DOI: 10.5061/dryad.stqjq2c1n
title: Ant-hemipteran mutualism: parasitic wasps use cuticular hydrocarbons of ants to avoid them

description: One of the most studied and best-known mutualistic relationships between
 insects is that between ants and phloem-feeding insects. Ants feed on
 honeydew excreted by phloem-feeding insects and, in exchange, attack the
 phloem feeders’ natural enemies, including parasitic wasps. However,
 parasitic wasps are under selection to exploit information on hazards and
 avoid them. Here, we tested whether parasitic wasps detect the previous
 presence of ants attending colonies of phloem feeders. Behavioural assays
 demonstrate that wasps left colonies previously attended by ants more
 frequently than control colonies. This behaviour has a cost for the
 parasitic wasp as females inserted their ovipositor in fewer hosts per
 colony. In a further bioassay, wasps spent less time on papers impregnated
 with extracts of the ant cues than on control papers. GC/MS analyses
 demonstrated that ants left a blend of cuticular hydrocarbons when they
 attended colonies of phloem feeders. These cuticular hydrocarbons are
 deposited passively when ants search for food. Overall, these results
 suggest, for the first time, that parasitic wasps of honeydew producers
 detect the previous presence of mutualistic ants through contact
 infochemicals. We anticipate such interactions to be widespread and to
 have implications in numerous ecosystems, as phloem feeders are usually
 tended by ants.
description: Mealybug colonies exposed to
 queenless ant nests To obtain the ant nests, eight
 nest fragments were collected in a citrus orchard at Instituto Valenciano
 de Investigaciones Agrarias (Moncada, Spain) by digging up the nest from
 the ground. In the laboratory, the experimental nests were placed in eight
 plastic boxes (30.5 × 24.5 × 20 cm). The inner walls of the boxes were
 lined with Fluon at 60% (Aldrich Chemistry) to prevent ants from escaping.
 All fragments of nests were queenless and composed of ~200 workers. Nest
 fragments were kept in the laboratory (23 ± 3°C, natural daylight) and
 provided with a solution of water, honey and yeast at 1:4:1 on a piece of
 aluminium foil twice a week. Water was provided in a glass vial (15 × 1.5
 cm in diameter) tapped with a piece of cotton wool in the middle of the
 vial. The vial was covered with a piece of aluminium foil and ants used it
 as a nest. Forty-eight hours prior to assays, food was removed to starve
 the ants and homogenize their feeding status. To obtain mealybug colonies of
 similar size, potato sprouts were infested with 20-25 second instar to
 pre-ovipositional adult mealybugs, as <i>A</i>.
 <i>vladimiri</i> shows a host preference for older mealybugs
 [35]. Potato sprouts were individually placed into plastic boxes (16.5 ×
 11 × 6 high cm) with walls coated with Fluon both inside and outside the
 boxes. Sprouts were maintained with centrifuge tubes filled with
 bacteriological agar (20 g/L), with the sprout base inserted directly into
 the agar. Tubes were sealed with Parafilm<sup>® </sup>to avoid
 ants digging. After infestation, mealybugs could settle and feed for 48
 h. To obtain
 mealybug colonies that had been in contact with ants (ant-exposed
 colonies), plastic boxes with the infested potato sprouts were connected
 to ant nests. Starved ants were allowed to forage in a mealybug colony by
 temporarily connecting the colony with the plastic box with a wire as a
 bridge (Fig. 1A). Twenty-four hours later the wire was carefully removed
 while ants were not using it. Immediately, potato sprouts were moved to an
 experimental arena to observe the behaviour of the female wasp. Arenas
 consisted of a polystyrene plastic box (10 × 14 × 14 cm) with a lateral
 hole (4 × 9 cm) covered with muslin. Inside the arena, the centrifuge tube
 with the infested potato sprout was placed vertically on a silicon base to
 elevate the colony and improve the accuracy of the observations. The same
 procedure was followed with mealybug colonies that had not been exposed to
 ants. Each treatment was replicated 55 times. 
 Mealybug colonies exposed
 to field queenright ant nests To obtain mealybug colonies of
 similar size, green bean pods were used as plant substrate for the
 mealybugs. Prior to inoculation, one side of the bean pods was submerged
 in red paraffin wax to minimize the area for the mealybugs to settle and
 facilitate the observations (based on the methodology described by [36]).
 Bean pods were infested with 20-25 second instar to pre-ovipositional
 adult <i>P. citri</i>. Mealybugs were allowed to settle and
 feed for 24 h. To
 obtain mealybug colonies that had been in contact with field ants, twelve
 citrus trees with high <i>L. grandis</i> activity were
 selected in a 15-year-old IVIA organic citrus orchard [<i>Citrus
 sinensis</i> (L.) Osbeck (Var. Navelate)]. A plastic box was used to
 expose the beans infested with mealybugs to foraging ants. Boxes were 38.5
 × 32 × 25 cm and had four small holes (0.5 cm diameter) in one side to
 allow the entry of ants, and two big lateral holes (15 × 10 cm) covered
 with mesh for ventilation. Boxes were placed near the base of a citrus
 tree’s trunk and the four holes of the boxes were placed next to the path
 of <i>L. grandis</i> that were foraging in the tree canopy
 (Fig. 1B). In each box, infested beans were kept with insect pins on
 flower sponge fixed with silicone to the base of the box. Ants had access
 to the infested beans for 24 h. Then, the box was carefully removed from
 the trunk while ants were not present. Immediately after collection,
 infested beans were transported back to the laboratory in sterile boxes
 using disposable nitrile gloves. The same procedure was followed to obtain
 mealybug colonies non-exposed to ants, but the holes were blocked to
 exclude ant attendance. Experimental arenas for the behavioural assays of
 the field queenright nests were the same as in the queenless assays.
