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fevo-10-773423 April 12, 2022 Time: 10:2 # 1
ORIGINAL RESEARCH
published: 13 April 2022
doi: 10.3389/fevo.2022.773423
Edited by:
Mathew Samuel Crowther,
The University of Sydney, Australia
Reviewed by:
Gabriele Rondoni,
University of Perugia, Italy
Tamara Szentivanyi,
Centre for Ecological Research,
Hungarian Academy of Science,
Hungary
*Correspondence:
Danny Haelewaters
danny.haelewaters@gmail.com
†
†
†Present Address:
Danny Haelewaters,
Department of Biology, Ghent
University, Ghent, Belgium
Specialty section:
This article was submitted to
Population, Community,
and Ecosystem Dynamics,
a section of the journal
Frontiers in Ecology and Evolution
Received: 09 September 2021
Accepted: 10 March 2022
Published: 13 April 2022
Citation:
Haelewaters D, Hiller T,
Ceryngier P, Eschen R, Gorczak M,
Houston ML, Kisło K, Knapp M,
Landeka N, Pfliegler WP, Zach P,
Aime MC and Nedv ˇ
ed O (2022) Do
Biotic and Abiotic Factors Influence
the Prevalence of a Common Parasite
of the Invasive Alien Ladybird
Harmonia axyridis?
Front. Ecol. Evol. 10:773423.
doi: 10.3389/fevo.2022.773423
Do Biotic and Abiotic Factors
Influence the Prevalence of a
Common Parasite of the Invasive
Alien Ladybird Harmonia axyridis?
Danny Haelewaters1,2,3†, Thomas Hiller4, Piotr Ceryngier5, René Eschen6,
Michał Gorczak7,8 , Makenna L. Houston2, Kamil Kisło8, Michal Knapp9,
Nediljko Landeka10 , Walter P. Pfliegler11, Peter Zach12, M. Catherine Aime2and
Oldˇ
rich Nedv ˇ
ed1,3
1Faculty of Science, University of South Bohemia, ˇ
Ceské Budˇ
ejovice, Czechia, 2Department of Botany and Plant Pathology,
Purdue University, West Lafayette, IN, United States, 3Biology Centre of the Czech Academy of Sciences, Institute
of Entomology, ˇ
Ceské Budˇ
ejovice, Czechia, 4Department of Ecology of Tropical Agricultural Systems, University
of Hohenheim, Stuttgart, Germany, 5Institute of Biological Sciences, Cardinal Stefan Wyszy ´
nski University, Warsaw, Poland,
6CABI, Delémont, Switzerland, 7Institute of Evolutionary Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland,
8Botanic Garden, Faculty of Biology, University of Warsaw, Warsaw, Poland, 9Department of Ecology, Faculty
of Environmental Sciences, Czech University of Life Sciences Prague, Prague, Czechia, 10 Public Health Institute of the Istrian
Region, Pula, Croatia, 11 Department of Molecular Biotechnology and Microbiology, University of Debrecen, Debrecen,
Hungary, 12 Institute of Forest Ecology, Slovak Academy of Sciences, Zvolen, Slovakia
Hesperomyces virescens (Ascomycota, Laboulbeniales), a fungal ectoparasite, is thus
far reported on Harmonia axyridis from five continents: North and South America,
Europe, Africa, and Asia. While it is known that He. virescens can cause mortality of Ha.
axyridis under laboratory conditions, the role of biotic and abiotic factors in influencing
the distribution of He. virescens in the field is unknown. We collected and screened
3,568 adult Ha. axyridis from 23 locations in seven countries in Central Europe between
October and November 2018 to test the effect of selected host characters and climate
and landscape variables on the infection probability with He. virescens. Mean parasite
prevalence of He. virescens on Ha. axyridis was 17.9%, ranging among samples from
0 to 46.4%. Host sex, climate, and landscape composition did not have any significant
effect on the infection probability of He. virescens on Ha. axyridis. Two color forms, f.
conspicua and f. spectabilis, had a significantly lower parasite prevalence compared to
the common Ha. axyridis f. novemdecimsignata.
