Effects of marigold on the behavior, survival and nutrient reserves of Aphidius Platensis

Abstract

Marigolds (Tagetes erecta L.) suppress nematodes and are attractive companion plants, but their role in biological control is unknown. We evaluated how exposure to marigold blooms impacts the aphid parasitoid, Aphidius platensis Brethes. Female wasps previously exposed to marigold spent more time walking and parasitizing Myzus percisae Sulzer or Schizaphis graminum Rodani aphids, and subsequently had higher parasitism rates. Meanwhile, completely starved wasps spent more time stationary and marginally more time grooming. Time spent probing, emergence rate, and sex ratio were not affected. Wasp survival was best on honey, followed by marigold, and lowest on non-blooming marigolds. Nutrient reserves of wasps given honey, aphid-infested marigold, or marigold alone were compared to newly-emerged unfed wasps. Their resulting lipid, glycogen and sugar levels were similar, suggesting that these foods helped wasps maintain reserves similar to emergence levels. These results suggest that marigold may improve biological control of aphids by A. platensis.

Full text

Effects of marigold on the behavior, survival and nutrient
reserves of Aphidius Platensis
Ivana Lemos Souza .Rosangela Cristina Marucci .Luis Claudio Paterno Silveira .
Na
´gila Cristina Paixa
˜o de Paulo .Jana C. Lee
Received: 21 November 2017 / Accepted: 4 April 2018
ÓInternational Organization for Biological Control (IOBC) 2018
Abstract Marigolds (Tagetes erecta L.) suppress
nematodes and are attractive companion plants, but
their role in biological control is unknown. We
evaluated how exposure to marigold blooms impacts
the aphid parasitoid, Aphidius platensis Brethes.
Female wasps previously exposed to marigold spent
more time walking and parasitizing Myzus percisae
Sulzer or Schizaphis graminum Rodani aphids, and
subsequently had higher parasitism rates. Meanwhile,
completely starved wasps spent more time stationary
and marginally more time grooming. Time spent
probing, emergence rate, and sex ratio were not
affected. Wasp survival was best on honey, followed
by marigold, and lowest on non-blooming marigolds.
Nutrient reserves of wasps given honey, aphid-
infested marigold, or marigold alone were compared
to newly-emerged unfed wasps. Their resulting lipid,
glycogen and sugar levels were similar, suggesting
that these foods helped wasps maintain reserves
similar to emergence levels. These results suggest
that marigold may improve biological control of
aphids by A.platensis.
Keywords Biological Control Flower Nectar 
Parasitoid Parasitism
Introduction
Aphids are among the most economically-important
crop pests worldwide (Giordanengo et al. 2010). They
are prolific due to their high reproductive capacity and
short developmental time (Bueno 2005). The green
peach aphid, Myzus persicae (Sulzer), attacks a wide
variety of agronomic crops including those in the
Solanaceae family (Capinera 2001). The greenbug or
wheat aphid, Schizaphis graminum Rondani, feeds on
a range of 70 graminaceous species in the Poaceae
family (Michels 1986; Nuessly and Nagata 2005).
In addition to plant feeding, these aphids cause
serious crop loss by vectoring plant viruses. In
neotropical regions, Aphidius platensis Brethes (Hy-
menoptera: Braconidae: Aphidiinae) is a parasitic
wasp that attacks aphids of economic importance
Handling Editor: Stefano Colazza.
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10526-018-9882-8) con-
tains supplementary material, which is available to authorized
users.
I. L. Souza (&)R. C. Marucci 
L. C. P. Silveira N. C. P. de Paulo
Departamento de Entomologia, Avenida Doutor Sylvio
Menicucci, Universidade Federal de Lavras (UFLA),
1001, Kennedy, Lavras, MG 37200-000, Brazil
e-mail: ilemossouza@gmail.com
J. C. Lee
USDA ARS Horticultural Crops Research Unit, 3420 NW
Orchard Ave, Corvallis, OR, USA
e-mail: jana.lee@ars.usda.gov
123
BioControl
https://doi.org/10.1007/s10526-018-9882-8

(Stary
´1975; Tomanovic et al. 2014). It is thought to
originate from India, and has since been introduced in
Africa, South America, and Australia. Aphidius
platensis is a solitary koinobiont endoparasitoid,
pupating within the body of the host.
Floral resources have been recommended to
enhance parasitism and biological control of pests
(Landis et al. 2000; Heimpel and Jervis 2005). For
example, floral diversification has led to higher
parasitism of pests in urban shrubs (Ellis et al.
