The suitability of native flowers as pollen sources for Chrysoperla lucasina (Neuroptera: Chrysopidae)

Rafael Alcalá Herrera; María Luisa Fernández Sierra; Francisca Ruano

doi:10.1371/journal.pone.0239847

. 2020 Oct 23;15(10):e0239847. doi: 10.1371/journal.pone.0239847

The suitability of native flowers as pollen sources for Chrysoperla lucasina (Neuroptera: Chrysopidae)

Rafael Alcalá Herrera ^1,^*, María Luisa Fernández Sierra ¹, Francisca Ruano ²

Editor: Manu E Saunders³

PMCID: PMC7584243 PMID: 33095792

Abstract

Green lacewings (Neuroptera: Chrysopidae) are key biological control agents found in a broad range of crops. Given the importance of enhancing their presence and conservation, in this study, we aim to identify and to determine the relative importance of the pollen consumed by Chrysoperla lucasina (Lacroix, 1936) from 29 pollen types offered by 51 native plant species sown in an experimental farm in Villarrubia in the south of Spain. For the purposes of this study, C. lucasina specimens were captured in the late spring of 2016 and 2017. The pollen types and other components in the alimentary canal of C. lucasina were microscopically identified using the transparency method, which is a novel technique applied to green lacewings captured in the field. The results show that (i) C. lucasina feeds on over half of the pollen types offered by the sown plant species, with no differences in behaviour by sex or year; (ii) Capsella bursa-pastoris was the most frequently identified pollen type in the alimentary canal; (iii) the majority of pollen types identified correspond to sown native plant species and not to surrounding plant species; and that (iv) most of the adults studied also consumed honeydew. Our feeding study has important implications for the selection of plant mixtures for ground cover restoration and flower vegetation strips in Mediterranean agroecosystems, which complements our previous findings on how C. lucasina use native plant species as host and reproduction sites. The plant species Capsella bursa-pastoris and Biscutella auriculata, which are best suited to provide pollen, host and reproduction sites for C. lucasina in late spring, should consequently be included in the proposed plant mixtures for Mediterranean agroecosystems.

Introduction

Agricultural intensification has increase global agricultural production, while also expanding agricultural land area and the use of external inputs such as pesticides, fertilizers and irrigation [1,2], as observed in Mediterranean vineyards, olive groves and fruit orchards [3–5]. However, these agricultural practices have had adverse environmental effects such as loss of natural habitats and biodiversity, leading to a decline in ecosystem services, particularly pest control and crop pollination, as well as in soil structure and fertility [6,7]. It is vitally important to minimize negative environmental effects through effective ecological intensification strategies which require a good understanding of the relationship between land use and communities of organisms [8]. For example, the European Union has developed agri-environmental policies to reduce pesticides use and to promote biodiversity in agricultural landscapes through the protection and creation of semi-natural habitats (SNH) defined as habitats composed of non-crop plant species, both within and/or outside crop areas [9–11].

Conservation biological control, an alternative to the use of pesticides, seeks to attract and maintain natural enemies in crops by modifying and managing SNH [12]. Agricultural landscapes are characterized by a diverse range of SNH such as hedgerows, woodlands and forests, cover crops, herbaceous ungrazed habitats and grassy linear habitats [11,13]. These types of SNH, with their higher insect abundance and diversity than crops [14,15], have the potential to attract and support natural enemies of insect pests [16,17]. The relationship between plant species and insects is linked to reproduction and feeding [18]. Flowering plants attract insects through their floral resources, including pollen, nectar and honeydew, which are important at certain stages in the life of many natural enemies, as well as alternative prey and hosts, shelter and oviposition sites [19–22]. Although several grass and wildflower seed mixtures are currently available to farmers to increase arthropod abundance and diversity [15], commercial seed mixtures are less adapted to Mediterranean climates [23,24]. The use of native species could have a beneficial impact on ground cover while avoiding the disadvantages of non-native plant species [24–27]. Thus, knowledge of the ways in which different native plant species attract natural enemies and pollinators and of how their floral resources are used could contribute to the development of more effective mixtures and to the promotion of effective landscape management practices [28–31].

Lacewings, which belong to the functional group of natural enemies, are present in virtually all field crops around the world. They are some of the most studied predators of pests [32] and are widely used by integrated and organic pest management systems within the framework of classical biological control [33]. Green lacewings, which can be captured in herbaceous, scrub and tree strata [34–36], are dependent on the presence of SNH [17,37]. Species belonging to the complex Chrysoperla carnea (Stephens) are the most frequently identified and abundant green lacewings in cultivated areas [32], whose wide ecological and geographical distribution is due to their ecological versatility [35]. C. carnea (Stephens) sensu Henry [= Chrysoperla affinis (Stephens) sensu Thierry] and Chrysoperla lucasina (Lacroix, 1936), the first green lacewings to appear in field crops, are attracted to large patches of flowering plants [38], which are used as oviposition and feeding sites [20,28]. Green lacewing larvae prey on a wide range of small, soft-bodied insects, as well as on the eggs and small larvae of lepidopteran insects [39], while the adults, generally palyno-glycophagous, feed on honeydew, pollen and nectar [38,40–42]. Laboratory [43–45] and field [46,47] studies have concluded that proteinaceous food is necessary for lacewing egg production, while other studies have shown that flowering resources can influence life history parameters such as survival, reproduction and development time [45,48,49].

Analysis of pollen grains in the alimentary canal of palynophagous insects has revealed the habitats and plants visited, what they eat, as well as dispersal movements in and around agroecosystems [50]. Different methodologies have been used to extract alimentary canal contents from insects [51–54]. These include a process called acetolysis, by which an insect is destroyed by artificial acid digestion [50], rendering it impossible to determine where the pollen is located in the alimentary canal and thus to determine when the pollen were consumed. However, the transparency method can be used to identify and locate pollen in the insect’s alimentary canal [55]. Analyses of pollen should be combined with plant inventories that provide the set of available pollen resources for chrysopids [56,57]. Previous studies have reported that chrysopids, which consume pollen where and when available, mainly feed on herbaceous plant species belonging to the families Apiaceae, Asteraceae, Brassicaceae, Chenopodiaceae-Amaranthaceae, Fabaceae, Liliaceae, Plantaginaceae, as well as tree and shrub species from the families Ericaceae, Cistaceae, Oleaceae and Pinaceae [38,58]. Hence, knowledge about how insects in general, and more specifically chrysopids, use floral resources, particularly in SNH, is critical for successful habitat management strategies aimed at improving natural biological control [29,38,59,60].