Keywords: community ecology, Hesperomyces, Laboulbeniales, parasite prevalence, precipitation, temperature,
spatial modeling, agricultural landscape
INTRODUCTION
Parasites may be the least studied life form on the planet (Price, 1980;Windsor, 1990, 1995).
In their call for a “global parasite conservation plan,” Carlson et al. (2020) proposed 12 major
goals within four themes. These themes are data collection and synthesis (aimed at describing
parasites and incorporating them into biodiversity surveys, among others), risk assessment and
prioritization (documenting drivers of parasite declines and develop regional and global Red
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Haelewaters et al. Biotic Factors Influencing Hesperomyces virescens
Lists), conservation practice (e.g., building parasite conservation
capacity), and outreach and education. One of the most common
parasites of the globally invasive harlequin ladybird Harmonia
axyridis (Coleoptera, Coccinellidae) is Hesperomyces virescens,
which was for the first time observed on this host in 2002
(Garcés and Williams, 2004).
Hesperomyces virescens (Ascomycota, Laboulbeniales) is a
biotrophic fungus that has a very wide distribution with
confirmed reports in North and South America, Europe, Africa,
and Asia (Haelewaters et al., 2017). Based on the results of an
integrative taxonomic approach, we know that He. virescens is
a complex of multiple species segregated by hosts (Haelewaters
et al., 2018a). Thus far, two species have been formally described
in the complex, He. halyziae (Haelewaters and De Kesel,
2020) and He. parexochomi (Crous et al., 2021). The other
species, including the one associated with Ha. axyridis, are
awaiting formal description. The parasite prevalence of He.
virescens on Ha. axyridis differs among geographic regions and,
exceptionally, may be as high as 96.5% in a given ladybird
population, as reported in Meise, Belgium (February 2012,
n= 107) and in Westmoreland, New Hampshire, United States
(December 2012; n= 83) (Haelewaters et al., 2017). As
a result, He. virescens has recently gained traction among
entomologists as a potential biological control agent against
Ha. axyridis.
Little information is available about the ecology of He.
virescens. A recent experimental study demonstrated that
infection with He. virescens affects the survival of Ha. axyridis on
its own and when ladybirds are co-infected with either of two
entomopathogenic fungi, Beauveria bassiana and Metarhizium
brunneum (Haelewaters et al., 2020). Thus far, however, potential
effects of biotic and abiotic factors on the distribution of He.
virescens and its parasite prevalence on Ha. axyridis are unknown.
This information is fundamental for studies in conservation,
applied ecology, and biocontrol strategies (Ferrier, 2002;Rushton
et al., 2004;Magan, 2021). In this study, we collected adult
specimens of Ha. axyridis across Central Europe and evaluated
how selected host traits and climate and landscape variables
affect infection patterns with He. virescens. Variables tested
included host sex, host color form, color of elytra, proportion of
agricultural and forested areas (European Environment Agency,
2020), temperature, and precipitation (Fick and Hijmans, 2017).
MATERIALS AND METHODS
Ladybirds were collected either by hand or using a mouth-
operated aspirator from October to November 2018 in different
Central European countries (Figure 1): Croatia (Istria County),
the Czech Republic (Central Bohemian Region, South Bohemian
Region, Plzeˇ
n Region), Germany (State of Bavaria), Hungary
(Hajdú-Bihar County), Poland (Mazovian Voivodeship),
Slovakia (Nitra Region), and Switzerland (Canton of Jura).
Contributors were asked to collect at least 100 specimens from
each ladybird population. Geographic coordinates were recorded
and can be found in Supplementary File 1. Specimens were
preserved in 70% ethanol until examination in the laboratory.
Ladybirds were screened under 40–50×magnification for
the presence of non-hyphal thalli of He. virescens (sensu De
Kesel, 2011;Haelewaters et al., 2018a). For each ladybird, the
following traits were recorded: sex (see McCornack et al., 2007);
color form [non-melanic f. novemdecimsignata (also referred
to as succinea), and melanic f. conspicua, f. axyridis, and f.
spectabilis]; color of elytra (for non-melanics) or spots (for
melanics) (yellow, orange, red; Fiedler and Nedvˇ
ed, 2019).
