THE FORMATION OF THE CONSORTIA RELATIONS OF MOLIPTERYX FULIGINOSA (UHLER, 1860) (HEMIPTERA, COREIDAE) WITH AMBROSIA ARTEMISIIFOLIA IN THE PRIMORSKII KRAI OF RUSSIA

Currently, invasive species spreading is becoming a key problem on the global scale. Scope of invasive species control get solved at governmental level in many countries; hundreds of billions dollars a year are allocated as financial support; global programs are created. For the first time, biological control of invasive species Ambrosia artemisiifolia using an introduced phytophage insect was applied in Russia (former USSR) in the 60-70s years of the 20th century and it remains a unique large-scale example until to nowadays. Ambrosia leaf beetle (Zygogramma suturalis (Fabricius, 1775) was brought to the Russian Far East for biological control of Ambrosia artemisiifolia in the 80-90s years of the 20th century for the first time, however the targeted result was not achieved. At present, Zygogramma suturalis were adapted at the Primorskii Krai in the south of the Far East, but due to climatic conditions its numbers are low and, as a result, the impact on ambrosia is minimal. The search for native insect species with capable of effectively suppressing the spread of aggressive quarantine weed in local conditions remains in the area of focus. At the moment, two species of native leaf beetles have been identified, using Ambrosia as an additional element in the diet. Climate changes and the shift of biogeographic boundaries cause the natural expansion of the areals of some East Asian insect species to the north within the Russian Far East. Bug-edge Molipteryx fuliginosa (Uhler, 1860) is one of such species. Expanding the area to the north and east, the species also corrects trophic preferences. As a result of our research in the south of Primorskii Krai, the trophic relations of Molipteryx fuliginosa were studied in the field conditions and in rearing cages. The same work was carried out in places where Ambrosia artemisiifolia were growing in a great number. For insects observing were used standard methods. As a result, firstly was established and confirmed that the bug-edge uses Ambrosia artemisiifolia as a feed plant only in select developmental stages, including imago. The feeding of larvae of IV – V ages was noted in natural conditions. The long-time Molipteryx fuliginosa development cycle on ambrosia leads to break the course of normal plant ontogenesis and causes wilting of individual organs.


Introduction
The problem of introduction and spread of invasive species is the key component of global changes taking place in the existing ecosystems and their functioning. Many invasive plants are highly allergenic. It has been estimated that annual medical costs for treatment of allergies inflicted by invasive plants amount to 290 million euro in Austria and Germany (Katza et al., 2014), 110 million euro in Hungary (Oude Lansink et al., 2018), and 120 million USD in the USA (Susaeta et al., 2016). Yield loss caused by common ragweed (Ambrosia artemisiifolia L.) can be as high as millions euro: for example, in Hungary this figure was 130 million euro per year (Oude Lansink et al., 2018).
Today, funds ranging from several dozens to several hundreds billion USD are annually spent to combat invasive species (Culliney, 2005). SMARTER, HALT-AMBROSIA, and NOVARA are the European government-supported projects launched to control the hotspots of aggressive invasive species, and many countries are currently participating in these projects (Oude Lansink et al., 2018). Their objective is to share experience with biological control over spread of invasive species. In 2013, 121 species of specialized insects were used for biological control of 34 weed species in the USA, and 94 insect species were used for 22 weed species in Canada (Zimdahl, 2018). However, the introduced biological agents are not always successful in new environments. Thus, the hawk moth (Hyles euphorbiae (L., 1758) (Lepidoptera, Sphingidae) imported from Austria, Hungary, and India had a negligible effect when used for weed control in the USA (Zimdahl, 2018).
In the USSR, the biological control method using introduced phytophagous insects was first employed in the 1960-70ss for combatting the invasive species A. artemisiifolia. Today, it is the only example of large-scale application of a biological method to control this weed species. For this purpose, the moth species Tarachidia candefacta Hübner, [1831] (Lepidoptera, Noctuidae), fruit fly Euaresta bella (Loew, 1862) (Diptera, Tephritidae), ragweed leaf beetles Brachytarsus tomentosus (Say, 1827) (Coleoptera, Anthribidae) and Zygogramma suturalis (Fabricius, 1775) (Coleoptera, Chrysomelidae) (Esipenko, 2012;Kovalev et al., 2013) were introduced to the European part of the USSR. Among the four candidates, only two were efficient for combatting common ragweed: Tarachidia candefacta and Zygogramma suturalis, which managed to acclimatize under conditions of Southern Russia (Esipenko, 2013).
