Entomofauna and potential pollinators of strawberry crop under semi-hydroponic conditions

Strawberry cultivation under semi-hydroponic conditions has increased in the Southern region of Brazil since it facilitates management, besides avoiding weather adversities, pests and diseases. However, protected environment can hamper the access of pollinator insects, essential for crop productivity. This work aimed to know the entomofauna associated with strawberry cultivar Albion cultivated under semi-hydroponic conditions and to identify potential species of native pollinating bees. Insects were captured at different times of the day (9:30 a.m., 12:30 p.m. and 3:30 p.m.) in three commercial crops located in Bento Gonçalves and Farroupilha, Rio Grande do Sul. In each study area, three days of sampling were carried out during January 2017. The flowers were visited by 47 species of insects. Apis mellifera (Hymenoptera: Apidae) was the most abundant, constant, dominant and frequent species. Twelve species of native bees were identified: Tetragonisca fiebrigi, Tetrapedia sp., Trigona spinipes, Schwarziana quadripunctata, Plebeia emerina, P. remota, Bombus pauloensis (Hymenoptera: Apidae), Dialictus sp.1, Dialictus sp.2, Augochloropsis sp.1, Augochloropsis sp.2, and Augochlora sp.1 (Hymenoptera: Halictidae). All these species are potential pollinators of the crop. The native species T. fiebrigi, P. emerina and P. remota present potential for directed pollination of the strawberry under protected cultivation due to abundance and ease of management.


Research
Hortic. bras., Brasília, v.37, n.3, July -September 2019 B elonging to the group of small fruits, strawberry (Fragaria x ananassa) is the most widespread crop in the world, being cultivated in nearly all temperate to tropical climate countries (Galvão, 2014). In Brazil, strawberry cultivation is popular in several regions, most notably in the states of Minas Gerais, Paraná, Rio Grande do Sul, São Paulo, Espírito Santo, Santa Catarina and Distrito Federal. The average productivity is about 30 t ha -1 , marked differences between regions, depending on the location and cultivation system adopted can be noticed, though (Antunes et al., 2015).
On strawberry crop, pollination is essential to reach physiological maturity, produce fruits and seeds. Although most cultivars are self-fertile, some show variations on their selfpollination ability (Witter et al., 2014). In many cases stigma becomes receptive before anthers release pollen, favoring cross-pollination (Roselino et al., 2009). Pollinating agents like bees are able to carry pollen among different plants, promoting pollination (Zebrowska, 1998).
H o w e v e r, c u l t i v a t i o n u n d e r protected environment conditions may be a physical barrier to the action of pollinating insects (Antunes et al., 2007). Insufficient amounts of pollen and lack of individuals to carry it among flowers result in imperfect egg fertilization, which decreases hormone production, especially auxins, which would promote the growth of the receptacle area near the achene (Abrol et al., 2017). These factors contribute to high percentages of fruit deformation and lower yield (Malagodi-Braga, 2002;Witter et al., 2012). Considering the global importance of this fruit and the high investment in its implementation and management, it is essential to take pollination into account. Some studies on species of floral visitors in strawberry were carried out under open field cultivation (Malagodi-Braga, 2002;Albano et al., 2009;Connelly et al., 2015;Abrol et al., 2017), whereas in protected environments, few studies can be found in literature (Antunes et al., 2007;Roselino et al., 2009, Witter et al., 2012.
The aim of this study was to know the entomofauna associated with strawberry flowers cultivated in semihydroponic system in Serra Gaúcha Region and identify potential species of native pollinating bees.
The authors used a semi-hydroponic system in the three areas, with strawberry plants grown on benches and in plastic bags with substrate. Nutrient availability was carried out through dripping irrigation system, 12-cm spacing between plants. Cultivation was in a protected environment with plastic cover (in a high tunnel greenhouse, arch type) (30 m length) with open sides. Flowering and fruiting of strawberry was noticed all over the period of this study, since the used cultivar is indifferent to the photoperiod (dayneutral).