2005), and even reduced crop damage and improved
yield in cabbage (Balmer et al. 2014). Floral feeding
may increase parasitism via non-exclusive mecha-
nisms: feeding improves a parasitoid’s ability to locate
hosts, extends its longevity, and increases its nutrient
reserves. The success of a parasitoid depends on its
capacity to recognize suitable hosts and parasitize
them. Yet, adult parasitoids must balance short-term
nutritional needs with host searching (Lewis et al.
1998). Sugar-feeding enables wasps to forage more
than starved wasps in a flight chamber (Wa
¨ckers
1994), so floral resources may also increase parasitoid
foraging in the field.
In a review of 104 plant species, many floral species
extend the longevity of parasitic wasps (Russell 2015).
The majority of adult parasitoids must consume plant-
derived foods such as floral nectar and pollen for
maintenance (Jervis et al. 2004). Floral nectar contains
amino acids, vitamins, minerals, sugar, glycogen, and
lipids (Baker and Baker 1983; Nicolson and Thorn-
burg 2007). Not surprisingly, parasitic wasps that
consume floral nectar have higher energetic reserves
of lipid, glycogen, and sugar than starved wasps (Eijs
et al. 1998; Lee et al. 2004). These nutrients are
essential for many functions. Glycogen and sugars
often fuel flight in insects (Hahn and Denlinger 2007),
and wasps fed dill nectar fly further than water or
sucrose-fed wasps in a flight mill (Wanner et al. 2006).
Lipid is essential for insect growth and reproduction
and provides energy during extended non-feeding
periods (Arrese and Soulages 2010). Sugar feeding
enables individual wasps to lay more eggs in hosts
while foraging freely in fields (Lee and Heimpel
2008b; Segoli and Rosenheim 2013). A food shortage
may cause females to resorb eggs and redirect energy
into survival, thus reducing fecundity (Rivero and
Casas 1999).
In selecting a floral resource, the flower must be
attractive and beneficial to the specific natural enemy.
For instance, sweet alyssum, Lobularia maritima (L.)
Desv., benefits some wasps (Berndt and Wratten 2005;
Balzan and Wa
¨ckers 2013; Irvin and Hoddle 2007) but
not others (Nafziger and Fadamiro 2011; Rahat et al.
2005). The marigold, Tagetes erecta Linn. (Aster-
aceae), is an annual herbaceous plant that is used for its
secondary metabolites that can suppress insects, mites,
nematodes, bacteria, fungi, and viruses (Salinas-
Sa
´nchez et al. 2012). ‘Nema-gone’ marigolds (Bur-
peeÓ, Warminster, Pennsylvania, USA) are marketed
for killing nematodes in the soil. Marigolds are also
grown as an agronomic crop because the petals are a
source of natural dye (Singh et al. 2003) and oil
extracts can be lethal to mosquito larvae (Namrata
et al. 2000). A few studies suggest that marigolds can
also benefit pest control. Marigolds planted adjacent to
onions promote greater richness and abundance of
parasitoids compared to onion monocrops (Silveira
et al. 2009). A large diversity of parasitoid families are
found in marigold lines (Sampaio et al. 2008), and
Asteraceae flower heads in Brazil (Nascimento et al.
2014). A related marigold, Tagetes patula, extends the
longevity of a Trissolcus wasp in a laboratory study
(Rahat et al. 2005).
To our knowledge, no studies have examined how
marigolds directly affect parasitism behavior, nor
focused on conservation of A. platensis. Therefore, our
objectives were to determine whether exposure to
blooming marigold: (1a) influences the behavior of A.
platensis among an aphid-host patch, and (1b)
increases the parasitism rate; (2) increases survival
of A. platensis; and (3) elevates the lipid, glycogen and
sugar reserves of A. platensis.
Materials and methods
Insects
The colony of A. platensis wasps was maintained at
the Biological Control laboratory at the Universidade
Federal de Lavras (UFLA), Lavras, Minas Gerais,
Brazil. Parasitoids were reared on Myzus persicae
aphids on sweet pepper Capsicum annuum L. Result-
ing mummies were placed individually in Petri dishes
of 10 cm diameter, sealed with plastic wrap, and held
at 25 ±1°C, 70 ±10% RH and L:D 12:12 h.
Mummies were checked daily for wasp emergence
for experimental use. A colony of Schizaphis
I. L. Souza et al.
123

graminum aphids were reared on sorghum Sorghum
bicolor (L.) Moench for the behavior study.
Marigold and sweet pepper plants
Marigold, sweet pepper and sorghum plants were
grown from seed on sterilized soil (Tropstrato HA
Hortalic¸as, Vida Verde, Mogi Mirim-SP, Brazil) and
watered daily in a greenhouse. When plants reached
10 to 15 cm in height they were transplanted to 2 l
plastic vessels containing a mixture of field-collected
soil (red Latosol, three parts soil and one part manure).
