Introduction {#sec1}
============

The multicolored Asian ladybeetle, *Harmonia axyridis* Pallas (Coleoptera: Coccinellidae), was introduced from Asia into Western Europe and other parts of the world to control aphid and coccid populations ([@bibr02]; [@bibr10]). In Belgium, *H. axyridis* was used as a biological control agent beginning in 1997 and was first observed in the wild in 2001. Since then *H. axyridis* populations have increased and gradually expanded into Belgium ([@bibr02]).

This species presents all the characteristics shared by an efficient aphid predator: large body size, high voracity, high predation efficiency ([@bibr35]), high colonization aptitude ([@bibr60]), rapid development, high fecundity, and low susceptibility to pathogens or natural enemies ([@bibr41]). *Harmonia axyridis* has become ubiquitous in many parts of the world, including America, Europe, and Africa ([@bibr37]; [@bibr12]), and has been reported in many different habitats, such as agroecosystems, gardens, and arboreal habitats ([@bibr40]).

Due to its large body and efficient physical and chemical defenses, *H. axyridis* has become an intraguild predator ([@bibr50]; [@bibr58]). Intraguild predation has been observed among other ladybeetle species ([@bibr45]; [@bibr58]); other aphid natural enemies, including syrphids, chrysopids, and parasitoids ([@bibr46]; [@bibr59]; [@bibr31]); and aphid pathogenic fungus ([@bibr45]). This intraguild predation behavior is thought to have led to a decrease in native species ([@bibr09]; [@bibr23]; [@bibr57]; [@bibr11]; [@bibr48]). In Belgian urban areas, Adriaens et al. ([@bibr03]) found a significant decline in native species, including the two-spot ladybird, *Adalia bipunctata* L. (Coleoptera: Coccinellidae), and the 10 spotted ladybird, *Adalia decempunctata* L., on pine, lime, and maple trees following the arrival of *H. axyridis.* The decline of native species can possibly be explained by the decline in number of their principal prey, resulting in reduced survivorship in local habitats and altered dynamics of habitat use and dispersal ([@bibr21]).

According to previous reports, the most dominant aphidophage in Belgian agroecosystems appear to be two coccinellids, the seven-spotted ladybug, *Coccinella septempunctata* L. (Coleoptera: Coccinellidae), and *H. axyridis*; one hoverfly, the marmalade hoverfly, *Episyrphus balteatus* De Geer (Diptera: Syrphidae); and one braconid, the parasitic wasp *Aphidius ervi* Haliday (Hymenoptera: Braconidae) ([@bibr20]; [@bibr02]; [@bibr05]). In arboreal habitats, four coccinellids were reported as abundant species: *A. bipunctata*.,the 14-spotted ladybird, *Propylea quatuordecimpunctata* L. (Coleoptera: Coccinellidae), the 22-spot ladybird *Psyllobora vigintiduopunctata* (L.), and *H. axyridis* ([@bibr02]). In 2001, the same results were observed by Francis ([@bibr22]), with the exception of *H. axyridis.*

Our study was conducted eight years after the first observation of *H. axyridis* in the wild in Belgium ([@bibr01]). Following aphidophagous decline highlighted by several studies, the current study was conducted in order to assess the relative abundance of *H. axyridis* through the quantification of the abundance of this exotic species and other aphidophages in four important Belgian crops (wheat, *Triticum aestivum* L. (Poales: Poaceae), corn, *Zea mays,* potato, *Solanum tuberosum* (Solanales: Solanaceae), and broad bean *Vicia faba* (Fabales: Fabaceae)) using a three-year inventory.

Materials and Methods {#sec2}
=====================

Study site {#sec2a}
----------

Aphidophagous insect populations were sampled from 2009 to 2011 in Hesbaye, an intensive agricultural production area in Wallonia, the southern region of Belgium (individual sites given in [Table 1](#t01_01){ref-type="table"}). Four crops were chosen for their agronomic importance: wheat, corn, potato, and broad bean *Vicia faba.* The sampling period ran from mid-May to late September. Every week, nine fields of each crop were sampled.