Traditional crop managements were kept, such as stolon removal, dry leaves and leaves in excess, application of insecticides and fungicides and fruit harvest. Management of these pesticides was similar in areas A and B; in area C, history of application along the development of the crop showed the adoption of more intense management in relation to the other areas, though. The active ingredients used in area C were: fluazinam, pyrimethanil, a z o x y s t r o b i n + d i f e c o n a z o l e , procimidone, boscalide + cresoximmethyl, abamectin, chlorfenapyr, azadiractin and spinetoram. The occurrence of strawberry borer Lobiopa insularis (Coleoptera: Nitidulidae) and thrips Frankliniella occidentalis (Thysanoptera: Thripidae) resulted in the need for frequent applications of chlorphenapyr and spinetoram in this place. In areas A and B, the following active ingredients were used: fluazinam, iprodione, azoxystrobin + difeconazole, abamectin, azadiractin and chlorfenapyr. In area A, besides these ingredients, the authors also used Bacillus subtilis, for managing Botrytis cinerea (Helotiales: Sclerotiniaceae).
The landscape surrounding the areas was characterized as follows: area A= existence of small native forest fragments near protected cultivation; area B= presence of large native forest fragments around cultivation; and area C= lateral fragment formed by eucalyptus and fruit monocultures (persimmon tree, vine and peach tree).

Sampling of floral visitors
To collect floral visitors, 25-meter four transects/cultivation lines were randomly established (adapted from Vaissiéri et al., 2011). Insects were collected directly from flowers with entomological net, totaling three sampling days per area in January 2017. Two previously trained collectors ran each predetermined transect at the same time for fifteen minutes (7.5 min round trip), three times along the day (09:30 a.m., 12:30 p.m. and 3:30 p.m.). Soon after collection, insects were stored in falcon tubes (50 mL) containing cotton moistened with ethyl acetate. In laboratory, insects were sorted, counted, mounted with entomological pins and labeled (Malagodi-Braga, 2002). Specimen identification was performed using a stereomicroscope, with the aid of dichotomous keys and specialists and later deposited in the Ceslau Biesanko Museum entomological collection, belonging to Universidade Federal de Pelotas, Pelotas-RS. The collections were performed on sunny days, with mild winds and temperatures above or equal 15ºC.

Data analysis
The diversity of insects in the study areas was evaluated by using Hill's diversity profile (1973) using R statistical program. Faunistic analysis was performed to define the abundance, constancy, dominance and frequency classes of bee species, according to described by Silveira Neto et al. (1976).

RESULTS AND DISCUSSION
A large variety of insects (47 species) was collected visiting strawberry
Comparing the study areas (A, B and C) using Hill's analysis, we observed a higher diversity of floral visitants in area B, followed by area A, when compared to area C (0= species richness; 1= Shannon Wiener index; 2= Simpson index) (Figure 1). However, regardless of insect diversity, we observed that as equability values were added to the Hill series (to the curve's right), communities ended up behaving similarly ( Figure  1), which indicates similar species distribution.
Differences in abundance and diversity values may be associated to the management adopted in each area and the inequality between landscapes near the crops. The scarcity of forest fragments and presence of monocultures of fruit trees around area C may have contributed to these results. According to Ricketts et al. (2008), landscape changes due to intensification of agriculture directly threaten biodiversity. In addition, frequent insecticide applications for strawberry borer and thrips management may have influenced the lower insect diversity in this area. Effects of pesticides on nontarget organisms have been portrayed in several studies (Tomé et al., 2015;Pitts-Singer & Barbour, 2016). The presence of larger forest fragments around area B may explain the higher diversity index, as these sites serve as shelter and nesting for various insect species, especially native bees. In area A, despite the existence of small fragments, they were present in larger quantities and closer to the protected cultivation. The surrounding landscape has an effect on the stability of ecosystem services, such as insect pollination (Halinski et al., 2015). Garibaldi et al. (2011) observed that floral visiting bee richness, visitation rate and fruit yield of 21 agricultural crops studied in 15 countries decreased with increasing distance from natural areas.