Plants were fertilized every 15 days according to
recommendations from the state of Minas Gerais
(Ribeiro et al. 1999). At the time of experimentation,
marigold plants were 65 days old and 1 m tall, and
sweet pepper and sorghum plants were 35 days old
and *0.6 m tall.
Parasitoid behavior
Parasitoid foraging was tested among four treatment
combinations with wasps having prior marigold
exposure or not, and presented one of two aphid
species: (1) marigold exposure then M.persicae, (2)
marigold then S.graminum, (3) M.persicae, and (4)
S.graminum. Each day, newly-emerged A.platensis
were released in acrylic cages (30 930 960 mm) to
allow mating, and either exposed to a potted blooming
marigold or no plant. After 24 h, wasps were removed
and sexed using a hand lens. Only females were used
in this behavior experiment with 15 replicate females
per treatment, and two replicates per treatment tested
per day.
One female was released in a foraging arena, a
sealed plastic Petri dish (50 915 mm) containing a
fitted leaf disc of sweet pepper with 20 second–third
instar M. persicae aphids on a bed of agar, or a
sorghum disc with 20 S. graminum aphids. Each wasp
was observed through a microscope at 8–1609
magnification for 15 min. Behaviors in real-time were
recorded in EthoLog 2.25 (Ottoni 2016), a software for
transcription and timing of behavioral observation
sessions. Behaviors included: (1) grooming—defined
as cleaning the antennae, legs or ovipositor; (2)
parasitizing–raising the antennae to a characteristic
vertical position, bending of the ovipositor below the
body, followed by prolonged touching of the ovipos-
itor to the aphid; (3) probing–quick touch of the
ovipositor on the aphid (\1 s, may be repeated); (4)
stationary–totally immobile; and (5) walking–moving
around the leaf. While these aphids can produce
honeydew, none of the presented aphids were
observed to have honeydew droplets nor were A.
platensis observed to feed on the aphids. After each
observation, the wasp was removed. The plates
containing aphids were transferred to new leaf discs
every two days. Aphids were checked daily for the
formation of the mummies and wasp emergence. Once
emerged, adults were sexed, and percent parasitism,
emergence rates from mummified aphids, and sex ratio
were tabulated.
An ANOVA was run for each of the eight
dependent variables: proportion of time spent groom-
ing, parasitizing, probing, stationary, and walking;
proportion parasitism, proportion emergence, and sex
ratio. Each dependent variable was tested with flower
exposure, aphid species, and the flower 9aphid
interaction as fixed effects with a normal distribution,
which had a lower AIC value (Akaike 1974; negative
values are smaller) relative to binomial or other
distributions. The AIC values with a binomial and
normal distribution were: grooming 25.6, -155.7,
parasitizing 15.0, -326.5, probing 49.6, -61.8,
walking 79.2, 9.3, stationary 51.3, -18.6, parasitism
73.6, -11.5, emergence 79.2, 50.4, and sex ratio 69.9,
58.0. While the model only included fixed effects,
PROC GLIMMIX was used in SAS 9.3 (SAS Institute Inc
2010) to test each model with various distributions.
Outcomes with normal distribution were identical to
models run in PROC GLM. Proportion parasitism was
regressed with the time spent parasitizing using PROC
REG.
Parasitoid survival
Survival of A.platensis was compared among three
treatments: (1) honey and non-blooming marigold, (2)
blooming marigold, and (3) non-blooming marigold.
Honey droplets were spread on the side of cages. To
prevent wasps from dehydration, cotton balls soaked
in water were placed in each cage to provide additional
humidity. Fifteen cages per treatment were arranged in
a completely randomized design. Ten newly emerged
wasps, five females and five males, were released in
each cage (15 921 923 cm) with a mesh-covered
top. Fresh water was provided every two days and
marigolds were replaced mid-experiment. Cut
Effects of marigold on the behavior, survival and nutrient reserves of Aphidius Platensis
123

marigolds were kept in water-filled 250 ml plastic
vials sealed with plastic wrap. Cages were monitored
daily to record wasp mortality until all wasps died.
Survival is presented as Kaplan–Meier curves, and
analyzed by log-rank using PROC LIFETEST. Since the
treatment effect was significant, subsequent means
separations were done by comparing each treatment
pair using a Sidak adjustment.