Sampling methods {#sec2b}
----------------

The sampling methods used to assess the numbers of aphidophagous predators and aphids consisted of whole-plant visual inspections, using 1 m^2^ quadrats distributed randomly throughout the whole field. In order to avoid the influence of surrounding crops, a 20 m buffer zone around the edge of each field was not sampled. Visual sampling was conducted, as it provides an easy and accurate method for the estimation of larval and adult densities of coccinellids in agroecosystems ([@bibr43]). Forty-eight quadrats were examined in each crop every week. Quadrats were located along transect lines across each field and spaced 20 m apart. All leaves and stems within the quadrat were examined, and all aphidophagous stages were recorded. Aphid species were also quantified on all leaves and stems. Larvae and pupae were brought to the laboratory to develop under laboratory conditions (24 ± 1° C, 75 ± 5% RH) for identification at the species level. All aphid predators were identified, with the exception of members of the common green lacewing, *Chrysoperla carnea* Stephens (Neuroptera: Chrysopidae) species complex, which were pooled together. This group comprises three cryptic species, *C. kolthoffi* Navas, *C. lucasina* Lacroix, and *C. carnea* Stephens, which can only be differentiated using molecular techniques ([@bibr07]; [@bibr38]).

Statistical analysis {#sec2c}
--------------------

In order to assess the species diversity, the collected data were analyzed by considering (1) species richness and (2) evenness according to the Shannon diversity index (H) ([@bibr39]), which considers both the number of species and the distribution of individuals among species. H is minimal if all individuals belong to only one species or if all species are represented by one individual; H is maximal if all individuals are evenly distributed. Evenness (J) varies from 0, if only one species dominates, to 1, if all species show similar abundance. Evenness (J) and the Shannon diversity index (H) were calculated as follows:

![](e01_01.jpg)

Because mean densities per m^2^ were low, these values are presented per 100 m^2^ The mean abundance per species was analyzed with an analysis of variance (ANOVA: general linear models) with crops (q = 4) and years (n = 3) used as factors (α = 0.05). Within crops, densities of species were compared using the least square difference (LSD; α = 0.05). To account for the variations of predator abundances, the abundance per species was analyzed using an analysis of covariance (ANCOVA: general linear models) with crops (q = 4) and years (n = 3) used as factors (α = 0.05) and aphid densities used as the covariable. Prior to analyses, a log~10~(x + 1) was used to transform the data distribution (counting) due to its asymmetry ([@bibr19]). Although statistical analyses were performed on transformed data, untransformed data are presented in [Tables 4](#t04_01){ref-type="table"} and [5](#t05_01){ref-type="table"}. Statistical analyses were performed using Minitab® release 1.5 ([www.minitab.com](www.minitab.com)).

Results {#sec3}
=======

Diversity of aphidophages {#sec3a}
-------------------------

During the three years (2009--2011), 11 aphidophagous taxa were observed on the four different cultures: the hoverfly *E. balteatus,* the coccinellids *C. septempunctata, C. quinquepunctata* L. (Coleoptera: Coccinellidae), *C. undecimpunctata* (L.), *H. axyridis, P. quatuordecimpunctata, A. bipunctata, A. decempunctata,* the cream-spot ladybird, *Calvia quatuordecimguttata* (L.), *Hippodamia variegata* Goeze; and the *C. carnea* species group ([Table 2](#t02_01){ref-type="table"}).