The principal objective of this study is to identify and to determine the relative importance of the pollen consumed by C. lucasina adults by observing the alimentary canal of individuals collected from sown native plant species. Specifically, we were interested in:

whether C. lucasina is a generalist or specialist pollen feeder,
the principal sown native species exploited,
the importance of surrounding natural plants species in relation to the pollen consumed,
whether the pollen consumed by both sexes show a similar pattern,
and the possibility of identifying other components (fungal spore, honeydew) in the alimentary canal.

This should enable us to advance our knowledge of the feeding behaviour and ecology of C. lucasina and to suggest the most effective plant species to attract and conserve this key predator in Mediterranean agroecosystems.

Material and methods

Study area

The study was carried out on an experimental farm in the village of Villarrubia (37°49′49″N, 4°54′20″W) in the southern Spanish province of Cordoba. The farm is bordered by a commercial orange orchard, an olive orchard and various irrigated crops, as well as, to the south, by the Guadalquivir river, with its riverbank vegetation. In addition, Pinus halepensis, Pinus pinaster and Pinus pinea trees can be found in the Sierra Morena mountain range, five kilometres to the north of the farm [61]. No specific authorizations were required for the field study, which did not involve endangered or protected species.

In the experimental farm, in order to test the way in which chrysopid species exploited the plant resources, a set of 51 plant species were planted (S1 Table) which need to [62]: (i) be native angiosperms, (ii) be self-sowing winter annuals which do not compete for water with orchards during the summer season and (iii) have a bloom period prior to olive blooming in order to attract natural enemies to the agroecosystem. The potential use of these plants as ground cover and for seed production [25], as well as their attractiveness to on chrysopid species, were tested [28] in previous studies. The farm was tilled in late November 2015 and 2016, and the seeds were planted on consecutive days. The plots were irrigated once during germination and several times during plant development when necessary.

We sampled three 3x3 and 1x9 metre sampling areas (a total of 9 m²) in 2016 and 2017, respectively, for every plant sown in each sampling event. In 2016, 40 plant species belonging to 13 families (S1 Fig and S2 Table) were randomly planted in three blocks of 40 plots. Each plant species was sown in three 3x3 m plots (a total of 120 samples). In 2017, although 35 plant species were sown in a single randomized plot configuration with different areas, we sampled only 20 plant species based on the chrysopid abundance results for 2016 sampling (S1 Fig and S2 Table). The three samples (9 m²) were located equidistantly from the centre of each plot to avoid border effect (60 samples in total).

Due to their poor development, five plant species sampled in 2016 (Anarrhinum bellidifolium, Helianthemum ledifolium, Tuberaria guttata, Aegilops geniculata and Aegilops triuncialis) and two species sampled in 2017 (Medicago orbicularis and Medicago polymorpha) were omitted from the analyses.

Insect collection

Insects were collected only on emerged and well-developed blooming plant species (S2 Table). Three samples (9 m²) per plant species were vacuumed twice in May 2016 and 2017 between 9 a.m and 2 p.m for 40 seconds using an InsectaZooka aspirator (BioQuip^® Products Inc., Rancho Dominguez, CA, US). Samples were stored at -20°C and cleaned in the laboratory. Chrysopid adults were identified under a stereomicroscope up to species level and were sexed according to the Iberian chrysopid key [63].

Alimentary canal content analysis

The alimentary canal contents of all chrysopid adults collected were analysed by slightly modifying the transparency method described by Bello et al. [55]. Though previously used in studies of aquatic insect feeding and benthic fauna [64–67], this was the first time that this method was used to analyse chrysopid specimens collected from the field. The adult chrysopids were defrosted at room temperature, washed three times with distilled water and then vortexed to remove external pollen on the insect’s surface. After removing the wings, legs and antennae, each individual was placed in a vial, covered with Hertwig´s solution and heated in an oven at 60°C for a period of 24 hours. Two specimens, which were lost during the transparency manipulation process, were excluded from the pollen identification analysis. Subsequently, the specimens were mounted on a slide for microscopic examination under a DMI600B inverted microscope (Leica, Wetzlar, Germany) at 1000x magnification to identify the pollen. In addition, photomicrographs of the most frequently occurring pollen types were taken using a C-1 confocal microscope system (Nikon, Japan) at 1000x magnification to obtain a 3D picture of the shape of the pollen grain and to observe the autofluorescence of the exine according to the method described by Castro et al. [68]. Before pollen was identified, we divided the alimentary canal into five segments: I–head, II–thorax, III–initial abdomen, IV–medium abdomen and V–final abdomen. This was done to show the percentage of the alimentary canal occupied by the pollen in each segment, which was classified into three different categories: absent (0% grains), medium (under 50%) and high (50–100% occupation of the alimentary canal) at 40x magnification. The presence of pollen in each segment provided a rough approximation of the intensity of consumption, of when the pollen was consumed and of any differences between sexes with regard to the amount of pollen consumed.

Given that other components, such as nectar, honeydew, fungal spores, other fungi and/or arthropod exuviae, in the chrysopids alimentary canal have been previously reported [38,40,69,70], we visually checked their presence under an inverted microscope according to the method described by Lacey et al. [71]. Based on the findings of Villenave et al. [38,72], honeydew consumption was indirectly identified by the presence of fungal spores Cladosporium sp. and Alternaria sp.

