INTRODUCTION

Regarding their role as biological control agents, the syrphid larvae, important natural enemies in agriculture, are largely found in the subfamily Syrphinae which has more than 1,800 species (Rojo et al., 2003). They feed on soft-bodied Hemiptera, mainly aphids, although some species can also feed on other arthropods such as thrips, whiteflies mites, young lepidopteran larvae, and psyllids (Rojo et al., 2003, Villa et al., 2016). Aphidophagous larvae are considered good biological control agents in several crops because of their voracity (Nelson et al., 2012; van Lenteren, 2012; Gomes Fidelis et al., 2018).

In Argentina, there are studies of taxonomy and biology of several species, mostly from Mendoza, Tucumán, Santa Fe and Entre Ríos (Greco, 1995; Driutti, 1999; Bertolaccini et al., 2008) and recently, more attention about their role as biological control agents has increased (López García & Maza, 2013; Maza et al., 2016; Díaz & Maza, 2017). In the province of Buenos Aires, Greco (1995) studied the phenology and habitat selection of 6 aphidophagous hoverflies species and found that they were present throughout the year, and distributed along an environmental mosaic with different crops and natural vegetation.

Plant diversity management is a technique of conservation biological control which consists of environmental manipulation to improve the effectiveness of established natural enemies (^{Barbosa, 1998}). In addition to this, habitat management can be considered as a conservation biological control method that alters habitats to improve availability of the resources required by natural enemies for optimal performance, conserving and enhancing favorable conditions or reducing unfavorable ones (Landis et al., 2000). Adult hoverflies need pollen, rich in protein, to mature the reproductive system in both females and males, and also females use pollen for eggs development (Gilbert, 1981; Haslett, 1989). In turn, flower nectar is an important energy resource both for flight and for the survival of adult hoverflies (Haslett, 1989). Floral resources and wild plants, that provide food and refugia to the hoverflies both within and around crops, are essential for their role as biological control agents (Pineda, 2011; van Rijn et al., 2013).

Moreover, one negative aspect for biological control is the presence of natural enemies of the control agents (Mills, 2006). Aphidophagous syrphids larvae are parasitized by Hymenoptera species of the

superfamilies Ichneumonoidea and Chalcidoidea. Parasitoid pressure can decrease hoverfly larval rate of predation on prey, and consequently could limit the biological control provided by them (Tinkeu & Hance, 1997; Hazell et al., 2005). However, the impact of parasitoids on hoverfly populations has not been widely studied, and in general, is highly variable ranging from nil to more than 50% of parasitization rates (Rodríguez-Gasol et al., 2020).

Aphids are a common pest in horticultural crops (van Emden & Harrington, 2017). Aphid predators and parasitoids are used as biological control agents through different strategies worldwide to control aphid populations. In horticultural systems, hoverflies are used through augmentative and conservation biological control, mainly in Europe (van Lenteren, 2012; van Lenteren et al., 2018). In Argentina, the knowledge of the biology and ecology of the most frequent species in this system is still incipient (Maza et al., 2016; Díaz & Maza, 2017; Díaz et al., 2020).

The aims of the current study were: a) to determine the most common predatory hoverfly species in organic crops of lettuce and broccoli in the horticultural belt of La Plata, b) to describe the numerical variations of immature hoverflies throughout the year and their relationship with the aphids’ abundance, c) to analyze the percentage of larval parasitism, and d) to identify wild plants species near to these crops that are frequently visited by hoverflies.

MATERIAL AND METHODSThis study was developed in one commercial horticultural farm located in Etcheverry, locality belonging to the horticultural belt of La Plata, Buenos Aires, Argentina (35°01’21.3” S; 58°03’25.5” W).

Horticultural farms of the region have several seasonal crops (tomato, sweet pepper, eggplant, artichoke, leaf vegetables, and strawberry) that are cultivated throughout the year under open fiel or greenhouse conditions. A random sampling design was used in lettuce and broccoli crops, which had approximately 300 plants each. Samples were taken fortnightly, between March 2019 and March 2020, and samples consisted of 30 sampling units (one leaf per plant) of each crop.