Nutrient analysis
The lipid, glycogen, and sugar contents of male and
female wasps were compared among four treatments:
(1) newly-emerged wasps, and wasps held with (2)
blooming marigold, (3) blooming marigold infested
with M. persicae, or (4) honey. Newly-emerged wasps
that have not fed served as a baseline control, 140
females and 112 males were frozen live at -80 °Cas
soon as they emerged. Meanwhile, other newly-
emerged wasps were placed in cages (30 930 9
60 mm) and given various foods for 24 h until
freezing. Potted marigolds and honey droplets were
presented in cages as in the survival study. The
marigold with M. persicae treatment was added in this
study to check whether A. platensis might obtain
additional nutrients from aphid honeydew with floral
nectar. Each treatment was replicated in seven cages,
with 20 live females and 16 males collected live per
cage, with 252 wasps per treatment. There was an
insufficient number of live wasps to collect after
one day of starvation to include this fifth treatment for
nutrient analysis. While it is possible that water
provisioning may have enabled some starved A.
platensis to live longer, it is unlikely that most of the
252 wasps would have survived to replicate this
treatment consistently. Other parasitic wasps with
access to water during starvation did not have
improved or only slightly improved longevity
under *25 °C and 50–75% RH or in field cages
(Idris and Grafius 1995; Leatemia et al. 1995; Lee and
Heimpel 2008a; Jacob et al. 2006; Krugner et al. 2005;
Dyer and Landis 1996).
Wasps were transported in vials of 95% ethanol on
ice to the USDA-ARS Horticultural Research Crops
Unit in Oregon, USA, and then stored at -80 °C.
Lipid levels of A.platensis were determined with a
biochemical assay using a vanillin reagent (van
Handel 1985b), and glycogen and sugar levels were
determined using an anthrone reagent (van Handel
1985a). These methods were first developed for
mosquitoes, and subsequently modified for hymenop-
teran parasitoids (Olson et al. 2000), and dipteran
parasitoids (Fadamiro et al. 2005).
Because of the small size of A. platensis,a
decreased volume of reagents was used to obtain
readings. Preliminary trials showed that
detectable readings occurred when two wasps were
assayed together, thus two females or two males from
the same cage treatment were assayed in pairs. Two
wasps were placed in a 1.5 ml microcentrifuge tube
with 13 ll of 2% sodium sulfate, and crushed with a
pestle. After adding 113 ll of chloroform–methanol
(1:2), the pestle was removed. Contents were vortexed
and then centrifuged for 2 min at 16,000 g to collect
the glycogen precipitate at the bottom of the tube. The
supernatant was transferred into 12 975 mm glass
tubes used for lipid or sugar analysis. With other
insects, such as Diadegma insulare Cresson (Hy-
menoptera: Ichneumonidae) (Lee et al. 2004), the
supernatant volume is divided in half for a lipid assay
and a sugar assay. Due to low nutrient content, half of
the samples with the entire supernatant were desig-
nated for a lipid assay, and the other half for a sugar
assay. Thus, all wasps were assayed for glycogen, and
only half of them were assayed for lipid or sugar
levels. This assay protocol could detect differences in
glycogen and sugar levels between honey-fed versus
starved wasps using a related wasp of similar size,
Aphidius colemani Viereck (Supplementary appendix
A).
For the lipid assay, the supernatant was evaporated
at 90 °C for 3 min until the liquid evaporated. Then,
16 ll of sulphuric acid was added and heated at 90 °C
for 30 s. Then, 190 ll of vanillin reagent was added,
mixed and kept at room temperature for 30 min. The
vanillin reagent contains 700 mg 99% vanillin (CAS
121-33-5, Sigma-Aldrich), 400 ml of 85% phosphoric
acid, and 100 ml of distilled water, with 100 mg more
vanillin than in van Handel (1985b) for increased
sensitivity. The solution was pipetted into a 96-well
ELISA plate, the absorbance was read on a plate
reader (ELX 808, BioTek, Winooski, Vermont, USA)
at 490 nm. Readings were compared to a calibration
line when reacting the vanillin reagent to 1, 5, 10, 20,
35 and 50 lg of the lipid standard (50 mg canola oil,
50 ml chloroform).
For the glycogen assay, 200 ll of anthrone reagent
was added to the precipitate, vortexed, and heated at
I. L. Souza et al.
123

90 °C for 3 min. The anthrone reagent contains
750 mg anthrone (CAS 90-44-8, Sigma-Aldrich),
380 ml sulfuric acid, and 150 ml distilled water (van
Handel 1985a). For the sugar assay, the tube contain-
ing the supernatant was evaporated for 2 min at 90 °C
until *20 ll of solution remained. Then, 200 llof
anthrone reagent was added, the solution vortexed,
and heated for 3 min at 90 °C. Both the glycogen and
sugar assays were cooled in ice, and solutions pipetted
into the ELISA plate. Absorbance at 630 nm was
recorded and compared to calibration lines as
described earlier using a glycogen standard (50 mg
D glycogen, CAS, 9005-79-2, Fisher Scientific, 50 ml
purified water) and sugar standard (50 mg sucrose,
50 ml 25% ethanol in water).