Pollen identification

We examined the pollen according to their morphological traits (polar and equatorial axes, shape, apertures and exine ornamentation), up to type (plant species and/or genus assemblages), family, genus and/or species level. We identified the pollen using the method described by Valdés [73] and our reference pollen collection. This collection was created by growing the native plant species sown in the field under controlled greenhouse conditions: 25°C, 50–60% humidity and a photoperiod of 16:8h (light:dark). The collection was deposited at the Department of Environmental Protection in the Estación Experimental del Zaidín, a Spanish Scientific Research Council (CSIC) centre in Granada.

Statistical analyses

All analyses were carried out using the R software packages agricolae, FactoMineR, Factoextra and vegan [74–77]. As the data did not follow a normal distribution, the differences in the number of pollen types identified and the percentage of the alimentary canal containing pollen by sex were determined using the Kruskal-Wallis test with Bonferroni adjustment. Multiple correspondence analysis (MCA) was used to summarize and visualize the presence or absence of each pollen type in each adult chrysopid. MCA was applied to the different types of pollen recorded more than four times in the adult chrysopids examined in order to minimize the effect of pollen types occasionally consumed. As the sown native plant species differed slightly each year, we carried out MCA annually. A permutational multivariate analysis of variance (PERMANOVA), with Jaccard distance and 999 permutations, was then performed each year to determine whether pollen type composition differed significantly by sex in each year. Differences between sexes in relation to each fungal spore identified were also determined using the Fisher test in each year.

Results

A total of 109 C. lucasina adults were collected (46 in 2016 and 63 in 2017), of which 71 were females (27 in 2016 and 44 in 2017) and 38 were males (19 in both 2016 and 2017). Overall, all adults examined were found to have pollen in their alimentary canal, with the highest percentages observed in the initial and medium abdomen (Fig 1). The percentage of alimentary canal containing pollen varied significantly: 39.2±2% in females as compared to 27.5±2% in males (Kruskal-Wallis test χ² = 9.849, p< 0.01). However, we observed very similar mean values for the number of pollen types by sex: 3.01±0.17 (n = 71 females) and 3.03±0.26 (n = 36 males) (Kruskal-Wallis test χ² = 0.1, p = 0.757).

The 51 native herbaceous plants sown between 2016 and 2017 in the experimental farm were pooled in relation to 29 pollen types (Table 1). C. lucasina consumed 17 of the 29 pollen types, with the following families being the most notable among the adults examined (Table 1): types Anthemis arvensis, Calendula arvensis, and Crepis capillaris on Asteraceae; types Borago officinalis and Echium plantagineum on Boraginaceae; types Capsella bursa-pastoris and Biscutella auriculata on Brassicaceae; and types Silene latifolia, Silene vulgaris and Vaccaria hispanica on Caryophyllaceae. Five pollen types from the following surrounding natural plants were also identified: Apiaceae, Ericaceae, Trifolium arvense, Castanea sativa and Pinus pinea.

Table 1. Presence (Y) or absence (N) of the different pollen types identified in C. lucasina adults collected in the experimental farm in 2016/2017.

Family	Pollen type	Pollen type identified in 2016/2017
Apiaceae	^*Type Apiaceae	Y(1)/Y(5)
	Type Orlaya daucoides	N/N
Asteraceae	Type Anthemis arvensis	Y(18)/Y(22)
	Type Calendula arvensis	Y(1)/N
	Type Crepis capillaris	N/Y(9)
Boraginaceae	Type Borago officinalis	Y(1)/Y(3)
	Type Echium plantagineum	Y(9)/Y(14)
Brassicaceae	Type Capsella bursa-pastoris	Y(38)/Y(57)
	Type Raphanus raphanistrum	N/N
Caprifoliaceae	Type Scabiosa atropurpurea	N/Y(1)
Caryophyllaceae	Type Silene latifolia	Y(4)/Y(13)
	Type Silene vulgaris	Y(8)/Y(18)
	Type Vaccaria hispanica	Y(9)/Y(16)
Cistaceae	Type Helianthemum ledifolium	N/N
	Type Tuberaria guttata	N/N
Ericaceae	^*Type Ericaceae	Y(2)/N
Fabaceae	Type Fabaceae	N/N
	Type Lotus creticus	N/N
	Type Trifolium arvense	Y(2)/Y(4)
	Type Trifolium repens	N/N
Fagaceae	^Type Castanea sativa*	Y(3)/Y(1)
Lamiaceae	Type Lamium amplexicaule	N/N
	Type Mentha aquatica	N/Y(3)
	Type Salvia verbenaca	N/Y(5)
Papaveraceae	Type Papaver rhoeas	Y(1)/Y(1)
Pinaceae	^Type Pinus pinea*	Y(8)/Y(16)
Plantaginaceae	Type Anarrhinum bellidifolium	N/N
	Type Plantaginaceae	N/N
	Type Plantago coronopus	Y(1)/Y(1)
Poaceae	Type Festuca arundinacea	Y(6)/Y(9)
	Type Poaceae	N/N
Ranunculaceae	Type Nigella damascena	N/N
Resedaceae	Type Reseda luteola	Y(2)/Y(10)
Rhamnaceae	^*Type Rhamnaceae	N/Y(1)

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The number of chrysopid specimens in which each type of pollen was identified in each year is in parenthesis.

*Pollen type identified from surrounding vegetation.

Type C. bursa-pastoris was the pollen type most frequently identified in the alimentary canal (Table 1 and Fig 2). Despite some difficulty, we managed to differentiate between B. auriculata and C. bursa-pastoris pollen up to plant species according to grain pollen size (S2 Fig). We found that the most abundant pollen in the alimentary canal was from the species C. bursa-pastoris, which was thus identified in 86 of the 95 adults captured (35 specimens in 2016 and 51 in 2017), followed by B. auriculata, identified in 40 of the 95 adults captured (15 specimens in 2016 and 25 in 2017). We also recorded both plant species pollen (C. bursa-pastoris and B. auriculata) in 31 of the 95 captured adults (12 specimens in 2016 and 19 in 2017). These results are particularly noteworthy when we take into account that both plant species share some months during their bloom period (S2 Table); however, a slight difference in the phenology of both species was observed in 2016, with C. bursa-pastoris showing signs of senescence, while B. auriculata was blooming at the time of sampling. This explains why the plant species C. bursa-pastoris was not sown in the experimental farm in 2017.