The order Hymenoptera was the most abundant in all study areas, followed by the order Diptera in areas A and C and the order Coleoptera in area B (Figure 2). The largest abundance of Coleoptera in this area was probably due to the high population of Diabrotica speciosa (Coleoptera: Chrysomelidae). The predominance of insects of these Table 2. Fauna analysis of bee species sampled in three sites with semi-hydroponic strawberry. Pelotas, UFPel, 2017.  three orders was also observed in flowers of 'Campinas' and 'Dover' strawberry cultivated in open system (Malagodi-Braga, 2002). This indicates that even under protected cultivation, as in the case of this greenhouse model adopted in the present study, insects go to the flowers for food resources, and bees, main representatives of the hymenoptera, stand out because they remove nectar and large amounts of pollen to feed the young (Malagodi-Braga, 2002).

Genus or species/area
In relation to species richness, the order Diptera stood out from the rest in areas A and B. In other studies, high richness of Diptera in strawberry flowers of cultivars Chandler and Camarosa was observed (Albano et al., 2009;Abrol et al., 2017). Most collected species of Diptera and Coleoptera are generalist insect predators, which can explain their presence in flowers, as besides prey which may appear, they also feed on pollen, an excellent protein source (Casari & Ide, 2012). Family Syrphidae (Diptera) was represented by several species, among them the genera Toxomerus, Eristalis, Palpada and Quichuana. In adulthood, these species mimic bees and wasps, feeding almost exclusively on pollen and nectar. During the search for these resources, they end up carrying pollen adhering to their bristles, and may complement the bees in pollinating flowers (Marinoni et al., 2007;Abrol et al., 2017). Other species of families Faniidae, Sarcophagidae, Calliphoridae and Sepsidae (Diptera) are considered only floral visitors, since most grow on decaying organic matter.
Coleoptera of families Curculionidae, Cantharidae, Chrysomelidae (D. speciosa), Tenebrionidae (Lagria villosa) and Scarabaeidae (Macraspis dichroa) were present in the flowers for food. Diabrotica speciosa was observed damaging the reproductive structures of strawberry. Eriopis connexa (Coleoptera: Coccinellidae) and Coleomegilla quadrifasciata (Coleoptera: Coccinellidae) are predators and, just like hemiptera of families Geocoridae and Miridae are present in the environment, playing an important role in biological control of insects-pests. Rhyparochromidae (Hemiptera) and the species Hylephila phylaeus phylaeus, Conga iheringii (Lepidoptera: Hesperiidae), as well as Duponchelia fovealis (Lepidoptera: Crambidae), were considered floral visitors, as they were present only for food and sometimes causing damages to strawberry plants, such as Rhyparochromidae and D. fovealis (Bernardi et al., 2015).
Richness of visiting floral species of the order Hymenoptera was similar in all study areas, with eight species in areas A and C, and seven species in area B, composed of bees belonging to families Apidae and Halictidae (Table 1). The most abundant species was Apis mellifera (Hymenoptera: Apidae), representing 62.7% of the hymenoptera sampled in area A, 72.4% in area B and 90.1% in area C (Table  1). According to faunal analysis, this species was classified as the most abundant, constant, dominant and very common in all sampled areas (Table 2). Similar results were found in studies evaluating the diversity of insects in cultivars Campinas, Dover and Chandler (Malagodi-Braga, 2002;Abrol et al., 2017). These bees live in colonies with hundreds to thousands of individuals and are easily found due to high foraging activity as they need to collect large amounts of pollen to feed their young (Malagodi-Braga, 2002). They currently represent the main species managed for pollination of agricultural crops worldwide (Rosa et al., 2019).