An analysis was run for each sex for each response
variable: lipid, glycogen and sugar. Each response
variable was tested with treatment as a fixed effect and
the cage as a random effect, with either a normal or
log-normal distribution where appropriate in PROC
GLIMMIX. If a treatment effect was significant, means
comparisons were done with Tukey HSD.
Results
Parasitoid behavior
The proportion of time A. platensis spent parasitizing
and walking significantly increased with prior expo-
sure to flowering marigold (Table 1). Wasps exposed
to marigold parasitized 190% and walked 50% more
than unexposed wasps [100 9(avg. marigold -avg.
no marigold)/avg. no marigold]. On the other hand,
wasps without prior marigold exposure spent signif-
icantly more time stationary, and marginally more
time grooming than marigold-exposed wasps
(Table 1). Stationary time was 440% and grooming
time was 130% longer in unexposed A. platensis.
Since time spent parasitizing was higher with prior
marigold exposure, it is not surprising that subsequent
parasitism on these aphids was also affected (Table 1):
parasitism was 155% higher with prior marigold
exposure. Parasitism rates were also affected by the
aphid species presented (Table 1): parasitism of S.
graminum was 67% higher than that of M. persicae.
Lastly, experimental treatments did not affect the time
spent probing, emergence rates for mummified aphids,
or sex ratio of offspring.
Table 1 Average ±SE proportion of time spent in the five behaviors, and reproductive outcomes of Aphidius platensis with/without
prior exposure to marigold in an arena with Myzus persicae or Schizaphis graminum
Behavior M. persicae S. graminum Flower Aphid Flower 9
aphid
Marigold No marigold Marigold No marigold d.f. FPFPFP
Grooming 0.019 ±0.01 0.055 ±0.02 0.023 ±0.01 0.041 ±0.01 1, 56 3.71 0.059 0.12 0.733 0.41 0.525
Parasitizing 0.015 ±0.01 0.003 ±0.01 0.018 ±0.03 0.008 ±0.02 1, 56 12.1 0.001 2.09 0.154 0.14 0.710
Probing 0.12 ±0.02 0.08 ±0.02 0.14 ±0.04 0.11 ±0.02 1, 56 1.56 0.216 0.68 0.41 0.06 0.81
Stationary 0.06 ±0.03 0.24 ±0.05 0.02 ±0.01 0.23 ±0.06 1, 56 19.2 <.0001 0.36 0.551 0.10 0.755
Walking 0.78 ±0.04 0.62 ±0.06 0.79 ±0.04 0.61 ±0.07 1, 56 9.22 0.0036 0.01 0.961 0.04 0.841
Outcomes
Parasitism 0.3 ±0.04 0.11 ±0.04 0.5 ±0.06 0.2 ±0.05 1, 56 27.0 <.0001 8.97 0.0041 1.3 0.260
Emergence 0.58 ±0.06 0.5 ±0.13 0.53 ±0.08 0.69 ±0.11 1, 49 0.262 0.611 0.47 0.495 1.52 0.224
Sex ratio 0.43 ±0.11 0.47 ±0.17 0.66 ±0.08 0.57 ±0.11 1, 42 0.047 0.829 2.15 0.150 0.31 0.579
Values in bold are significant (P\0.05) are from a two-way ANOVA. d.f. are identifical for the three effects in a given test
Parasitism = mummies/aphids, emergence = parasitoids/mummies, sex ratio = female/all parasitoids
Effects of marigold on the behavior, survival and nutrient reserves of Aphidius Platensis
123

Parasitoid survival
Providing A. platensis various foods affected their
survival (Fig. 1,v
2
= 139.7, d.f. = 2, P\0.0001).
Survival on honey was longer than on blooming
marigold, and survival on either food was greater than
on non-blooming marigold. On day 5, all parasitoids
from non-blooming marigold were dead. Honey-
provisioned wasps lived up to 18 days and blooming
marigold provisioned wasps lived up to 16 days.