From 2009 to 2011, species richness increased in broad bean, corn, and wheat, reaching 6, 8, and 7 species respectively ([Table 3](#t03_01){ref-type="table"}). Species richness did not evolve in potato crops, remaining at five species during the entire period. Overall, five aphidophages were continually observed during the three-year period in each crop and represented 95% of all the aphidophage observed in 2009 and 99% in both 2010 and 2011: *E. balteatus, C. carnea* s.l., *C. septempunctata, P. quatuordecimpunctata,* and *H. axyridis.*

The evenness value was low in wheat with J = 0.35, 0.25, and 0.38 in 2009, 2010, and 2011, respectively, with the numerical dominance of two species: *C. septempunctata* and *E. balteatus* ([Tables 3](#t03_01){ref-type="table"}, [4](#t04_01){ref-type="table"}, and [5](#t05_01){ref-type="table"}). In broad bean and potato, evenness varied from year to year. In broad bean, *C. septempunctata* was the most abundant in 2010 (J = 0.45) and 2011 (J = 0.59). In potato, *C. carnea* s.l. and *C. septempunctata* numerically dominated the aphidophagous guild in all three years (J = 0.65, 0.79, 0.74) ([Table 3](#t03_01){ref-type="table"}). In corn, the evenness during the three years decreased from 0.82 (2009) to 0.61 (2010).

Relative abundance of aphidophage in four crops {#sec3b}
-----------------------------------------------

The relative abundance of both adult and larval stages of the five aphidophage within each crop showed significant differences ([Tables 4](#t04_01){ref-type="table"} and [5](#t05_01){ref-type="table"}), with the exception of potato in 2010, in which densities of larvae populations of different predators were not significantly different *(F~4.3570~* = 1.4*; p* = 0.25) ([Table 5](#t05_01){ref-type="table"}).

Corn {#sec3c}
----

The most abundant adult species in 2009 and 2010 was *C. carnea* s.l. and *C. septempunctata* (2009: *F~4,1065~* = 10.7, *p* \< 0.001 ; 2010: *F~4,3425~* = 14.3, *p* \< 0.001) *(p* \< 0.05; LSD) ([Table 4](#t04_01){ref-type="table"}). In 2011, *H. axyridis* and *C. septempunctata* densities were both significantly higher than those of other species *(p* \< 0.05; LSD) *(F~4,4055~* = 24.2, *p* \< 0.001) ([Table 4](#t04_01){ref-type="table"}). *H. axyridis* larvae densities were significantly higher in 2009 and 2011 (2009: *F~4,1065~* = 5.4, *p* \< 0.001 ; 2010: *F~4,3425~* = 25.5, *p* \< 0.001 ; 2011: *F~4,4055~* = 57.0, *p* \< 0.001) than those observed for the other species ([Table 5](#t05_01){ref-type="table"}).

Wheat {#sec3d}
-----

Adult populations of *H. axyridis* remained lower than other species during the three-year sampling (2009: *F*~*4,1765*~ = 5.4, *p* \< 0.001 ; 2010: *F~4,2925~* = 24.1*, p* \< 0.001; 2011: *F~4,2625~* = 38.0, *p* \< 0.001) *(p* \< 0.05; LSD) and did not exceed 1.1 ± 0.6 adults per 100 m^2^ ([Table 4](#t04_01){ref-type="table"}). *Episyrphus balteatus* was the most frequently encountered adult species in 2009, whereas in 2010 and 2011 *C. septempunctata* was the most abundant species *(p* \< 0.05; LSD) ([Table 4](#t04_01){ref-type="table"}). Larvae of *E. balteatus* were the most abundant *(p* \< 0.05; LSD) of the aphidophage during the three years (2009: *F~4,1765~* =11.7, *p* \< 0.001 ; 2010: *F~4,2925~ =* 91.1*, p* \< 0.001; 2011: *F*~*4,2625*~ = 213.6, *p* \< 0.001) ([Table 5](#t05_01){ref-type="table"}).