Lettuce and broccoli crops

The collected leaves were analyzed under a stereoscopic microscope in the laboratory. Aphids and immature stages of syrphids (larvae and pupae) were identified following Malais & Ravensberg (2004) and Maza (2018), respectively, and the number of individuals of each species was recorded. The syrphids were placed individually in Petri dishes and conditioned in rearing chambers (25 °C, 55-65% RH and 14:10 L:D) until the emergence of adults or parasitoids. The pupae from which no insects emerged were kept for one month and then they were dissected to detect the presence of developing parasitoids. Adults of syrphids identification were carried out following the specific keys of Thompson et al. (2010), and parasitoids identification with the keys of Fitton & Rotheray (1982) and Gibson (2001). The percentage of parasitism was estimated as the number of

parasitoids in relation to the total number of larvae and pupae collected. The number of parasitoids was estimated as the number of emerged adults plus the number of individuals of the parasitoid that did not complete their development.

Wild plants

The wild plants present in one or two patches adjacent to each crop, depending on the time of the year and the cultural management of the field were surveyed. The coverage of each wild plant species was evaluated in order to consider the most abundant species. The coverage estimation was made in a rough way and those species that visually covered more than 30% of the total patch area were considered abundant. Two whole plants of each abundant species were taken for their identification at the laboratory. The plants were botanized and deposited in the Laboratory of Evolutionary and Systematic Plant Anatomy Studies (LEAVES) of the Faculty of Natural Sciences and Museum of La Plata.

At the same time, three points (around 1 m diameter) from each patch were randomly selected, and 10 minutes observations were made in each point to collect adults of syrphid using plastic boxes. The plant on which they were, as well as whether the plants were in bloom, were recorded by direct observation. The specific identification of syrphids was carried out, in the laboratory, following Thompson et al. (2010). The frequency of syrphid adults of each species in the different seasons of the year was compared separately using one-way ANOVA or ANOVA with permutation test when normality and homoscedasticity assumptions were not fulfilled. Analyses were performed using the software R, version 3.5.1 (R Core Team, 2018).

RESULTS

The hoverfly adults recorded in the wild plants in this study were Allograpta exotica Wiedemann, Toxomerus duplicatus Wiedemann and Syritta flaviventris

Macquart (Diptera: Syrphidae). The last one

belongs to the subfamily Eristalinae and it has saprophagous habits, whereas A. exotica and T. duplicatus belong to the subfamily Syrphinae and their larvae have predatory habits, feeding mainly on aphids.

Lettuce crop

In this crop immature stages (eggs, larvae and pupae) were found from August, all of them belonging to A. exotica. The greatest abundance was recorded in spring, coinciding with the greatest abundance of aphids (Fig. 1a).

Three aphid species were recorded throughout the study period, Myzus persicae (Sulzer), Macrosiphum euphorbiae (Thomas) and Nasonovia ribis-nigri (Mosley). Myzus persicae was the most abundant, in the autumn, winter and spring, followed by M. euphorbiae which had higher relative abundance in the summer (Fig. 2a).

In lettuce crop, the species Diplazon laetatorius (Fabricius) (Hymenoptera: Ichneumonidae), a larval parasitoid of A. exotica, was recorded in October and February, causing 33% and 100% of parasitization, respectively (Fig. 3a).

Broccoli crop

The presence of immature stages of syrphids were recorded from October to February and, as in the lettuce crop, all belonged to A. exotica. In the spring and summer, two increases in the abundance of A. exotica were observed and coincided with the greatest abundance of aphids in the crop (Fig. 1b). The species of aphids recorded in this crop were M. persicae, M. euphorbiae and Brevicoryne brassicae (L.), the latter being predominant followed by M. persicae (Fig. 2b).

Two species of syrphid parasitoids, D. laetatorius and Pachyneuron aff. nelsoni (Hymenoptera: Pteromalidae), were recorded during the spring and summer. Diplazon laetatorius caused a parasitism that ranged between 20% and 100%, and the parasitism by P aff. nelsoni ranged between 8 and 100% (Fig. 3b).