For novemdecimsignata specimens, we described whether spots
were well-circumscribed (0), missing or fewer in number than
typically present (−), or large and touching each other (+)
(Fiedler and Nedvˇ
ed, 2019). For each population, no matter the
number of sampled ladybirds, we screened 100 randomly selected
specimens. When available, we screened and processed additional
specimens of the melanic forms to avoid statistical restrictions
due to these forming in low percentages. Screening results for
all processed ladybirds are available in Supplementary File 1.
After processing, voucher specimens were deposited in the
Purdue Entomology Research Collection (West Lafayette, IN,
United States) under the following accession numbers: PERC
0147670–0147680.
All statistical analyses were performed using the R software,
version 3.6.3 (R Core Team, 2020). To identify the variables
influencing the infection probability of He. virescens on Ha.
axyridis, we used generalized mixed effect models (GMEM) with
a binomial data distribution (infected yes/no) [function glmer(),
R package lme4;Bates et al., 2015]. We included host sex, host
color form, color of elytra, the proportion of agricultural, and
forested areas in a buffer surrounding each sampling location
(100 m, 300 m, 600 m, 1 km, and 2.5 km), and climate variables.
Urban area was excluded from the analysis because it was highly
collinear with the other predictor variables. For each buffer
radius, a separate model was calculated, resulting in five distinct
models. The landscape variables were obtained by extracting the
landscape composition of Copernicus Corine Land Cover images
taken in 2018 (European Environment Agency, 2020) with the
help of R package raster, using the function extract (Hijmans
et al., 2020). We pooled values in the categories “broad-leaved
forest,” “coniferous forest,” and “mixed forest” into the forested
area variable, whereas the agricultural area variable consisted
of values for the categories “non-irrigated arable land,” “fruit
trees and berry plantations,” “pastures,” “complex cultivation
patterns,” and “land principally occupied by agriculture, with
significant areas of natural vegetation” (Supplementary File 2).
We further calculated three models using a “ring buffer” or
annulus (sensu Rey et al., 2020), one for each of the following
radius combinations: 100 m inner and 300 m outer radius,
300 m inner and 600 m outer radius, and 600 m inner and
1 km outer radius.
Climate variables were extracted from WorldClim with a
resolution of 30 arc seconds (ca. 1 ×1 km) (Fick and Hijmans,
2017) (Supplementary File 2). As variables for temperature
and humidity are generally collinear, we decided to summarize
the following variables using a principal component analysis
(PCA) [prcomp(), R package stats;R Core Team, 2020]: annual
mean temperature, mean maximum temperature in the hottest
month, mean minimum temperature in the coldest month,
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Haelewaters et al. Biotic Factors Influencing Hesperomyces virescens
FIGURE 1 | Parasite prevalence by sampled population.
annual precipitation, precipitation during the wettest month,
and precipitation during the driest month. We extracted the
values for the first two dimensions for all sampling sites
(Supplementary File 2). Statistical processing revealed two
principal dimensions that explained 64 and 30.1% of the climatic
variation, respectively. Finally, we included the random intercept
collection nested within sampling region to address repeated
sampling at the same location and also spatial autocorrelation as
suggested by Zuur et al. (2010).
Hypothesis testing was done using likelihood ratio tests, with
pvalues calculated based on χ2distributions, declaring an effect
significant when p≤0.05. Nine models were compared, namely,
the Null Model and the model with variables of interest within
the different buffer radii (100 m, 300 m, 600 m, 1 km, and
2.5 km) and annulus radii (100–300 m, 300–600 m, and 600 m–
1 km). Model selection happened using the Akaike Information
Criterion (Akaike, 1974). For all models, we calculated pseudo-
R2values to estimate model fit by accounting for the variation
explained by both fixed and random effects [function r2(), R
package performance;Lüdecke et al., 2020].
