemic Italian species Jordanita (Jordanita) tenuicornis (Zeller, 1847) also reacted to two attractants: EFETOV-S-2 and EFETOV-S-S-2.The sex attractants for these two species were found for the first time.
It is worth mentioning about the study of diurnal rhythms of pheromone communication of some species of Procridinae in Italy.It is known that the time of the day when the males react to the sex pheromone is usually synchronized with the time when the conspecific females release pheromones.Therefore observations on calling females or the attraction of males to a pheromone (or synthetic attractant) source could be used for these investigations.On 08-09-VI-2015 a twenty four hour experiment with the attractant EFETOV-S-2 was carried out by G. M. Tarmann in the province Pordenone, Magredi di Cellina, Vivaro S. The researcher slowly crossing the biotope testing the attractant attached to his hat for 30 minutes, with a break for one hour thirty minutes.In total, 12 exposures of the sex attractant were carried out.The males of three species, viz. A. (T.) mannii, Jordanita (Tremewania) notata (Zeller, 1847), and J. (J.) globulariae, were found in the biotope and attracted to EFETOV-S-2 (Table 4).The males of J. (T.) notata and J. (J.) globulariae reacted to the lures from 2 to 5 a.m. while the males A. (T.) mannii were attracted from 6 to 7 p.m.In our further field observations, most of A. (T.) mannii males (27 from 33) were attracted between 3 and 7 p.m.This fact does not contradict the results received in the described experiment.Thus, the sexual activity of A. (T.) mannii is high late in the afternoon.The peak of the searching behaviour of J. (T.) notata and J. (J.) globulariae occurs at night before sunrise.
New localities of the Italian Procridinae species were found, including the hybridisation zone between A. (A.) italica and A. (A.) alpina.
The presumed extinct species Rh. (Rh.) pruni was rediscovered after more than 100 years in Piemonte.

Table 4 .
-The diurnal rhythms of sexual activity of the three Procridinae species studied with the help of the attractant EFETOV-S-2 in province Pordenone on 08-09-VI-2015.