Fig. 1: Mean number of the hoverfly Allograpta exótica and aphids per leaf in the (a) lettuce crop and (b) broccoli crop. E: eggs, L: larvae, P: pupae. Brackets indícate SE. 

Fig. 2: Relative abundance of aphids in the (a) lettuce crop and (b) broccoli crop. 

Fig. 3: Total number of healthy immature stages of A. exotica and parasitized by D. laetatorius and Pachyneuron sp. in the (a) lettuce crop and (b) broccoli crop.  

Wild plants

Hoverfly adults were recorded on five wild plants in vegetation patches adjacent to lettuce and broccoli crops, all of them Asteraceae, and the most frequent throughout the year were Galinsoga parviflora Cav. “wild basil” and Matricaria chamomilla L. “chamomile”. A seasonal alternating occurrence of these plants (i.e. some replacement of these species over time), which were visited in their flowering period by both species of predatory syrphids, was observed (Fig. 4). Adults of A. exotica on wild plants were more abundant in the winter (F = 8.35; d.f. = 3, 11; P = 0.003) meanwhile T duplicatus abundance was similar in every season (F = 0.35; d.f. = 3, 11; P = 0.83) (Fig. 5).

Fig. 4: Presence of Allograpta exótica (black squares) and Toxomerus duplicatus (white circles) on wild plants in bloom (black lines) adjacent to the lettuce and broccoli crops throughout the year in one horticultural farm in La Plata, Buenos Aires, Argentina. 

Fig. 5: Mean number of Allograpta exotica (left) and Toxomerus duplicatus (right) adults throughout the year in the wild vegetation near the lettuce and broccoli crops. Brackets indicate the SE. 

DISCUSSION

Allograpta exotica and T duplicatus are predatory hoverflies that were found on wild plants and in the lettuce and broccoli crops. Both A. exotica y T. duplicatus are widely distributed in the Neotropical region, from Mexico to southern Chile and Argentina. In

Argentina, these species have been recorded from Jujuy to Río Negro provinces in many agricultural and natural systems (Maza, 2018). In horticultural crops of Entre Ríos province, Díaz & Maza (2017) found T duplicatus with T watsoni (Curran) in lettuce crop as well as adults of the same species and A. exotica in alyssum strips Lobulada marítima (L.) Desv. (Brassicales: Brassicaceae), added to enhance the biological control by conservation.

The predominant aphids associated with Toxomerus species in Argentina have been Uroleucon sonchi (L.) and M. euphorbiae (Díaz & Maza, 2017), while in our study, the most abundant aphid associated with A. exotica was M. persicae, followed by M. euphorbiae. On the other hand, the lettuce aphid N. ribisnigri, that is predominant in North America, Europe, New Zealand and Australia (Díaz & Fereres, 2005) was recorded, in Argentina, as an important species only in greenhouse lettuce crop in La Pampa (^{Baudino et al., 2007}). The relative abundance of N. ribisnigri in our study system was low and there are no records of its population density in Buenos Aires crops.

In relation to the crucifer crops in Argentina, syrphid larvae have been found associated with B. brassicae in cabbage (Dubrovsky Berensztein et al., 2017), since this species is the most frequent and abundant in plants belonging to that group. Gomes Fidelis et al. (2018) found that one of the main causes of B. brassicae mortality in Brazil was the predation of nymphs and adults by larvae of A. exotica. Larvae of other syrphid species are known to be effective predators of B. brassicae and could be considered an important factor to reduce their population growth (Jankowska, 2005).