We recorded similar pollen types in the alimentary canal of C. lucasina in both years studied; only seven pollen types, C. arvensis, C. capillaris, Ericaceae, Mentha aquatica, Rhamnaceae, Salvia verbenaca and Scabiosa atropurpurea, were absent from one of the sampling years, while twelve pollen types were not recorded in either of the sampling years (Table 1). The consumption of each pollen type by C. lucasina individuals showed different patterns of association, with no significant differences by sex in either year (Fig 3); PERMANOVA 2016 R² = 0.038, d.f. = 1, p = 0.15; 2017 R² = 0.024, d.f. = 1, p = 0.173. In 2016, the pollen consumed by C. lucasina were grouped in the following three pollen assemblages: E. plantagineum, V. hispanica and S. vulgaris; C. bursa-pastoris and Festuca arundinacea; and P. pinea, A. arvensis and S. latifolia isolates (Fig 3A). Meanwhile, in 2017, there were four groups: C. capillaris, S. vulgaris, Apiaceae, F. arundinacea and V. hispanica; P. pinea, E. plantagineum and A. arvensis; Reseda luteola and S. verbenaca; S. latifolia, C. bursa-pastoris and T. arvensis isolates (Fig 3B).

Fig 3 — MCA biplot shows the association between the pollen types identified (▲) and sex of chrysopids (□) in 2016 (a) and 2017 (b).

In addition to pollen, we identified fungal spores in the alimentary canal contents of 98 of the 107 C. lucasina adults examined. Alternaria sp. and Cladosporium sp. were the most frequently identified spores, with no significant differences by sex observed in either year (2016 Fisher tests: Alternaria sp. p = 0.640; Cladosporium sp. p = 0.359; Spore1 sp. p = 0.355; Spore2 sp. p = 1, and 2017 Fisher tests: Alternaria sp. p = 0.224; Cladosporium sp. p = 0.780; Spore1 sp. p = 0.785; Spore2 sp. p = 1) (Fig 4).

Fig 4 — Fungal spore identified on chrysopid adults by sex in 2016 (a) and 2017 (b).

Discussion

In this study, the alimentary canal contents of C. lucasina provided great insight into their late spring feeding behaviour. The transparency method also enabled us to show that C. lucasina had mainly fed on the plant species sown and to investigate their previous visits to plants.

We show that 17 of the 29 pollen types on the 51 native plants sown were consumed by C. lucasina, which have a rich polyphagous diet without any differences observed by sex. Furthermore, multiple correspondence analyses found no stable pattern of association in either year among the pollen types consumed, thus confirming that C. lucasina is not a specialist pollen feeder. We also observed that the principal pollen types in the alimentary canal of C. lucasina were type C. bursa-pastoris (including B. auriculata and C. bursa-pastoris plant species) belonging to the Brassicaceae family, as well as type A. arvensis from the Asteraceae family which, according to previous studies, are important sources of pollen for C. carnea s.l. especially in spring [38,58]. Most of the pollen types identified have been reported in previous studies of the feeding patterns of chrysopids, whose feeding habits were shown to be opportunistic and eclectic, although, in some cases, their diet was found to specialize in particular pollen types [29,36,38,57,58,78]. These feeding differences in chrysopids could be explained by the variety of plant species in certain habitats and their availability for chrysopids, but might also be due to different chrysopid species, with probably slightly different feeding behaviours, present in the C. carnea complex [29,57,58]. It might be necessary to carry out further research into the 12 pollen types in our study that did not appear in the chrysopid alimentary canal in order to determine feeding preferences when type C. bursa-pastoris is unavailable in the landscape.

C. lucasina emerge in spring and is one of the most frequently identified and abundant chrysopid species during the blooming stage of herbaceous vegetation [36,79]. Green lacewing populations have different patterns of activity and levels abundance depending on the species and stage of development and habitat, as observed in vineyards, with fewer captures observed during the month of May [17]. Thus, the provision of sown plants which bloom when chrysopid populations decreasing could contribute to increasing predator/prey ratios at key moments in order to improve biological control.

In a previous study, we selected 42 native plant species to evaluate their attractiveness to chrysopids based on chrysopid captures from sown plant species and on their use as reproduction sites [28]. By linking these previously published findings to the current results, we observed that one of the most attractive plant species for chrysopids is B. auriculata, whose pollen is the second more abundant in the alimentary canal of C. lucasina. Immature and adult stage C. lucasina were found on B. auriculata, which was visited for feeding, reproductive and refuge purposes. On the one hand, the lack of captured adult or immature stage chrysopids in 2016 from C. bursa-pastoris can be explained by signs of plant senescence at the moment of sampling. Although C. bursa-pastoris was not on the sown plant list for 2017 [28], its pollen was consumed by 81% of the chrysopid adults examined as compared to 40% from B. auriculata in that year. We therefore concluded that C. bursa-pastoris selectively attracts C. lucasina. We also observed that the plant species such as Nigella damascena, Moricandia moricandioides and Silene arvensis [28], most visited by chrysopids are not used as pollen sources by C. lucasina. However, other plant species, such as B. officinalis, E. plantagineum and P. rhoeas were abundantly consumed in certain chrysopid specimens despite the presence of bees and bumblebees in these plant species at the time of sampling. The present study demonstrates that, given the feeding preferences of C. lucasina, the plant species C. bursa-pastoris and B. auriculata should be included in conservation biological control programs.

Our findings could also facilitate the selection of suitable plant species in the context of a probable shift from woody pollen sources in spring to herbaceous pollen sources in summer according to the study by Bertrand et al. of C. carnea [29] given that the blooming period of the sown plant species in our study coincides with C. lucasina activity in Mediterranean agroecosystems [80]. Villa et al. [58] found that C. carnea s.l. mainly feeds on Olea europaea (woody strata) in all seasons and to a lesser extent on herbaceous plants in spring and summer. As suggested by Villenave et al. [38], this highlights the importance of providing plant species with different blooming periods that correspond to seasonal chrysopid flight activity and, as recommended by Bertrand et al. [29] that of increasing plant and vegetation diversity to attract chrysopid populations to agroecosystems. Despite the availability of several flowering plant species over time, Villenave et al. [38] have shown that C. lucasina is attracted to large patches of flowering plant species. In our study, although the pollen type C. bursa-pastoris was consumed, the plant species was sown in small 9 m² patches and even in surrounding non-sown plants.