Nutrient analysis
Overall, lipid, glycogen, or sugar levels of wasps that
were given food were not different from newly-
emerged wasps. Lipid levels of A. platensis females
and males did not differ among the four treatment
groups (Table 2). Newly-emerged females had
numerically the highest lipid level of 2.2 lginan
assay combining two females, or an estimated 1.1 lg
per wasp (Fig. 2a). Individual wasps had on average
0.6–1.1 lg of lipid. Glycogen levels of females and
males also did not differ significantly, although trends
were marginally significant in females (Table 2). Both
Fig. 1 Survival of Aphidius platensis provided honey ?non-
blooming marigold, blooming marigold, or non-blooming
marigold. Different letters show significant treatment differ-
ences following Sidak adjustment: honey versus marigold
P\.0001, honey versus water P\.0001, marigold versus
water P= 0.0041
Table 2 Statistical tests between marigold, aphid-infested
marigold, honey and newly emerged treatments for the lipid,
glycogen and sugar levels of female and male Aphidius
platensis
Sex Nutrients d.f. FP
Females Lipid 3,110 0.1 0.962
Glycogen 3,252 2.17 0.092
Sugar 3,112 4.19 0.008
Males Lipid 3,79 0.23 0.877
Glycogen 3,193 0.81 0.492
Sugar 3,74 0.62 0.602
Significant (P\0.05) values are from a generalized linear
mixed model
Fig. 2 Average (±SE) levels of lipid (a), glycogen (b) and
sugar (c)ofAphidius platensis from four treatments, values are
from two wasps assayed together. Letters indicate a significant
difference between treatments with Tukey HSD, P\0.05
(Table 2), ns no differences between treatments for a given sex
I. L. Souza et al.
123

honey-fed females and males had numerically higher
glycogen levels than other wasps (Fig. 2b). Individual
wasps had on average 0.6–0.95 lg of glycogen.
Interestingly, sugar levels were not affected by
treatment for males, but were for females (Table 2).
Females given access to marigold infested with M.
persicae aphids had 180% higher sugar levels than
females with marigold only (Fig. 2c). Individual
wasps had on average 0.15–0.6 lg of sugar.
Discussion
This study showed that A.platensis previously
exposed to blooming marigold spent more time
walking on the leaf and parasitizing M.persicae and
S.graminum aphids than completely starved wasps.
Consequently, they parasitized a greater proportion of
aphids (Table 1): on average six M.persicae aphids
were parasitized by floral-exposed wasps versus 2.2 by
starved wasps, and ten S.graminum versus four,
respectively. This increase in host search could be due
to the parasitoid having more energy, and is supported
by the observation that starved wasps spent more time
stationary. Alternatively, the increased host search
could be due to marigold-exposed wasps having more
eggs available, which was not assessed in this study.
Starved A. platensis probed for a similar amount of
time as fed wasps, and parasitized 11–20% of the 20
aphids, indicating that they had some eggs and some
interest in hosts. In future studies, it would be
worthwhile to see how various diets affect egg load
by dissecting the ovaries of these small 1.7–2.3 mm
wasps. Numerous past studies support that floral
sources increase parasitoid host foraging. Olfactome-
ter studies reveal that sugar-fed wasps orient towards
host cues (Jacob and Evans 2001; Siekmann et al.
2004; Takasu and Lewis 1993), although high levels of
satiation reduce movement (Lightle et al. 2010).
Wasps fed sugar or nectar have higher egg loads
(Aduba et al. 2013; Araj and Wratten 2015; Tylianakis
et al. 2004). While sugar feeding is expected to
increase fecundity via increasing parasitoid lifespan, it
has also increased the short-term activity of wasps to
parasitize three times more hosts in 24 h than unfed
wasps (Mitsunaga et al. 2004), and 8% more in 5 h
(Ge
´neau et al. 2013).
This study also revealed a difference in host
suitability between two aphid pests, something not
known previously. Female A.platensis spent similar
time in ‘parasitizing’ behavior with both aphid
species, but ultimately parasitized more S.graminum
aphids than M.persicae during 15 min of observation.
This may be related to the smaller 1.3–2.1 mm body
size of S.graminum (Nuessly and Nagata 2005)
compared to the larger 1.7–2.0 mm body size of
M.persicae (Capinera 2001). During observations, the
ovipositor of A. platensis would touch the body of
M.persicae and slide off, suggesting that the para-
sitoid had more difficulty handling this aphid. Another
parasitoid, Aphidius ervi (Hymenoptera: Braconidae),
attacked Acyrthosiphon pisum more than Sitobion
avenae aphids (Hemiptera: Aphididae), and size was
suggested to affect encounter rate and stabbing ability
(Daza-Bustamante et al. 2003). In our study, both
aphid species may have been suitable in terms of
quality. Among mummified aphids, 50–69% success-
fully emerged into parasitoids, and 43–66% of
offspring were female. Other Aphidius species showed
similar trends. Emergence rates of Aphidius gifuensis
(Ashmead) were similar among M. persicae and S.
avenae (Pan and Liu 2014). Emergence of A. colemani
was similar among Aphis gossypii Glover and M.
persicae even though A. gossypii was attacked more
frequently (Sampaio et al. 2001).