Regarding larval-pupal parasitism, of the two A. exotica parasitoid species identified D. laetatorius was the predominant species in both lettuce and broccoli crops. This is a solitary parasitoid with a worldwide distribution (Bordera et al., 2001). The association of this parasitoid with A. exotica has been mentioned by Korytkowski (1967) and Greco (1997) in Peru and Argentina, respectively, and A. exotica is regarded as one of its main hosts. The other parasitoid recorded in our study is gregarious and belongs to the genus Pachyneuron, although it has not been identified to species level it was possible to determine that it is closely related to P nelsoni (Díaz Lucas et al., 2019). Pachyneuron is a cosmopolitan genus that includes about 60 species distributed mainly in the Palaearctic region (Gibson, 2001; Noyes, 2020), and its position in the trophic network is very wide. Most species of Pachyneuron are hyperparasitoids of Aphididae or other phytophagous Hemiptera (Coccoidea, Psylloidea) through their Braconidae (Ichneumonoidea), Aphelinidae, and Encyrtidae (Chalcidoidea) primary parasitoids. Furthermore, they can be primary parasitoids or hyperparasitoids of predators belonging to different taxonomic groups (Diptera: Syrphidae, Chamaemyiidae; Coleoptera:

Coccinellidae; Neuroptera: Chrysopidae) (Noyes, 2020). Pachyneuron nelsoni is a widely distributed species that has not been recorded yet in the Americas and is generally known as a parasitoid of syrphids (Gibson, 2001; Noyes, 2020). The absence of taxonomic specific keys for the Chalcidoidea of the Neotropical region makes the determination of the species found in Argentina difficult. The right identification of the species found in this study and further investigation to know its biology will allow us to understand its position in the trophic network and its potential effect on the biological control of aphids. Therefore, if it were a primary syrphid parasitoid its effect would be negative, while if it were a hyperparasitoid -i.e. parasitoid of a syrphid parasitoid-, its presence in the system could be positive for biological control.

The two more frequent plant species, M. chamomilla and G. parviflora, in the patches of wild plants adjacent to the lettuce and broccoli crops, have annual growth. Matricaria chamomilla is a plant native to Europe and Iran, introduced in our country in 1916. In the studied area, the observed flowering period -from winter to spring- differs from that recorded by Wojciechowicz-Zytko & Jankowska (2017), who mention that the flowering period in the northern hemisphere is from early summer to early autumn. Several authors found that M. chamomilla flowers were more attractive to different species of syrphids than other wild, aromatic, and flowering plants in several countries (Sadeghi, 2008; Wojciechowicz-Zytko & Jankowska, 2017). Galinsoga parviflora grows in temperate and subtropical zones, is native to Central and South America and was later introduced to Europe where it is now widely distributed. It is usually found in gardens and agricultural areas associated with various crops such as tomatoes, cabbage, potatoes, strawberries and corn. The flowering period occurs in the warm months of the year (Damalas, 2008), as was observed in this study.

The flowering plants are essential to provide pollen, nectar and refuge to pollinators and biological control agents; however, it is necessary to evaluate eventual negative aspects of these plants, mainly as host potential pests. It is known that colonies of the aphid Aphis fabae Scopoli are frequently associated with M. chamomilla (Wojciechowicz-Zytko & Jankowska, 2017), as well as other aphids and cicadellids are associated with G. galinsoga (Batra, 1979).

The temporal occurrence of the adults throughout the year and the immature stages of hoverflies only in the warmest months observed in this study is concordant with the finding of Villa et al. (2016) and Greco (1995), and could be due to facultative reproductive diapause of females in the winter, as was observed in some species (Rodríguez Gasol et al., 2020). The wild plants surrounding the crops will probably favor overwintering hoverflies by providing both undisturbed habitat and more overwintering sites (Rodríguez Gasol et al., 2020). Therefore, the temporal alternation of the flowering between M. chamomilla and G. galinsoga would offer resources to hoverfly adults to remain in this horticultural system throughout the year.

The results of this study contribute to the identification of the wild plants adjacent to agricultural crops frequently visited by syrphids. We also present new information on the parasitoid species attacking syrphid larvae within lettuce and broccoli crops. Wild flowering plants and parasitoids can be viewed as positive and negative factors for Syrphids, respectively and should be considered to design aphid biological control strategies in these crops.

ACKNOWLEDGMENTS

We thank Eliana Nieves for the maintenance of the insect colonies. This research was supported by grants from Facultad de Ciencias Naturales y Museo (Universidad Nacional de La Plata) Proyectos de Investigación Básica y Aplicada (PRIBA) 2018.