RESULTS
We screened a total of 3,568 ladybirds, resulting in a mean
infection prevalence of 17.9%, ranging from 0 to 46.4% among
sampled populations (Figure 1). The population with the highest
prevalence of He. virescens was from Levice in southwestern
Slovakia. Two populations showed no visible signs of He.
virescens infection, both of which were from Poland. Forty-seven
individuals were excluded from statistical analyses, including
a single f. intermedia specimen from Warsaw, Poland and 46
specimens with missing information on elytral color. Likelihood
ratio tests confirmed that each of the eight models explained
the observed variance better than chance (Table 1), while the
conditional pseudo R2-values estimated model fit at around 0.33,
indicating good fit (Table 1). We only found variables on host
individual characters to have significant effects on the parasite
prevalence of He. virescens on Ha. axyridis (Tables 2,3). The
significant effect of color form was consistent in all five models,
with the color forms f. conspicua and f. spectabilis being less often
infected compared to the common form f. novemdecimsignata(0).
There was a trend for f. novemdecimsignata(-) to be less
likely infected by He. virescens compared to the common
form f. novemdecimsignata(0). This trend was consistent over
all candidate models but not significant. Finally, the color of
elytra had a significant effect on the infection probability, with
individuals with red elytra being more likely and individuals with
yellow elytra less likely infected compared to individuals with
orange elytra. Host sex, climate, and habitat composition resulted
in not having any significant effect on the infection probability of
He. virescens on Ha. axyridis.
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Haelewaters et al. Biotic Factors Influencing Hesperomyces virescens
TABLE 1 | Results of model (dependency of Hesperomyces virescens prevalence
on Harmonia axyridis on coloration and climate and habitat properties) evaluation
showing candidate models being significantly better than the null model and
estimated pseudo-R2values indicating model fit.
Model AIC χ2pvalue pseudo-R2
Null Model 2,934.0
Buffer 100 m 2,880.7 77.3 <0.001*** 0.35
Buffer 300 m 2,880.1 77.9 <0.001*** 0.33
Buffer 600 m 2,879.3 78.6 <0.001*** 0.32
Buffer 1 km 2,879.6 78.4 <0.001*** 0.32
Buffer 2.5 km 2,880.8 77.2 <0.001*** 0.35
Annulus 100–300 m 2,880.1 77.9 <0.001*** NA
Annulus 300–600 m 2,879.2 78.8 <0.001*** 0.32
Annulus 600 m–1 km 2,879.8 78.2 <0.001*** 0.33
Significance levels at: ***p <0.001.
DISCUSSION
Ha. axyridis–He. virescens
The first published record of He. virescens on Ha. axyridis in
Europe was made in the winter of 2006–2007, from Meise
in Belgium (De Kesel, 2011). Other country records followed
quickly, from the Netherlands (2008), Germany (2008–2009),
Croatia and the Czech Republic (2013), Hungary and Poland
(2014), Slovakia (2015), Bulgaria and Greece (2017), and most
recently European Russia and Switzerland (2018) (Herz and
Kleespies, 2012;Ceryngier and Twardowska, 2013;Ceryngier
et al., 2013;Pfliegler, 2014;Gorczak et al., 2016;Ceryngier
and Romanowski, 2017;Haelewaters et al., 2017;van Wielink,
2017;Orlova-Bienkowskaja et al., 2018; this paper). Many of
these papers show that the parasite prevalence of He. virescens
differs significantly over time and in space (Raak-van den
Berg et al., 2014;Haelewaters et al., 2017). Differences in
Laboulbeniales prevalence among locations have been attributed
to host population density and habitat type (Scheloske, 1969;De
Kesel, 1996), but to date, no data were thus far available with
regard to the He. virescens–Ha. axyridis association.
Effect of Biotic Factors
A remarkable finding from our study is that the melanic color
forms f. conspicua and f. spectabilis were less often infected
with He. virescens compared to the common nineteen-spotted
f. novemdecimsignata(0). Two other studies investigated the
relationship between the degree of melanization and infection
patterns. Haelewaters et al. (2018b) observed a slight trend to
higher intensity of parasitism in more melanic males of Ha.
axyridis f. novemdecimsignata.Fiedler and Nedvˇ
ed (2019) found
(i) a positive association between putative age groups of Ha.