Among native species, T. fiebrigi, P. remota and P. emerina are easily found in biomes in the state of Rio Grande do Sul and, in relation to their good adaption to rational management, they show a great potential to use in largescale in protected crops. Tetragonisca angustula and P. nigriceps, belonging to the same genera of the species observed in this work, were efficient in pollinating strawberry flowers cultivated in protected environment, reducing mainly the percentage of deformed fruits (Antunes et al., 2007;Witter et al., 2012). Trigona spinipes and B. pauloensis were also found, however, some authors report difficulties of managing these pollinating species, since they build their nests in trees and pre-existing cavities, respectively (Marsaro Júnior et al., 2017).
Dialictus sp.1 was the most abundant native bee in the three areas. Genus Dialictus, as well as genera Augochlora and Augochloropsis, comprise solitary or semi-social bees, known as metallic bees. These bees are easily found pollinating several crops, belonging to the genera with the highest species richness in Southern Brazil (Halinski et al., 2015). Despite the ease of being found in agroecosystems, and the important pollination service they deliver, little is known about their biology, and the fact that they are socially poor makes rational management of pollination difficult. Therefore, it is necessary to adopt conservation measures for these species in their natural habitat, by maintaining forest fragments close to strawberry crops.
The sampling time with the highest abundance and richness of flowervisiting bees was at 12:30 p.m. in all areas (Table 1). The decreased visitation throughout the afternoon may have been influenced by the reduced availability of floral resources. According to Polatto et al. (2014), after a certain period of the day, the scarcity of floral resources produced by most plants can stimulate bees to forage flowers early in subsequent days. Imperatriz-Fonseca et al. (1985) highlight that resource availability is one of external factors which regulate the flight activity of bees.
Higher number of native bees and individuals visited flowers until 12:30 p.m., with declining activities after this period (Table 1). Similar behavior was verified in melon plants (Cucumis melo), where native bees preferred to visit inflorescences in order to collect pollen and nectar during morning time (until 12 p.m.), when temperatures tend to be milder (Tschoeke et al., 2015). The abundance of individuals of species A. mellifera was higher at 12:30 p.m., remaining more active than the other species after 3:30 p.m. (Table  1); according to Chang et al. (2001), their foraging pattern is between 10 and 4 p.m. Tschoeke et al. (2015) verified that A. mellifera maintained its activity during afternoon, only to collect nectar, though. Interspecific differences in foraging activities may be directly related to abiotic factors (temperature, luminosity and relative humidity) and the generalist habit of A. mellifera, which has populous swarms with a high need for food resources, making it efficient in recruitment of individuals (Giannini et al., 2015).
Knowing some particularities among species may contribute to adopt management strategies for strawberrycrop pollination. One possibility to optimize this ecosystem service is the association of A. mellifera, managed in hives near the crops, with native bees, which can be installed inside protected environments and preserved in surrounding forests. The complementary action of different bee species makes the pollination process even more effective, assisting mainly in improving fruit formation (Malagodi-Braga & Kleinert, 2007).
To simultaneously benefit crop and preserve pollinator species, it is recommended that insecticides and/or fungicides should be applied, when necessary, in the late afternoon, trying to coincide with the lowest pollinator activity times. In addition, the maintenance of native forests around protected cultivations is fundamental for the natural reproduction of the species.
In summary, strawberry cultivation in semi-hydroponic system is visited by a wide entomofauna, including insects of the orders Hymenoptera, Diptera, Coleoptera, Hemiptera and Lepidoptera, being Hymenoptera the most frequent and abundant. The 13 bee species belonging to the Apidae families (A. mellifera, T. fiebrigi, Tetrapedia sp., T. spinipes, S. quadripunctata, P. emerina, P. remota and B. pauloensis) and Halictidae (Dialictus sp.1, Dialictus sp.2, Augochloropsis sp.1, Augochloropsis sp.2, Augochlora sp.1), are potential pollinators of the crop, since they present adaptations for transporting pollen. In the municipalities of Bento Gonçalves and Farroupilha, the introduction of T. fiebrigi, P. emerina or P. remota hives into protected strawberry crops may be an alternative to the pollination deficit due to abundance and easy handling of these species.