We also found that the alimentary canal of C. lucasina females contained significantly more pollen than that of males. The number of pollen grains observed by Villenave et al. [36] in the diverticulum, an alimentary canal structure of adult chrysopids where the pollen is stored before being digested, is similar to that reported in our study. In our view, the differences between sexes could be related to the higher feeding requirements of female green lacewings as compared to those of males. A previous study found that protein titre levels significantly affect the reproductive physiology and fecundity of C. carnea [81]. Females reach protein titre levels during egg laying on roughly the fourth or fifth day following emergence, while males reach this point at the end of the second day [81]. Thus, the proteinaceous food provided by flowers is essential for the reproduction of adult lacewings [40,43–47]. However, the floral architecture and nectaries need to be accessible to ensure that these food resources can be reached [49]. Furthermore, as found for bees by Liolios et al. [82], the nutritional requirements of chrysopids are probably satisfied by a small number of abundant plants with accessible pollen present in the landscape and blooming for long periods of time in a manner unrelated to pollen protein content. This could be related to the attraction of green lacewings to large patches of flowering plants [38], the reported consumption by C. carnea s.l. of mostly O. europaea pollen in olive agroecosystems [58] or our results regarding its feeding on C. bursa-pastoris and B. auriculata. Nevertheless, Liolios et al. [82] have observed that the protein content of O. europaea pollen is lower than that of other common plant species surrounding olive orchards, such as Reseda sp., Papaver rhoeas, Trifolium sp. and S. verbenaca, which were also sown for our study.

Given the high capacity of chrysopid movements [83–85], we also identified mixtures of entomophilous and/or anemophilous pollen types from surrounding trees and herbaceous vegetation such as Ericaceae, C. sativa, P. pinea, Foeniculum vulgare and Rhamnaceae which were also reported as chrysopid resources in previous studies [36,58]. The presence of these pollen types was low in the C. lucasina alimentary canal as compared to the pollen types offered by the sown plant species. Anemophilous plant species, such as type P. pinea, whose pollen may have been consumed on vegetation surfaces, were at the blooming stage in our study area and at the time of sampling [86]. Furthermore, as C. lucasina had already been known to use P. halepensis for reproductive purposes [80], its use of this tree stratum for feeding and refuge cannot be ruled out.

With respect to honeydew, Villenave et al. [38,72] managed to indirectly identify its consumption through the presence of fungal spores, arthropod exuviae and pollen from anemophilous plant species in the alimentary canal of chrysopids. We found that most chrysopid adults have fungal spores in their alimentary canal, while previous studies have reported that Alternaria sp. and Cladosporium sp. fungal spores reaching peak abundance during the months of May and June in our study area [87,88]. We therefore concluded that a large proportion of C. lucasina adults feed on honeydew in late spring given the presence of fungal spores and anemophilous pollen in the alimentary canal of chrysopids.

Finally, in our view, although flower visitation studies are an important aid to selecting suitable plant species as refuges and reproduction sites, they need to be supplemented by flower foraging analyses. Only then can a full picture be obtained of how chrysopids exploited plant species and of how to guarantee their presence in the required habitat and time.

Conclusions

This study shows that the C. lucasina captured feed mainly on pollen from sown native species (>77%), with no differences observed by sex or year, and that C. bursa-pastoris was the most abundant pollen type consumed by C. lucasina. The identification of anemophilous pollen and fungal spores in its alimentary canal, confirms that C. lucasina feeds on honeydew on vegetation surfaces. Our recommendation at the conclusion of this study is to include C. bursa-pastoris and B. auriculata in the list of the most attractive plant species for C. lucasina. These native plant species are associated with the most frequently consumed pollen types identified in its alimentary canal, are able to attract and maintain C. lucasina populations and also promote a more heterogeneous landscape in Mediterranean agroecosystems. Finally, analysis of floral visits by chrysopids should be complemented with feeding studies in order to provide a more complete picture of plant resources use by chrysopids.

Supporting information

S1 Fig

Field layout with sown plant species distribution in the experimental farm in 2016 (A) and 2017 (B).

(PDF)

Click here for additional data file.^{(8.6MB, pdf)}

S2 Fig

Biscutella auriculata (A and B) and Capsella bursa-pastoris (C and D) flowers; photomicrographs of B. auriculata (E) and C. bursa-pastoris (F) pollen grains taken by a confocal microscope at 1000x magnification. Both images (E and F) show exine autofluorescence after merging multiple optical sections; microscopic images of B. auriculata (G) and C. bursa-pastoris (H) pollen grains at 1000x magnification.

(PDF)

Click here for additional data file.^{(359.3KB, pdf)}

S1 Table. List of plant species used in the study.

(PDF)

Click here for additional data file.^{(88.8KB, pdf)}

S2 Table. Complete list of plant families, pollen types, plant species, year sown, year sampled, chrysopid collected, bloom period, plant type and lacewing feeding studies references.

Y–Yes, N–No.

(PDF)

Click here for additional data file.^{(70.9KB, pdf)}

Acknowledgments

We wish to thank Dr. Mercedes Campos for assistance obtaining funding, laboratory support and encouraging us to publish this study, Luis Plaza and Joaquin Chocano for assistance in the field, Dr. Manolo Tierno for transparency methodology training and Dr. Cándido Galvez for resolving pollen identification issues. Finally, we would like to thank Michael O’Shea for proofreading the manuscript.

Data Availability

Data has been uploaded to Digital CSIC with the following DOI and link: http://dx.doi.org/10.20350/digitalCSIC/12637.