Our survivorship study revealed that access to
honey or blooming marigold extends the longevity of
A.platensis. Wasps lived up to 18 days on honey,
16 days on marigold, and five days on non-blooming
marigold. Access to flowers usually enhances para-
sitoid survival as reported in a meta-analysis (Russell
2015) and more recent studies (Araj and Wratten
2015; Zhu et al. 2015). Honey may have been a
superior food source since it is comprised of carbo-
hydrates (95%), proteins including enzymes and free
amino acids (0.5%), and the rest in vitamins, minerals,
aroma compounds and polyphenolic compounds
(Alavarez-Suarez et al. 2009). Other lab studies have
also found honey to benefit parasitoids more than
floral nectar (Charles and Paine 2016; Davies et al.
2004; Irvin et al. 2007; Nafziger and Fadamiro 2011).
Marigolds may have intermediate benefits because the
nectar is oftentimes concealed, which limits access. In
a floral survey, Asteraceae flowers had less consistent
effects on extending parasitoid lifespan because of its
concealed anthers and nectar (Tooker and Hanks
2000). In addition, Tagetes spp. flowers could have
Effects of marigold on the behavior, survival and nutrient reserves of Aphidius Platensis
123

secondary metabolites that are toxic to insects (Sali-
nas-Sa
´nchez et al. 2012).
Lastly, nutrient levels of A. platensis were exam-
ined to see if exposure to different sugar sources
elevated their energetic storage. Parasitoids that feed
on sugar solutions often have elevated glycogen and
sugar (Fadamiro et al. 2005; Lightle et al. 2010; Zhang
et al. 2011) and sometimes lipid levels compared to
unfed parasitoids (Lee and Heimpel 2007). In this
study, A. platensis exposed to various sugar-rich foods
for 24 h had similar nutrient levels as newly-emerged
wasps. This suggests that feeding on these three food
sources allowed wasps to maintain reserves similar to
emergence levels. Additional nutrient assays of
starved A. platensis at one day could further confirm
the benefits of feeding. Nevertheless, assays with the
related A. colemani show that starved wasps have
lower glycogen or sugar levels than honey-fed wasps
(Supplementary appendix A). Previous studies have
not always found higher nutrient levels in wasps
following floral exposure. Two braconid wasp species
lived longer with buckwheat, Fagopyrum esculentum
Moench, but their carbohydrate levels were not
statistically different from starved wasps (Aduba
et al. 2013; Nafziger and Fadamiro 2011). In those
studies and our study, it is possible that ingested food
is metabolized quickly before differences are detected,
or prolonged exposure to food may be needed to build
energetic stores. Lastly, one treatment stood out in our
study. The sugar levels of females exposed to aphid
honeydew and marigold (aphid-infested marigold)
were two-fold higher than females with only marigold.
Since aphid honeydew may be widely accessible in the
field, it may be important for conserving A. platensis.
Honeydew has increased the longevity of other
parasitoid species, although it is often inferior to other
sugar sources (Wa
¨ckers et al. 2008).
In summary, this study supports that marigolds may
have a beneficial impact on A. platensis survival and
parasitism activity, and that additional access to aphid
honeydew leads to higher sugar reserves in A.
platensis. Future studies are needed to determine
whether honeydew also enhances longevity and par-
asitism, whether other floral species are more benefi-
cial, and how attractive marigold and other flowers are
to A. platensis. Based on a model incorporating the
energy status and spatial distribution of parasitoids,
floral attractiveness is key to enhancing biological
control in the field (Bianchi and Wa
¨ckers 2008).
Further studies could also test marigolds in a banker
system for increasing control of S. graminum aphids in
cereal fields. Marigolds could provide nectar for adult
A. platensis and M. persicae aphids for parasitoid
reproduction and honeydew. This aphid species does
not feed on cereal crops, and represents a viable way to
continually maintain A. platensis parasitoids in the
field for biological control.
Acknowledgements We thank all people that work on DEN,
UFLA especially to the Biological control and Conservation
biological control laboratories. We thank the USDA-ARS
Horticultural Crops Research Unit for facilitating a visit to
perform part of the experiments, and J. Wong for comments,
Hanna McIntosh and Katerina Velasco-Graham for reviewing
the manuscript. This work was supported by CAPES
(Coordenac¸a
˜o de Aperfeic¸ oamento de Pessoal de Nı
´vel
Superior) funding, travel by the Universidade Federal de
Lavras Sandwich program, and nutrient assays by base funds
USDA CRIS 2072-22000-040-00D.