Potato {#sec3e}
------

Trends for *H. axyridis* were the same in potato as in wheat: *H. axyridis* was not the most abundant species, and its density did not exceed 2.5 ± 0.7 adults per 100 m^2^ ([Table 4](#t04_01){ref-type="table"}). Two species were more abundant than others: *C. carnea* s.l. in 2009 and 2011 (2009: *F*~*4,500*~ = 7.7, *p* \< 0.001 ; 2011: *F~4,3250~* = 10.8, *p* \< 0.001), and *C. septempunctata* in 2010 *(F~4,3570~* = 12.8, *p* \< 0.001) and 2011 *(p* \> 0.05; LSD). In 2009, larvae of *C. carnea* s.l., *P. quatuordecimpunctata,* and *H. axyridis* were the most abundant species but densities remained low ([Table 5](#t05_01){ref-type="table"}). In 2010, larvae densities of the five above-mentioned species were not significantly different from each other *(F~4,3570~* = 1.4, *p* = 0.25) ([Table 5](#t05_01){ref-type="table"}). In 2011, only *C. carnea* s.l. numerically dominated the aphidophages community (*F~4,3250~* = 5.5, *p* \< 0.001) *(p* \> 0.05; LSD) ([Table 5](#t05_01){ref-type="table"}).

Broad bean {#sec3f}
----------

In 2009, *H. axyridis* and *C. septempunctata* adults were the only adult species observed in broad bean (*F*~*4,160*~ = 0.7, *p* \> 0.05). *Coccinella septempunctata* was the most abundant species in 2010 *(F~4,2415~* = 22.1, *p* \< 0.001), while in 2011 both *C. septempunctata* and *C. carnea* s.l. were profusely observed (*F~4,280~* = 37.5, *p* \< 0.001) *(p* \< 0.05; LSD) ([Table 4](#t04_01){ref-type="table"}). In 2009, three species were present at the larval stage: *P. quatuordecimpunctata, C. carnea* s.l., and *E. balteatus* (*F~4,160~* = 1.0 *,p* = 0.43) *(p* \< 0.05; LSD). In 2010 (*F~4,2415~* = 8.35*, p* \< 0.001) and 2011 *(F~4,2480~* = 21.2, *p* \< 0.001), all species were observed, and *C. septempunctata* was the most abundant ([Table 5](#t05_01){ref-type="table"}).

Effect of aphid densities and sampling year on relative abundance of aphidophage {#sec3g}
--------------------------------------------------------------------------------

Abundances of *H. axyridis* in wheat and potatoes were not analyzed, due to very low numbers of individuals observed during the three-year inventory. Over the three-year sampling, adult populations of *H. axyridis* in corn significantly increased (*F*~2,1709~ = 14.5, *p* \< 0.001) ([Table 6](#t06_01){ref-type="table"}) from 7.8 ± 2.0 in 2009 to 19.9 ± 1.8 individuals per 100 m^2^ in 2011 ([Table 4](#t04_01){ref-type="table"}). Larval populations in the same crop also increased statistically (*F*~2,1709~ = 39.9, *p* \< 0.001) ([Table 6](#t06_01){ref-type="table"}), rising from 11.9 ± 4.2 to 70.8 ± 6.6 larvae per 100 m^2^ ([Table 5](#t05_01){ref-type="table"}). In broad bean, relative abundance of *H. axyridis* was not significantly different among the three years, neither at the adult ([Table 6](#t06_01){ref-type="table"}) nor larval ([Table 7](#t07_01){ref-type="table"}) stages.

*Coccinella septempunctata* larvae declined in broad bean (F~2,1011~ = 4.7, *p* = 0.009), wheat (*F*~2,1463~ = 14.4, *p* \< 0.001), and corn (*F*~2,1709~ = 31.9, *p* \< 0.001) ([Table 7](#t07_01){ref-type="table"}); densities decreased by 10 and 29.6 times in corn and broad bean respectively. In wehat, no larvae were observed in 2011, while 13.4 ± 3.9 larvae per 100 m^2^ were observed in 2009.

The abundances of three other aphidophage showed variable changes ([Tables 6](#t06_01){ref-type="table"} and [7](#t07_01){ref-type="table"}).