Funding Statement

This study was funded by a grant awarded to FR and Mercedes Campos from the Junta de Andalucía (project P12-AGR-1419) and a grant from Consejo Superior de Investigaciones Científicas (project 201840E055) to Mercedes Campos. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0239847.r001

Decision Letter 0

Manu E Saunders

18 May 2020

PONE-D-20-10818

The suitability of native flowers as pollen source for Chrysoperla lucasina (Neuroptera: Chrysopidae)

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Reviewer #1: In the current manuscript, the authors investigated the feeding behaviour of the chrysopid species Chrysoperla lucasina for different types of pollen obtained from 51 native plant species from an experimental farm in Spain. The authors found that this chrysopid species fed on various plants, consuming 17 out of the 29 pollen types available. Some methods and results presented by the authors are not reliable and some details are missing. In the method section, the sampling area and the plant species used by the authors are not mentioned. They also don’t mention the number of plants used (replicates) and how plants were distributed in the farm. Something that also needs be noted is the number of insects that the authors sampled. In 2016, they sampled 45 individuals and in 2017, they sampled 62. If the authors sown 51 plant species in the farm, sampling only 45 chrysopid is not enough. Furthermore, in several parts of the manuscript, the authors claimed they have tested the feeding preference of the chrysopid, which is also misleading. They did not perform any preference test. Table and figures need to be improved (need to be more informative and should align with the goals of the manuscript). Therefore, due to all these issues, I am rejecting the publication of this manuscript.

I recommend the authors to be focused on their goals and on the scientific contribution of the manuscript while re-writing the text and I hope my comments bellow help with that.

COMMENTS TO THE AUTHORS:

Abstract:

Line 14: Species name should be writing without abbreviation when cited for the first time.

Line 24: Can you be sure that the insects consumed honeydew?

Introduction:

The manuscript is related to native plants of a specific region in South of Spain, but in the introduction, the authors don’t provide info for questions such as: Why is it important to study this region? Why is it important to study green lacewings (more specific, C. lucasina) in this area? What is the problem you can try to solve? I suggest to author include some info about this. Write more clearly the aims of the study and follow it through the text.

Line 36: I suggest:"…to minimize negative effects in the environment through ..."

Line 40: What are semi-natural habitats (SNHs)? Maybe include just one sentence to explain it better, because right after that, you talk about “low SNHs.

Line 45: I suggest “Flowering plants attracts insects through its floral resources, ...”

Line 47: When the author says entomophagous insects, I suggest to add some explanation or synonym such as predator, carnivorous insects or natural enemies.

Line 51: The conclusion of the sentence (“depending on the target arthropod”) seems loose.

Line 54: Is it correct to say “complex Chrysoperla carnea (Stephens)”?

Line 64: I suggest you to remove this sentence (C. carnea require a floral architecture sufficiently large for their thorax and mouthpart to reach the pollen and well-exposed nectaries to gain access to nectar [27-29].).

Line 76: “species-richness inventories” are referring to plant species or insect species? This paragraph needs a conclusion sentence.

Material and Methods

I suggest you to split the topic “Study area” in two: “Study area” and “Insect collection” (or something like that). Again, why is it important to study green lacewings in this area? What is the problem to be solved? In the topic “Insect collection” write more details about the sampling effort, how the sampling was made….

Include details about the native species that were sown and why they were selected?

Results

The results are confused, as well as the Table 1. The figure 1 have two variables mixed: pollen type and plant species, is it correct? Line 179 to 185: In the first part, you used “first, second and third” to refer to groups. Then, you use numbers “1, 2, 3 and 4”. I suggest you to choose only one way to refer to groups.

Discussion

In general, I suggest you to explore more your results.

Lines 224 to 232: this paragraph could be in the result section. It is simple and clear.

Conclusion

Line 272: “We specifically suggest that the sources of pollen include the following native plant species … which are the most frequently consumed by C. lucasina.” Do you suggest the use of these species everywhere or only at your study region? Be more cautious with your conclusions and keep the focus.

Reviewer #2: I have reviewed the article entitled “The suitability of native flowers as pollen source for Chrysoperla lucasina (Neuroptera: Chrysopidae)”. This is an interesting article addressing an important topic for conservation biological control of pests. Despite that there are other similar studies published, I believe it is important that this study is focused on C. lucasina, which was previously neglected or confounded with C. carnea, so any advance on knowing the feeding behavior and ecology of the species from the carnea complex will help us understand more about their ecology, the differences between and, from an applied point of view, how to enhance their abundance in cultivated fields.

I congratulate the authors for their work, given that identifying the pollen grains is complicated, and the use of the transparency method seems like a good option, easier than the acetolysis process that is more commonly used. Nevertheless, I think that the article would benefit from some changes before being published. Below, I provide my main concerns and some minor suggestions to the authors.

Major comments:

-The introduction is good and deals with relevant topics for the study system. However, at some points, it can be improved. The first paragraph would benefit from more connectivity between the sentences, as some of them are a bit disconnected, leading to the ecological intensification of agriculture. At the end of the second paragraph, the authors jump directly to chrysopids when they were talking about biological control and natural enemies in general. I believe that the link with lacewings can be included directly in the next paragraph or that the last sentence should be slightly modified to incorporate them in this general context. The third paragraph is a bit disorganized and would benefit from a better structure when talking about different aspects of lacewings biology and feeding preferences.

-The objectives of the article focus on pollen feeding preferences but do not mention the differences between sexes and the importance of honeydew for lacewings. However, both topics are addressed in the results and specific figures are shown. I think both topics are relevant, especially honeydew consumption, so the authors could include these as specific objectives or questions. During the introduction and methods, the fact that some lacewings feed on honeydew in briefly mentioned, but only at the end of the discussion the authors explain that this can be identified by the presence of fungal spores. I think this should be clarified before, in the introduction or methods section.

-The plant species sown are listed in table 1 in the results section and, in methods, this is only briefly mentioned (L. 98). However, the selection and sowing of the species is part of their methods, so I think that the reasons for their choice can be included before. The authors could refer to table 1 in methods, but explain if the plant species included flowers with different traits, flowering periods, previous reports on lacewing feeding, etc.