Application of the classical biocontrol method can be an important and even the only option for managing aggressive invasive species in the localities where chemical or mechanical control is infeasible (Culliney, 2005;Lommen et al., 2017). Therefore, searching for native insects to be used for biocontrol is less threatening for ecosystems compared to the alien species that can run out of control and become invasive, and sometimes in a totally unpredictable way. Furthermore, application of native species is less cost demanding.
The climate change taking place over the past decades has naturally expanded the habitats of many East Asian insect species in the Russian Far East (Koshkin et al., 2015). Leaf-footed bug Molipteryx fuliginosa (Uhler, 1860) (Hemiptera, Coreidae) is one of such species. The genus Molipteryx Kiritshenko, 1916 comprises three species commonly occurring in the southeastern Palearctic region (Vinokurov et al., 2010). It was reported in literature that this species is noxious (Wang et al., 2002;Chen et al., 2014;Luo et al., 2014). In China, M. fuliginosa was included in the list of economically important insects (Zhang, 1985). In Russia, this species was first encountered in the late 1980s in the Khabarovskii Territory (Kerzhner & Kanyukova, 1998). Later, the species was gradually spreading to the Primorskii Krai (Kanyukova & Vinokurov, 2009;Kanyukova, 2012), where it can be found today.
Application of A. artemisiifolia as a feeding plant for M. fuliginosa has been reported in neither foreign (Zhang, 1985;Wang et al., 2002;Chen et al., 2014;Luo et al., 2014;Zhou et al., 2017) nor Russian literature (Kerzhner & Kanyukova, 1998;Vinokurov, 2009;Kanyukova, 2012;Markova et al., 2016a). In most studies discussing the measures for reducing the common ragweed population, the primary focus was placed on its natural enemies, the insects (Esipenko, 1998;Kovalev & Medvedev, 1983;Kuznetsov & Esipenko, 1991;Kovalev et al., 2013;Zhou et al., 2017;Lommen et al., 2018). However, the climatic conditions of the region or country where A. artemisiifolia is currently found are not always favorable for the insects recommended for introduction. Therefore, it is extremely important to study the native insect species that can be used for biocontrol of invasive plants. Among all the phytophagous insects found in the Russian Far East (Kuznetsov & Storozhenko, 2010;Esipenko, 2014;Aistova et al., 2014), M. fuliginosa has the longest life cycle on A. artemisiifolia, which disrupts the natural ontogenesis of A. artemisiifolia.
This paper presents the results of the studies demonstrating the features of M. fuliginosa development on A. artemisiifolia in natural plant communities and stationary cages in the southern Primorskii Krai.

Materials and methods
The studies were carried out in the southern Primorskii Krai near the villages of Kaimanovka and Kamenushka (forest tree nursery), in the specially protected natural area of the natural landmark of regional importance "Barsukovskaya groove" (the Ussuriysk urban district), in the Ussuri State Nature Reserve Preferential localities of A. artemisiifolia (verges of dirt and forest roads, anthropogenically transformed and waste areas, gardens, and woodland areas) were inspected every ten days. The standard methods (visual inspection and hand picking) were used to collect insects. Photos were taken. A GPS navigation unit was utilized for the route examination of plant communities; the data were plotted on a GPS map using the MapSours Trip Waypoint Manager software (Fig. 1). All the photos presented were made by the authors of this paper.
The trophic associations were studied under field conditions and in stationary cages (Fig. 2). The stationary cages allow one to simulate near-natural housing conditions for insects (with feeding quality of plants retained) and to observe the insects for a long period, between the oviposition stage and adult emergence (Markova et al., 2017 a, b;2018). We conducted three variants of studies using different food plants to prove that the leaf-footed bug uses A. artemisiifolia during its life cycle. In the control variant, we used only the food plants Cirsium pendulum Fischer (Asteraceae)and Agrimonia striata Michaux (Rosaceae); in the first variant, Ambrosia artemisiifolia, C. pendulum and A. striata; and A. artemisiifolia only was used in the second variant.
Ten M. fuliginosa adult specimens were placed into each stationary cage containing undamaged plants pre-planted into the soil. Since M. fuliginosa nymphs feed on a more constant and narrow range of plants than adults do (Puchkov, 1956), similar experiments were also conducted using II-V instar nymphs.

The features of the biology of Molipteryx fuliginosa in the Primorskii Krai
The leaf-footed bug Molipteryx fuliginosa is a species native to East Asia and is widespread in the northeastern China, the Korean Peninsula, and Japan (Kyushu and Hokkaido islands). In Russia, this species has been found in the southern parts of the Khabarovskii and Primorskii Krai (Park, 1996;Kerzhner & Kanyukova, 1998;Kanyukova & Vinokurov, 2009;Vinokurov et al., 2010;Kanyukova, 2012;Markova et al., 2016b).