axyridis estimated as carotenoid content and infection with
He. virescens and (ii) a negative association between elytral
melanization of Ha. axyridis f. novemdecimsignata specimens
and infection. The latter was explained by the fact that younger
ladybirds emerged later in the year, with lower temperatures
inducing extensive melanization; since they were younger, they
had less opportunities to be parasitized by He. virescens. Our
results are in line with the findings of Fiedler and Nedvˇ
ed (2019),
but a definitive answer as to how elytral melanization affects the
susceptibility to infection with He. virescens remains unclear. The
black coloration of melanic forms of Ha. axyridis is negatively
correlated with the total content of alkaloids (Bezzerides et al.,
2007), which serve as defense against predators and pathogens
(Röhrich et al., 2011). Then, heavier melanization should result in
more infection with He. virescens, but our results are inconsistent
with this hypothesis. Future work—performing bioassays in
controlled settings and analyzing expression levels of immune
genes—is needed to shed light on the susceptibility of different
color morphs to He. virescens.
The results of our modeling approach show a significant
correlation between elytral color and the infection probability of
Ha. axyridis with He. virescens. Carotenoid accumulation, and
thus red color intensity, is a function of ladybird age (Bezzerides
et al., 2007;Nedvˇ
ed et al., 2019). In our study, older individuals
of Ha. axyridis accumulated more He. virescens inoculum.
Similar observations were made by Fiedler and Nedvˇ
ed (2019),
particularly that individuals with red elytra are more likely to
be infected. Hesperomyces virescens transmits among ladybirds
through physical contacts (during mating and in overwintering
aggregations), but auto-transmission by grooming or cleaning
also occurs. These factors contribute to parasite prevalence (at the
level of population) and thallus density (at the individual level)
being positively correlated with host age (Riddick and Schaefer,
2005;Nalepa and Weir, 2007;Haelewaters et al., 2017) and thus,
incidentally, with elytral color.
Effect of Abiotic Factors
Mean parasite prevalence did not significantly change according
to any of the temperature variables tested [Kruskal–Wallis test,
function kruskal.test(), R package stats; R Core Team, 2020], but
our sampling scheme was somewhat limited, with only localities
in Central European countries. For a broader understanding of
the associations between He. virescens and Ha. axyridis within
Europe, we recommend incorporation of data from northern and
southern European countries in order to compile a dataset with a
larger range in temperature. Collections also need to be expanded
to city centers (e.g., in parks, community gardens, edges of
playgrounds). A negative correlation was found between parasite
prevalence of He. virescens sensu lato on Adalia bipunctata and
distance from the city center of London (Welch et al., 2001).
Prevalence in central London was as high as 40% (n= 105),
whereas it was 0% outside of the urban area at a distance of
25 km. This could be linked to increased temperatures in urban
environments (urban heat island effect), but this has not yet been
tested. Adriaens et al. (2008) highlighted the idea that Ha. axyridis
is less frequently found in natural landscapes compared to more
urbanized and anthropogenic landscapes. Habitat preference may
also be an important factor in the parasitism with He. virescens.
Factors promoting Ha. axyridis may indirectly promote He.
virescens. Particularly, a higher dominance of Ha. axyridis in
a given ladybird community will be beneficial for ascospore
transmission among individuals. The number of generations
of Ha. axyridis in Central Europe varies from two to three
and is probably dependent on habitat summer temperatures
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TABLE 2 | Obtained parameters of each candidate model addressing the prevalence of infection by Hesperomyces virescens for different buffer radii (100 m, 300 m,
600 m, 1 km, 2.5 km).
100 m buffer 300 m buffer
Estimate Std. Error z value pvalue Estimate Std. Error z value pvalue
(Intercept) −1.597 0.507 −3.149 0.002 ** −1.652 0.480 −3.441 0.001 ***
f. axyridis 0.299 0.768 0.389 0.697 0.303 0.769 0.394 0.693
f. conspicua −0.944 0.282 −3.347 0.001 *** −0.942 0.282 −3.344 0.001 ***
f. spectabilis −0.469 0.156 −2.996 0.003 ** −0.469 0.156 −2.999 0.003 **
f. novemdecimsignata(-) −0.224 0.122 −1.846 0.065 . −0.225 0.121 −1.853 0.064 .