-In the multivariate analyses of pollen consumption, the authors refer to the overlap between diets in results, but this could be explored explicitly. A specific test such as an ANOSIM or similar could be done to determine if the differences between sexes on the diet composition were significantly different or not.

-As with the objectives section, I think the results section could be organized to refer to different specific objectives. The section has some order, but I have the impression that results from different questions are mixed and can be organized better. In my opinion, different parts that can be separated better are the description of the diet including the presence of surrounding plants, feeding preferences (including the multivariate analyses), differences by sex, presence of spores, and honeydew.

-In the discussion (l. 226-231) and conclusions (l. 272-275) the mention of a lot of plant species names becomes too repetitive. Furthermore, the temporal variation in the diet of lacewings should be discussed more, as the authors only sampled in May, and the diet changes through time. Regarding this, a recent and very relevant paper (Bertrand et al. 2019, J Appl Ecol, 56(11), 2431-2442) should be also mentioned.

-The article is, in general, well written, but would benefit from the language edit by a native English speaker. The parts where corrections are necessary are just a few, but it will improve the text flow. Some of these details are highlighted below in my minor comments.

Minor comments:

-The title should say "as pollen sources" or "as a pollen source" instead.

-L. 23: the sentence from point (iii) of the results section of the abstract is not so clearly written. I believe it would be more correct to say that the majority of the pollen types identified correspond to the sown native plants, and no that the pollen was found on the plant species.

-Throughout the manuscript, the authors refer to semi-natural habitats as SNH or SNHs, not always because they refer to singular or plural mentions of these habitats. I believe that SNH is widely used in the literature with no need to add the ‘s’ in the end and the same criteria should be applied across the text.

-L. 52: the family of natural enemies sounds strange, as it can be confounded with taxonomical families. The group or functional group might be more appropriate.

-L. 83: The sentence would be improved by adding “to identify which/the sown native species”.

-L. 58: Many references are cited like Villenave, Deutsch in this line. Please check the journal guidelines as I believe that in these cases only the first author should be mentioned before the number of the reference.

-L. 90: the section describes the study area but also lacewing samplings. The title could then be modified to make this clear?

-L. 108: is “peeling off” the best term to describe this? Taking off wings, legs, and antennae might be better.

-L. 116: the division of the alimentary canal in sections is interesting, but it is not clear if the authors did it after identifying the pollen or if the identification was done without contemplating the location of the pollen grains and the division was done just to show the proportions of the locations. A better explanation of the reasons can be helpful here.

-L. 137-138: instead of “in addition to”, the authors might refer to “using the packages”.

-L. 145: I would not say that including the species that were consumed only a few times would cause disturbances in the analysis, but they could be influential. Thus, perhaps “to minimize the influence of species that were only consumed occasionally” or something similar could be a better expression.

-Table 1 could be organized in a different way to show the relative importance of plant families. Instead of organizing it alphabetically by pollen type names, can the authors organize it alphabetically by plant family and then by pollen type within each family?

-L 271-272: pollen sources and resources could be changed to pollen resources or plants providing pollen resources.

-Fig. 2 would benefit from titles on each panel for better identification of what it is shown.

**********

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Reviewer #1: No

Reviewer #2: Yes: Ezequiel Gonzalez

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PLoS One. 2020 Oct 23;15(10):e0239847. doi: 10.1371/journal.pone.0239847.r002

Author response to Decision Letter 0

3 Jul 2020

We have answered the reviewers’ comments in a separate file titled "Response to Reviewers".

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PLoS One. doi: 10.1371/journal.pone.0239847.r003

Decision Letter 1

Manu E Saunders

13 Aug 2020

PONE-D-20-10818R1

The suitability of native flowers as pollen sources for Chrysoperla lucasina (Neuroptera: Chrysopidae)

PLOS ONE

Dear Dr. Alcalá Herrera,

Please address the comments from Reviewer 1, and especially ensure the Introduction and Discussion sections are clear and well-organised.

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We look forward to receiving your revised manuscript.

Kind regards,

Manu E. Saunders

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #2: In the revised version of the manuscript “The suitability of native flowers as pollen source for Chrysoperla lucasina (Neuroptera: Chrysopidae)”, the authors have replied to the comments made by the two reviewers during the first submission. I thank the authors for their work on the revision. They have placed more emphasis on the importance of the study for the Mediterranean region, which helps the reader to understand why this research was relevant. English writing is also improved, but still, some changes are necessary, especially in parts of the discussion. The manuscript is clearer now and the objectives are clearly defined. Nevertheless, I think that the discussion needs more structure and some important topics are omitted. Plus, I found many minor changes that would improve the text. Therefore, I am recommending another major revision.

Major comments:

-Despite that their previous article (Alcalá Herrera et al. 2019, Biol. Control) is cited in the text, I feel that more links to this paper are necessary, as both works are clearly part of the same project and are highly connected. Particularly, in the discussion, it could be mentioned if the plants that were frequently visited by lacewings in their previous article are the same that were found to be consumed here. By looking at the highlights and abstract, it seems that not all species are shared, at least within the most important. What are the implications of this? Could the recommendations of sown plant species based only on flower visitation be inaccurate?

-In general, the discussion structure seems unorganized. The first paragraph is quite long and mixes their main findings with the discussion about other aspects of lacewings’ identification, temporal dynamics, and movement. I suggest to keep the first paragraph short and with a clear mention of the contribution of their study and discuss the other topics in other parts. Furthermore, on lines 267-269 the question of whether C. lucasina is a generalist or specialist pollen feeder appears, and this was not mentioned before, at least explicitly. The specific objectives could introduce this question more clearly, perhaps. The paragraph on the competition with other insects such as pollinators on lines 306-313 is disconnected from the rest of the article, as this was not mentioned before, so some changes to improve the connection are needed. Finally, the result of significant differences between sexes in the percentage of alimentary canal with pollen are not discussed in detail, and this could be important due to the implications of flower resources on reproduction (which is mentioned in lines 295-305, where this difference could be discussed).

Minor comments:

-L 13-16: I think that the revised description of the objective in the abstract can still be improved. The authors did not only want to identify the consumed pollen but also to quantify their relative importance. I agree with reviewer #1 that there are not measuring preferences explicitly, but at least the relative consumption of the different species was analysed and it would be good to highlight it here.