The experiments conducted in the Primorskii Krai have demonstrated that overwintered M. fuliginosa imagines were found in nature starting with the late second ten-day interval of May; mating and oviposition were observed between the last ten-day interval of May until the first ten-day interval of August (Markova et al., 2017b). According to foreign literature sources, there is only one generation of the leaf-footed bug per year in Korea; this species is found there from early May until late September, and overwinters as adults (Park, 1996). In the stationary experiments in cages conducted in the Primorskii Krai, four to eighteen repeated copulations lasting between two and 48 hours were reported for one female bug. A total of five bug pairs were kept in the stationary cages. The observations were carried out continuously, both visually and using a photo camera. The average number of eggs laid between the copulations was 3.8; the average number of oviposition events was 7.8; and female fertility was 28.4 (n = 5) (Markova et al., 2017b). Copulation of M. fuliginosa was also observed under natural conditions. After mating, the females were leaving the plants in search of a suitabble place for oviposition. Single batches of eggs laid by M. fuliginosa were found on plants Solanum lycopersicum L., Carex sp., Elytrigia repens (L.) Nevski, and Taraxacum officinale Wigg. It seems that female leaf-footed bugs are not selective to oviposition spots; i.e., it is not always associated with food plants even if they are abundant and easily accessible (Markova et al., 2017b).

Trophic associations between Molipteryx fuliginosa and plants
Whether an individual insect species is a good candidate for being used as a biological agent or not depends on ability of instar nymphs to complete the cycle of development to the adult stage or at least spend the individual stages of their life cycle on an invasive plant, as well as on the rate of ontogenesis (Lommen et al., 2017). Different species of phytophagous insects (the ones naturally feeding on the common ragweed) are used in different countries to eradicate A. artemisiifolia hotspots. Leaf beetles belonging to the genus Ophraella Wilcox, 1965 (Coleoptera, Chrysomelidae Latreille) have been used most successfully in Europe. Species within this genus have consortive ties with plants in the family Asterаceae (Lommen et al., 2018). The studies conducted in Switzerland and focused on simulation of climatic suitability of O. slobodkini (Futuyma, 1991) andO. communa LeSage, 1985 have demonstrated that the former species can be used only in the subtropical climate, while the latter one has a broader habitat and can be utilized as a biological agent for biocontrol in Europe (Lommen et al., 2017). Today, O. communa has established in northern Italy (Lommen et al., 2018). The moth Epiblema strenuana (Walker, 1863) (Lepidoptera, Tortricidae Latreille, 1803) and leaf beetle Zygogramma bicolorata Pallister, 1953 (Coleoptera, Chrysomelidae Latreille, 1802) (Lommen et al., 2017(Lommen et al., , 2018 are widely used to control A. artemisiifolia in Australia. Epiblema strenuana (Walker) and Ophraella communa LeSage are the species efficiently utilized to control A. artemisiifolia in South China. As demonstrated by the Chinese researchers (Zhou et al., 2017), cold-tolerant populations of O. communa have been revealed there. Their succeeding generations might be able to establish in North China, where the common ragweed is also spreading rapidly. Today, chemical control is the only method used in North China to control A. artemisiifolia (Zhou et al., 2017). In Russia, O. communa is planned to be introduced in the southern part of the country as a potential agent for controlling common ragweed (Esipenko, 2013).
Our studies aimed to confirm the trophic associations between the leaf-footed bug and common ragweed. Detailed observations of the leaf-footed bug living on A. artemisiifolia have been conducted since 2014. In June 2016, we first found a V instar nymph of M. fuliginosa feeding on the base of A. artemisiifolia inflorescences (Fig. 3). In July-August 2018, the evidence supporting the fact that M. fuliginosa at the nymph and adult stages feeds on common ragweed under natural conditions was obtained. The studies carried out in natural biocenoses demonstrated that in wood habitats, the insects choose moderately insolated ecotonic plant associations (preferring the mesophyticones ones), as well as anthropogenically disturbed areas (forest roads, fringes, overgrowing wood glades, etc.). It was observed that the M. fuliginosa species was feeding on A. artemisiifolia in these biotopes. No leaf-footed bugs were found on A. artemisiifolia in xerophytic biotopes and monodominant communities. After the leaf-footed bugs were feeding on A. artemisiifolia during one week, inflorescences of this plant started to slowly dry out, without passing through the full development cycle. Although the number of damaged A. artemisiifolia plants was insignificant yet, it is fair to say that this bug species gradually establishes trophic associations with A. artemisiifolia. In July-August 2018, we conducted an experiment to confirm the existence of trophic association between M. fuliginosa at the nymph or imago stages and A. artemisiifolia.