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I. L. Souza et al.
123

Luis Claudio Paterno Silveira studies conservation of preda-
tors and parasitoids with flowers support.
Na
´gila Cristina Paixa
˜o de Paulo is an undergraduate student
whom assisted with trials.
Jana C. Lee studies pest management in small fruit and
ornamental crops. This work was carried out at the Universi-
dade Federal de Lavras in Brazil (Led by Silveira and
Marucci), and nutrient assays at the USDA ARS in Oregon,
USA (led by Lee).
Effects of marigold on the behavior, survival and nutrient reserves of Aphidius Platensis
123

Supplementary appendix A: Nutrient assay of Aphidius colemani with various foods
Main paper: Effects of marigold on the behavior, survival and nutrient reserves of Aphidius
platensis
Objective: To determine the lipid, glycogen, and sugar levels of A. colemani exposed to
different food treatments, and confirm that the modified protocol can detect differences in
nutrient levels in this small sized wasp.
Rationale: The main study with A. platensis, held wasps with various diets and compared their
nutrient levels to the newly emerged controls. Most starved A. platensis died before 1 day and
could not be included in the nutrient comparisons with the other treatments. Since A. platensis is
present in Brazil and not available in the USA, where the nutrient assays were done, we
conducted a study with A. colemani, which is related to A. platensis and a similar size.
Methods: Aphidius colemani wasps (Evergreen Growers, Portland, Oregon, USA) were shipped
as mummies to the USDA lab. Wasps that already emerged were not used in the experiment and
were removed from the transport vial. On the same day, newly emerged wasps were isolated in
glass vials and sexed under the microscope. Some wasps were immediately frozen at -80°C,
while others were transferred to 23 x 23 x 25 cm plastic cages with a mesh sleeve. Cages
contained either: 1) cut flowering marigold in water flask, 2) honey streaked on sides, 3) water
wick, or 4) nothing (complete starvation). Wasps were held in cages with marigold, honey or
water for 24 hours, and in cage with nothing for 12 hours before wasps were frozen. Cages were
set up from June 22 to August 2, 2017 on three separate days from three different parasitoid
shipments. For nutrient analyses, wasps were assayed in pairs, the same sex from the same
treatment cage using the same protocol described in the main paper. Sixteen pairs, or 32 females
and 32 males per treatment group, were assayed for glycogen, and 8 pairs per treatment group
were assayed for lipid and sugar levels.
Analyses were done separately for females and males for each response variable: lipid, glycogen
and sugar. Treatment was a fixed effect, and cage was a random effect. A generalized linear
mixed model with a lognormal distribution was fit using PROC GLIMMIX in SAS 9.3 (SAS
Institute 2010).
Results: The variously treated A. colemani did not vary in lipid levels. Honey-fed wasps often
had higher glycogen and sugar levels than starved wasps given water or nothing (Table A1).
Newly emerged A. colemani generally had intermediate levels of nutrients, with no statistical
difference from honey-fed wasps in four out of six comparisons.

Table A1 Average nutrient levels (± SE) of paired A. colemani from diet treatments, and statistical results.
Females
Males
Treatment
Lipid (µg)
Glycogen
Sugar
Lipid
Glycogen
Sugar
Emerged
4.37 ± 0.93
1.83 ± 0.32 b
0.99 ± 0.18 b
4.30 ± 0.69
1.65 ± 0.19 a
2.01 ± 0.64 ab
Honey
3.71 ± 0.73
3.98 ± 0.59 a
6.55 ± 1.89 a
2.02 ± 0.37
2.41 ± 0.36 a
5.40 ± 1.49 a
Marigold
5.98 ± 1.22
1.30 ± 0.14 ab
1.01 ± 0.34 b
2.77 ± 0.71
1.49 ± 0.19 ab
1.08 ± 0.24 ab
Nothing
3.84 ± 1.05
1.43 ± 0.48 ab
0.86 ± 0.26 b
3.56 ± 0.56
1.46 ± 0.24 ab
2.42 ± 1.10 ab
Water
4.02 ± 0.97
1.78 ± 0.24 b
2.72 ± 0.85 ab
2.43 ± 0.39
0.86 ± 0.10 b
0.43 ± 0.16 b
d.f.
4,20
4,60
4,20
4,20
4,60
4,20
F
0.30
5.31
4.85
1.67
6.05
4.85
P
0.877
0.001
0.0067
0.197
0.0004
0.0067