f. novemdecimsignata(+) 0.020 0.170 0.117 0.907 0.022 0.170 0.128 0.898
red 0.599 0.124 4.839 0.000 *** 0.594 0.124 4.797 0.000 ***
yellow −0.663 0.154 −4.310 0.000 *** −0.665 0.154 −4.318 0.000 ***
sex m −0.003 0.102 −0.032 0.975 −0.003 0.102 −0.030 0.976
agricultural 0.036 0.123 0.293 0.769 0.090 0.108 0.835 0.403
forest 0.029 0.097 0.304 0.761 0.044 0.096 0.453 0.651
Dim1 0.327 0.210 1.556 0.120 0.279 0.205 1.360 0.174
Dim2 −0.090 0.127 −0.713 0.476 −0.100 0.122 −0.822 0.411
600 m buffer 1 km buffer
Estimate Std. Error z value pvalue Estimate Std. Error z value pvalue
(Intercept) −1.679 0.461 −3.644 0.000 *** −1.654 0.466 −3.549 0.000 ***
f. axyridis 0.303 0.768 0.395 0.693 0.302 0.768 0.393 0.694
f. conspicua −0.944 0.282 −3.352 0.001 *** −0.948 0.282 −3.364 0.001 ***
f. spectabilis −0.469 0.156 −3.005 0.003 ** −0.471 0.156 −3.017 0.003 **
f. novemdecimsignata(-) −0.223 0.121 −1.839 0.066 . −0.221 0.121 −1.824 0.068 .
f. novemdecimsignata(+) 0.023 0.170 0.136 0.892 0.023 0.170 0.133 0.894
red 0.591 0.124 4.771 0.000 *** 0.593 0.124 4.780 0.000 ***
yellow −0.666 0.154 −4.331 0.000 *** −0.668 0.154 −4.341 0.000 ***
sex m −0.002 0.101 −0.018 0.985 −0.002 0.101 −0.016 0.987
agricultural 0.135 0.105 1.291 0.197 0.123 0.104 1.178 0.239
forest 0.039 0.090 0.432 0.666 0.027 0.088 0.304 0.761
Dim1 0.248 0.198 1.249 0.212 0.264 0.200 1.321 0.187
Dim2 −0.113 0.120 −0.946 0.344 −0.120 0.122 −0.981 0.327
2.5 km buffer
Estimate Std. Error z value pvalue
(Intercept) −1.571 0.502 −3.129 0.002 **
f. axyridis 0.300 0.769 0.390 0.697
f. conspicua −0.946 0.282 −3.356 0.001 ***
f. spectabilis −0.470 0.156 −3.005 0.003 **
f. novemdecimsignata(-) −0.224 0.121 −1.841 0.066 .
f. novemdecimsignata(+) 0.021 0.170 0.122 0.903
red 0.598 0.124 4.808 0.000 ***
yellow −0.665 0.154 −4.319 0.000 ***
Sex m −0.002 0.101 −0.020 0.984
Agricultural 0.030 0.157 0.189 0.850
Forest 0.034 0.113 0.299 0.765
Dim1 0.334 0.216 1.544 0.123
Dim2 −0.086 0.136 −0.634 0.526
Significance levels at: .p<0.1, **p <0.01, ***p <0.001.
and prey availability. Population densities can increase due to
the use of various prey patches enabled by high mobility in
combination with the weak tendency for diapause extending
the breeding period (Honek et al., 2018). In urbanized areas,
the breeding season of Ha. axyridis is advanced by 2–3 weeks
(Honek et al., 2021).
In addition to our buffer radius models, which are often
used for forest management on a landscape scale (Brouwers
et al., 2010), we also employed annulus radii to better model
the behavior of Ha. axyridis in autumn. Our sampling localities
are not where ladybirds became infected with He. virescens. As a
result, the habitat variables might not be accurate for the habitats
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TABLE 3 | Obtained parameters of each candidate model addressing the prevalence of infection by Hesperomyces virescens for different annulus radii (100–300 m,
300–600 m, 600 m–1 km).