Aphid populations and correlation with aphidophage densities {#sec3h}
------------------------------------------------------------

In 2009, 2010, and 2011, seven, nine, and 10 species of aphids were identified, respectively: the pea aphid, *Acyrthosiphon pisum* Scopoli (Hemiptera: Aphididae); the cowpea aphid, *Aphis craccivora* Koch; the black bean aphid, *Aphis fabae* Scopoli; *Aphis frangulae* Walker; the buckthorn aphid, *Aphis nasturtii* Kaltenbach; the potato aphid, *Macrosiphon* *euphorbiae* Thomas; the vetch aphid, *Megoura viciae* Buckton; the rose grain aphid, *Metopolophium dirhodum* Walker; the green peach aphid, *Mizns persicae* Sulzer; *Rhopalosiphum* sp.; and *Sitobion* sp. ([Table 8](#t08_01){ref-type="table"}). The mean number of observed aphids increased in corn (*F~2,2589~* = 39., *p* \< 0.001) and potato (*F~2,1410~* = 17.11, *p* \< 0.001) from 2009 to 2011. Aphid densities also statistically varied in broad bean (*F~2,974~* = 8.7, *p* \< 0.001) and wheat (*F~2,1392~* = 102.7, *p* \< 0.001) from 2009 to 2011, but without any general evolution ([Table 8](#t08_01){ref-type="table"}).

The ANCOVA analyses showed a linear relationship between aphid and predator populations in 55% of adult populations ([Table 6](#t06_01){ref-type="table"}) and 35% of larvae populations ([Table 7](#t07_01){ref-type="table"}) (*p*~aphids~ \< 0.05, ANCOVA). In these cases, aphid densities influenced the predator abundance. Results (*p*~years~) comparison between ANOVA and ANCOVA showed that the influence of aphid populations on predator abundance variations between years was not statistically significant.; *p~years~* of the two statistic analyses showed the same results.

Discussion {#sec4}
==========

Since the invasive coccinellid *H. axyridis* spread over Europe ([@bibr10]), imposing negative impacts on native aphidophage and affecting composition of several guilds ([@bibr53]; [@bibr48]), studies have evaluated the population spread of this coccinellid. The current study\'s sampling of aphid predators in Belgian agroecosystems from 2009 to 2011 showed that *H. axyridis* lives and reproduces more efficiently in corn and broad bean than in wheat and potato. In corn, the evenness during the three years decreased when *H. axyridis* population increased strongly and was higher than the population of other species.

During the three-year sampling, 11 aphidophage were observed in these agroecosystems, but five of them predominated: *E. balteatus, C. septempunctata, P. quatuordecimpunctata, H. axyridis,* and *C. carnea* s.l. Five dominant species in agroecosystems is a common observation ([@bibr26]). Observations on predator densities highlight that *H. axyridis* was not the numerically dominant species in every crop: in wheat and potato, *C. septempunctata* was more abundant than *H. axyridis.* In many European agricultural crops, both *C. septempunctata* and *P. quatuordecimpunctata* were dominant prior to the arrival of *H. axyridis* (Honěk 1979; [@bibr06]; [@bibr14]), and it appears that these two species have maintained their dominance in spite of being prone to intraguild predation by *H. axyridis* in the field ([@bibr24]).

In our study, larvae of *E. balteatus* were the most abundant observed predators in wheat, which has already been reported by Tenhumberg and Poehling ([@bibr55]) prior to the arrival of *H. axyridis. Episyrphus balteatus* has also been previously reported as one of the most abundant aphidophage in vegetable crops, such as broad beans ([@bibr17]; [@bibr16]). This could be explained by abiotic conditions (high density cereal crop, with high humidity and low temperature) that are more favorable to the larvae of *E. balteatus* (Honěk 1983).

The fact that *H. axyridis* is not the most abundant aphidophage in agrosystems is probably due to its generalist behavior and arboreal habitat selection ([@bibr25]; [@bibr15]; [@bibr36]; [@bibr09]; [@bibr34]). However, it has been reported that *H. axyridis* can also thrive in agrosystems such as wheat, corn, and potato ([@bibr36]; [@bibr13]; [@bibr16]; [@bibr42]; [@bibr08]; [@bibr44]; [@bibr50]; [@bibr32]), as well as in herbaceous habitats ([@bibr36]; [@bibr33]; [@bibr04]).