-L 19-20: Not clear. Did the authors mean that it feeds on more than half of the pollen types offered by the sown plants?

-L 32-33: Agricultural intensification is also very evident in annual crops, perhaps even more than on vineyards and orchards. Maybe changing “especially” for something like “as observed in” can retain the same meaning without emphasizing on the difference between different production systems.

-L 38: A better connection with the sentence about the European agri-environment schemes would improve the end of the paragraph, such as “For example, the European…”.

-L 40: delete the comma after “defined as habitats”.

-L 73-74; the expression “are subject to” is not ideal for this sentence. I would say that flowering resources can influence or affect these life-history parameters rather than being subject to them.

-L 82-83: this sentence is not clear, particularly the species-rich flowering plants part. Analyses of pollen should be combined with plant inventories or samplings that provide the set of available pollen resources.

-L 92-93: I would replace “To achieve this goal, we needed to determine” for a shorter expression such as “Specifically, we were interested in”, or something similar that makes a better connection between the general and specific objectives because those four objectives are specific questions asked by the authors.

-L 104-105: I understand that this information is necessary for the methods section according to the journal. But I believe that the beginning of the section is not the best option. Perhaps at the end of the paragraph, it would be better?

-L 110: can be found is repeated twice in this sentence.

-L111: plant species for what? This is the first time the authors mention the sowing of the flowering plants in methods, so the paragraph should start by saying that in this experimental farm, a set of X plant species were planted, following those criteria.

-L 119: squares should be replaced by plots or something alike, as the 1x9 m sampling areas are not squares. This is also true for the rest of the paragraph and in other parts of the methods section.

-L 122: some plots were not well developed? It should be 120.

-L127-130: the writing on this short gives the idea that only those five species were sampled in 2016 and those two in 2017, because of the addition of “the” before the species number. I suggest to change it for something like “Due to their poor development, five species sampled in 2016 (species names) and two species sampled in 2017 (species names) were omitted from the analyses”.

-L 191: Please correct, you did not check for differences among years, but among sexes on each year (at least that is shown in results).

-L 200: the “also” gives the impression that no differences were found for the percentage of the alimentary canal in the previous sentence, but you did found differences.

-L 209: remove the comma after C. lucasina.

-L 224: confusing redaction, rephrase “the pollen types most recorded among the ten most frequently identified”.

-L 239-243: what information is provided by these groups? This is not discussed later in the article and it is probably linked with some individuals sharing some pollen types, but if it doesn’t represent something relevant, I would remove this description of the groups from the results.

-L 250-253: again, no differences between years were tested, rephrase. Also, the start of the parenthesis is missing and those are several Fisher tests, in the plural.

-L 288-289: this sentence is not clear.

-L 314-324: were these anemophilous plants represented by only a few pollen grains per individual lacewings or by many? In previous articles and my experience, C. sativa could be used as a direct pollen source because many grains can be observed sometimes. But Pinus and other coniferous are sometimes represented by very few grains and this could be a clear indication that lacewings consumed these species on the surface of other plants or with honeydew.

Reviewer #3: The authors appear to have carefully addressed all queries and comments raised in review and provision of a marked copy enabled efficient assessment of resubmission

**********

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Reviewer #2: No

Reviewer #3: Yes: Linda J. Thomson

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PLoS One. 2020 Oct 23;15(10):e0239847. doi: 10.1371/journal.pone.0239847.r004

Author response to Decision Letter 1

10 Sep 2020

Please see Response to Reviewers letter at the end of this PDF

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PLoS One. doi: 10.1371/journal.pone.0239847.r005

Decision Letter 2

Manu E Saunders

15 Sep 2020

The suitability of native flowers as pollen sources for Chrysoperla lucasina (Neuroptera: Chrysopidae)

PONE-D-20-10818R2

Dear Dr. Alcalá Herrera,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Manu E. Saunders

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0239847.r006

Acceptance letter

Manu E Saunders

14 Oct 2020

PONE-D-20-10818R2

The suitability of native flowers as pollen sources for Chrysoperla lucasina (Neuroptera: Chrysopidae)

Dear Dr. Alcalá Herrera:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

Field layout with sown plant species distribution in the experimental farm in 2016 (A) and 2017 (B).

(PDF)

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S2 Fig

(PDF)

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S1 Table. List of plant species used in the study.

(PDF)

Click here for additional data file.^{(88.8KB, pdf)}

S2 Table. Complete list of plant families, pollen types, plant species, year sown, year sampled, chrysopid collected, bloom period, plant type and lacewing feeding studies references.

Y–Yes, N–No.

(PDF)

Click here for additional data file.^{(70.9KB, pdf)}

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Attachment

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Click here for additional data file.^{(29.8KB, docx)}

Data Availability Statement

Data has been uploaded to Digital CSIC with the following DOI and link: http://dx.doi.org/10.20350/digitalCSIC/12637.

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PERMALINK

The suitability of native flowers as pollen sources for Chrysoperla lucasina (Neuroptera: Chrysopidae)

Rafael Alcalá Herrera

María Luisa Fernández Sierra

Francisca Ruano

Roles

Abstract

Introduction

Material and methods

Study area

Insect collection

Alimentary canal content analysis

Pollen identification

Statistical analyses

Results

Fig 1. Estimated percentage of alimentary canal containing pollen (mean ± SE) in each segment (I–head; II–thorax; III–initial abdomen; IV–medium abdomen and V–final abdomen) by sex.

Table 1. Presence (Y) or absence (N) of the different pollen types identified in C. lucasina adults collected in the experimental farm in 2016/2017.

Fig 2. Pollen type most frequently identified in the alimentary canal of C. lucasina adults by sex.

Fig 3.

Fig 4.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Manu E Saunders

Roles

Author response to Decision Letter 0

Decision Letter 1

Manu E Saunders

Roles

Author response to Decision Letter 1

Decision Letter 2

Manu E Saunders

Roles

Acceptance letter

Manu E Saunders

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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