100–300 m 300–600 m 600 m–1 km
Estimate Std. Error z value pvalue Estimate Std. Error z value pvalue Estimate Std. Error z value pvalue
(Intercept) −1.651 0.478 −3.454 0.001 *** −1.671 0.460 −3.636 0.000 *** −1.634 0.472 −3.464 0.001 ***
f. axyridis 0.304 0.769 0.395 0.693 0.302 0.768 0.393 0.694 0.301 0.768 0.393 0.695
f. conspicua −0.943 0.282 −3.346 0.001 *** −0.946 0.282 −3.357 0.001 *** −0.949 0.282 −3.369 0.001 ***
f. spectabilis −0.469 0.156 −3.001 0.003 ** −0.470 0.156 −3.010 0.003 ** −0.472 0.156 −3.021 0.003 **
f. novemdecimsignata(-) −0.225 0.121 −1.853 0.064 . −0.222 0.121 −1.832 0.067 . −0.221 0.121 −1.820 0.069 .
f. novemdecimsignata(+) 0.022 0.170 0.129 0.898 0.023 0.170 0.135 0.892 0.022 0.170 0.130 0.897
red 0.594 0.124 4.795 0.000 *** 0.591 0.124 4.773 0.000 *** 0.594 0.124 4.789 0.000 ***
yellow −0.665 0.154 −4.320 0.000 *** −0.667 0.154 −4.333 0.000 *** −0.668 0.154 −4.341 0.000 ***
sex m −0.003 0.101 −0.030 0.976 −0.002 0.102 −0.015 0.988 −0.002 0.101 −0.016 0.987
agricultural 0.093 0.106 0.877 0.381 0.139 0.104 1.336 0.181 0.111 0.105 1.058 0.290
forest 0.046 0.097 0.472 0.637 0.037 0.089 0.419 0.675 0.024 0.087 0.269 0.788
Dim1 0.275 0.204 1.348 0.178 0.249 0.197 1.262 0.207 0.279 0.201 1.388 0.165
Dim2 −0.101 0.121 −0.829 0.407 −0.114 0.119 −0.957 0.338 −0.119 0.124 −0.964 0.335
Significance levels at: .p<0.1, **p <0.01, ***p <0.001.
that the ladybirds occupied when they became infected with
the fungus. Generally, we estimate that Ha. axyridis ladybirds
fly 500 m during autumn migration. Detailed observations of
ladybirds in ˇ
Ceské Budˇ
ejovice, Czech Republic allowed the exact
measurement of migration distance from before flight and after
flight, which was 200–500 m toward the north (O. Nedvˇ
ed,
unpublished). The longest flights measured were around 1,800 m
in laboratory flight mills (R˚
užiˇ
cka, 1984). When Nalepa et al.
(2005) conducted their experiments to test the role of visual
contrast in autumn behavior of Ha. axyridis in 4 ha of open
pasture, the required flight was about 200 m (Nalepa et al., 2005).
In open fields in Japan, the median flight distance was around
400 m (Seko et al., 2008). We tested three different annulus radii
(100–300 m, 300–600 m, and 600 m–1 km), but the results of
our candidate models were highly similar to the buffer radius
models. Aggregations of ladybirds are often formed on walls of
building oriented toward the south or west (Kidd et al., 1995;
Raak-van den Berg et al., 2012;Haelewaters et al., 2018b). As
a result, we suggest that the annulus modeling approach could
be made even more specific by obtaining landscape variables
for an annulus section (or ring pie chart) facing the direction
where ladybirds likely migrated from. This is methodologically
complex and out of the scope of this paper, but is a consideration
for future studies.
Community ecology research of Laboulbeniales is still
in its infancy, with thus far only two published studies.
Szentiványi et al. (2019) investigated whether climatic variables
(temperature, humidity) influenced the distribution of ant-
and bat fly-associated Laboulbeniales. They found that both
the presence and prevalence of Laboulbeniales on their hosts
were positively associated with low annual mean temperature
and humidity. In addition, based on the study of more
than 9,374 workers of the invasive ant Lasius neglectus in
66 colonies, Gippet et al. (2021) found that the presence
of Laboulbenia formicarum on the ants was positively linked
to warmer and dryer conditions at lower elevations. These
are seemingly contrary results, and our data render drawing
general conclusions for these microfungi even more complex.