There were evident changes in the abundance of aphidophages in crops through the years, but this study does not propose to identify a global evolution (increase or decline) in any of the species that were observed in this study. The causes of such fluctuations are diverse and may include factors such as the diversity and abundance of aphid species ([@bibr61]; Honěk 1982; [@bibr56]). The results of the ANCOVA showed that there was a linear relationship between prey and predator populations, but additional biotic and abiotic factors contribute to the annual variability of predator abundance. Climate could be one such factor, due to its influence on natural enemies, overwintering mortality, and aphid populations ([@bibr26]; Szentkirályi 2001; [@bibr47]). Several other factors could also explain the variation between crops: insolation, humidity (Honěk 1985), quantity and quality of host plants ([@bibr05]), and adjacent habitats ([@bibr17]; [@bibr05]).

A particularly interesting finding is that although *H. axyridis* breeding occurred in all four inventoried crops to some extent, adults of this species are not ubiquitous; few immature individuals were recovered from potato and wheat. Assuming that declines in native species are caused by *H. axyridis* ([@bibr48]), this suggests that certain crops, such as wheat and potato, could act as refuges from *H. axyridis* at certain times, while native species, such as *E. balteatus* and *C. septempunctata,* are able to breed with a lower risk of intraguild predation or other forms of competition from the invaders. Such habitats could become even more important as native species adapt to the invader by evolving to avoid habitats where *H. axyridis* occurs in high numbers, as has been seen in co-occurring aphidophages in their native habitats ([@bibr51]).

In conclusion, our study indicates that *H. axyridis* was not the most frequently observed aphidophage in the four most important Belgian agronomical crops. In future studies, longer samplings would be preferable in order to eventually identify quantitative changes in the native fauna suggested from other studies. Agroecosystems may even constitute an ecological reservoir for certain native aphidophage.

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Sites surveyed for aphidophage from 2009 to 2011 in Belgium.

![](t01_01)

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Aphidophage diversity in four crops (broad bean, wheat, corn, and potato) from 2009 to 2011.

![](t02_01)

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Species richness and diversity index (H= Shannon-Weiner diversity index, where absolute diversity = 1.00; J = evenness or relative diversity (H/Hmax), where absolute evenness = 1.00).

![](t03_01)

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Abundance (means and SE) of aphidophage (adults) per 100 m^2^ in four agroeco system s from 2009 to 2011. Means within a crop followed by the same letter were not significantly different for the same field (*p* \> 0.05; LSD test).

![](t04_01)

###### 

Abundance (means and SE) of aphidophage (larvae) per 100 m^2^ in four agroecosystems from 2009 to 2011. Means within a crop followed by the same letter were not significantly different for the same field (*p* \> 0.05; LSD test).

![](t05_01)

###### 

ANOVA and ANCOVA summary of the effects of aphid abundance and year sampling (2009, 2010, 2011) on the abundane of five predators at the adult stage in four crops (corn, wheat, potato, and broad bean).

![](t06_01)

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ANOVA and ANCOVA summary of the effects of aphid abundance and year sampling (2009, 2010, 2011) on the abundance of five predators at the larval stage in four crops (corn, wheat, potato, and broad bean).

![](t07_01)

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Mean numbers and SE of aphids/100 m^2^ observed in four fields (wheat, broad bean, corn, and potato) from 2009 to 2011.

![](t08_01)

We thank V. Sibret and A.M. Buset for their technical assistance, Dr Y. Brostaux for his advice on statistical analysis, and P. Leroy and C. De Clerck for their helpful comments on previous versions of the manuscript. The authors are grateful to Professor B. Bodson and F. Vancutsem of the Unité de Phytotechnie des Régions Tempérées (ULg, GxABT) for the availability of fields. This research was funded by the Service Public de Wallonie (SPW - DGO3, project n°D311197). Delphine Durieux was financially supported by a Ph.D. grant from the Fonds pour la formation à la Recherche dans l\'Industrie et l\'Agriculture (FRIA), Belgium.

[^1]: **Editor:** Xinzhi Ni was editor of this paper.