The stationary experiments demonstrated (adult species, n = 30; II-V instar nymphs, n = 30) that in a cage containing a combination of plants A. artemisiifolia, Cirsium pendulum and Agrimonia striata, some leaf-footed bugs preferred the habitual food plants: Agrimonia striatа on days 1 and 2 and C. pendulum on day 3. By day 8, groups of leaf-footed bugs were found only on Cirsium pendulum; individual bugs were feeding on A. artemisiifolia and Agrimonia striata. One egg was laid on the lamina of C. pendulum (Fig. 4a). In the cage containing A. artemisiifolia, M. fuliginosa adults damaged the laminae, the top portions of stems (suction of phloem from vascular bundles), the base of shoots with leaves and those with developing inflorescences. Two eggs were laid on the stem and the lamina of A. artemisiifolia (Fig. 4b).
In the control cage containing C. pendulum and Agrimonia striata, we observed clusters of feeding leaf-footed bugs at 9:1 and 10:0 ratios. The feeding patterns slightly differed for the nymph and adult stages of M. fuliginosa. In the cage containing a combination of A. artemisiifolia, C. pendulum, and Agrimonia striata plants, II instar nymphs preferred feeding on Agrimonia striatа, while III-V instar nymphs preferred C. pendulum. Although the plant condition and its feeding quality remained rather good (no wilting, drying off, or significant visual destruction was observed at the perforation sites), single individuals of leaf-footed bugs were feeding on A. artemisiifolia and Agrimonia striata (with the 6:2:2 ratio, respectively, on day 8 of being kept in the cage). Feeding bugs do not migrate over the plant during the daylight hours and stay in one part of the plant. In the control cage containing C. pendulum and Agrimonia striata, III-V instar nymphs also preferred C. pendulum. In the stationary cages containing A. artemisiifolia only, II instar nymphs were feeding on the laminae (suction from the rachis was observed). III-V Instar nymphs were feeding on the laminae and stems (suction of phloem from vascular bundles), with preference given to the top parts, bases of shoots with a leaf, and some shoots with developing inflorescences. Furthermore, it was also noted that when feeding on the laminae, II instar nymphs did not damage the plant. We revealed no visual changes in plant appearance (perforation marks, dark spots emerging because of feeding, wilting or drying of the laminae or their fragments) after the insects had been removed. When III-V instar nymphs and adult bugs were feeding, the overall wilting of the plant was observed starting on day 4 (Fig. 5b). Perforation marks on leaf veins could be seen, while no visible marks of damage were revealed on the stems. Once the experiment was completed, the cages were removed, and the plants remained on their spots until the end of the season. By day 8 of constant feeding of bugs on A. artemisiifolia, parts of the plant continued to wilt and dry out at the sites of insect clustering (10 insects per plant). When the insects were feeding on A. artemisiifolia inflorescences, those were slowly wilting and subsequently drying out (Fig. 5 a, b).
Our experiments demonstrated that M. fuliginosa adults and nymphs at different instar stages feed on common ragweed without any harm to themselves. No insect death was observed in the cages; the nymphs developed normally and emerged to the next nymph stage after molting. This was possibly related to the chemical composition of the plants (namely, phytosterols and alkaloids, which play a crucial role in cellular metabolism of the phytophage (Telitchenko & Ostroumov, 1990; Useful wild plants of Russia, 2001). Accumulation of these substances protects the insects against predators (both vertebrate and invertebrate ones) and can be useful for them (Telitchenko & Ostroumov, 1990).

Conclusions
The studies conducted under natural conditions demonstrated that M. fuliginosa feeds on common ragweed in the ecotone plots of forest cenoses and is preferentially found in moderately insolated mesophytic plant cenoses. No leaf-footed bugs were found in the xerophytic biotopes and monodominant communities of common ragweed. The observations in stationary cages showed that nymphs of the leaf-footed bug feed on A. artemisiifolia only at some nymph instars, while this bug does not dwell on A. artemisiifolia during the full life cycle. By day 15-20 of cage keeping, the plant wilted because of the loss of turgor in cells, which makes further feeding of bugs impossible and may lead to their death. Under natural conditions, this forces M. fuliginosa switch to other plants. It is worth mentioning that ten M. fuliginosa individuals placed in a stationary cage cause death of the whole plant, while in nature two or three nymphs of leaf-footed bug (nymph instar V) make normal ontogenesis of the plant impossible as they cause wilting of individual plant parts, disturb the vegetative functions and inflorescence development, and lead to death of generative organs of the plant.