However, the direction of the effects of some of these
variables may be species-specific, as suggested by Dumolein
(2021). One could make the case that the combined analysis
of presence/absence data in Szentiványi et al. (2019) may
obscure true interactions, and thus that separate analyses would
give a more accurate picture of how bioclimatic variables
affect the distribution of the two assessed study systems—the
bat fly-associated Arthrorhynchus spp. vs. the ant-associated
Rickia wasmannii. We note that the studies of Szentiványi
et al. (2019) and Gippet et al. (2021) used outside climatic
data as obtained from MERRAclim, but many of these ant–
Laboulbeniales and bat fly–Laboulbeniales interactions and the
host dynamics resulting in fungal transmission take place in
ant nests and bat roosts, respectively. Ant nest and bat roosting
environments are characterized by their own microclimatic
conditions, which likely play a role in shaping the distribution
of these species of Laboulbeniales. Efforts should be redirected at
collecting temperature and relative humidity data (e.g., through
automated readers) within these environments to test for the
effect of these microclimate-specific abiotic traits on parasitism
with Laboulbeniales.
Our results are the first for the Ha. axyridis–He. virescens
study system based on specimens collected during autumn
migration. Understanding the factors influencing the infection
of Laboulbeniales on invasive ladybirds—including climatic and
landscape variables as well as seasonality and host behavior (e.g.,
Raak-van den Berg et al., 2014;Haelewaters et al., 2015, 2017;
Markó et al., 2016)—will help understand their global spread
as they cross many different ecosystems and environmental
conditions. Resolving this question will also inform potential
biocontrol strategies because it will inform us under which
conditions He. virescens may (or may not) thrive. The collection
of standardized multi-year, multi-site field data will help in this
regard in addition to controlled laboratory experiments.
Frontiers in Ecology and Evolution | www.frontiersin.org 6April 2022 | Volume 10 | Article 773423
fevo-10-773423 April 12, 2022 Time: 10:2 # 7
Haelewaters et al. Biotic Factors Influencing Hesperomyces virescens
DATA AVAILABILITY STATEMENT
The original data from this study are included in the
article/Supplementary Material, further inquiries can be
directed to the corresponding author.
AUTHOR CONTRIBUTIONS
DH and ON designed the study. DH, TH, PC, RE, MG, KK,
MK, NL, WPP, PZ, and ON collected the data. DH, TH, PC,
and MH performed data analysis. DH, MCA, and ON acquired
funding. DH and TH drafted the manuscript. DH revised the
manuscript. All authors edited and approved the final version
of the manuscript.
FUNDING
This work was supported by a Junior Postdoctoral Fellowship
from the Research Foundation–Flanders (1206620N to
DH); the Polish Ministry of Science and Higher Education
(grant no. DI2014012344 to MG); funds from the project
Improvement in Quality of the Internal Grant Scheme at CZU,
CZ.02.2.69/0.0/0.0/19_073/0016944 (students grant 71/2021);
the Scientific Grant Agency of the Ministry of Education,
Science, Research and Sport of the Slovak Republic and the
Slovak Academy of Sciences (VEGA, grant no. 2/0032/19 to PZ);
a USDA National Institute of Food and Agriculture Hatch project
(1010662 to MCA), and the Czech Science Foundation (grant
no. 20-10003S to ON). RE was supported by CABI with core
financial support from its member countries (and lead agencies)
including the United Kingdom (Foreign, Commonwealth and
Development Office), China (Chinese Ministry of Agriculture
and Rural Affairs), Australia (Australian Centre for International
Agricultural Research), Canada (Agricultural and Agri-Food
Canada), Netherlands (Directorate-General for International
Cooperation), and Switzerland (Swizz Agency for Development
and Cooperation).
ACKNOWLEDGMENTS
We thank Aaron D. Smith (Purdue Entomological Research
Collection) for curatorial support, Thomas E. Martin (Operation
Wallacea) for textual edits, Michal ˇ
Reˇ
richa (Czech University
of Life Sciences Prague) for help with the processing of
Czech specimens, and two reviewers for critical feedback that
considerably improved the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fevo.2022.
773423/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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