 Inside the arena, the beans infested with mealybugs were placed in two
 acrylic cylinders (5 cm diameter, 1 cm height) to elevate the colony and
 improve the accuracy of the observations. Each treatment was replicated 63
 times. Effect of previous ant attendance
 on parasitoid behaviour For both types of ant colonies,
 parasitic wasp searching behaviour was recorded in mealybug colonies i)
 non-exposed to ants and ii) ant-exposed. A female wasp was released in an
 arena with the mealybug colony and the following behaviours were recorded:
 i) arrival of the wasp in the mealybug colony (i.e. spent more than 3 sec
 in the colony), ii) total time spent in the colony, iii) whether it left
 the colony (i.e. spent more than 3 sec out of the colony), iv) number of
 times it left the colony, and v) the total number of times she inserted
 her ovipositor in a host. Each observation begun 1 min after the parasitic
 wasp was released in the arena and lasted a minimum of 30 min and, after
 this period, the observation ended when the wasp rested or walked for more
 than 5 min without contacting hosts. To account for potential temporal
 effects, equal numbers of each treatment were tested each day in both
 assays, randomizing the order of testing between days. Arenas were used
 once per day, cleaned with alcohol and left to dry for at least 24 h. All
 observations were carried out between 10:00 and 15:00 h. 
 Effect of ant cue
 infochemicals on parasitoid behaviour The effect of <i>L.
 grandis</i> cue extracts on the behaviour of <i>A.</i>
 <i>vladimiri </i>was further investigated with a non-choice
 bioassay and extracts of the ant cues. Five extra trail extracts (ca. 960
 ×5 = 4,800 ant-equivalents), obtained in the laboratory as described above
 from queenless ant nests, were gathered and used to treat filter paper
 squares for the bioassays. Papers treated with ant cues were impregnated
 with 50 µL of the pentane solution of trail extracts (ca. 320
 ant-equivalents). Control papers were impregnated with the same volume of
 pentane (50 µL). Papers were used for experiments 5 min after they were
 impregnated to allow the solvent to evaporate. A drop of honey (75%) was
 provided on treated and control paper squares (1.5 × 1.5 cm). Papers were
 left in the middle of a glass Petri dish (5 cm diam.), into which a single
 wasp was then released. Petri dishes were used once per day, cleaned with
 alcohol and left to dry for at least 24 h. After allowing the parasitic wasp
 to settle (1 min), the proportion of wasps that contacted the paper and
 the time spent on the paper were measured for a period of 10 min. Each
 treatment was repeated 15 times. To account for potential temporal
 effects, equal numbers of each treatment were tested each day, randomizing
 the order of testing between days. Composition of ant
 infochemicals <i>Collection of chemical
 trails</i> The collection of chemical trails
 left by <i>L. grandis</i> was performed using Teflon coated
 wires [37], with slight differences between the queenless ant nests and
 field ant nests. For the queenless ant nests, metal wires (25 x 0.5 cm
 diameter) previously washed with ethanol were coated with Teflon tape and
 were employed as bridges to connect ant nests (total 300 ants) with boxes
 containing a sucrose solution feeder. To obtain control Teflon coated
 bridges, the same procedure was followed but boxes did not have
 ants. For the
 field queenright ant nests, we used a similar methodology as for the
 behavioural observations. The same boxes were placed on the foraging path
 of the ants, but bean pods infested with mealybugs were placed inside a
 smaller plastic box (16.5 × 11 × 6 cm) coated with
 Tangle-Trap<sup>®</sup> (Tanglefoot, Grand Rapids, Michigan).
 Metal wires (25 x 0.5 cm diameter) coated with Teflon tape were also
 employed as bridges to connect the inside of the small plastic boxes with
 the outside bigger box. Therefore, ants searching in the plastic
 containers had to walk over the Teflon coated bridges to reach the
 mealybug colonies. To obtain control Teflon coated bridges, the same
 procedure was followed but the entrance holes to the plastic container
 were blocked with clay to exclude ants. In both experiments, ants were
 allowed to forage and cross the coated bridges for 24 h. Considering 8 h
 of effective activity of the total 24 h that the bridge was coated and one
 ant crossing the bridge each 30 s, each trail extract was considered to
 contain ca. 960 ant-equivalents. This is likely a conservative estimation
 as ants remain active during the night. Bridges were carefully removed
 when ants were not crossing the bridges and Teflon tapes were extracted
 with 3 mL of pentane (HPLC grade, Sigma-Aldrich, Madrid, Spain) to obtain
 the cues left behind by the ants, which were subsequently analysed by gas
 chromatography coupled to mass spectrometry (GC/MS). The control Teflon
 coated bridges were extracted in an identical way. Five replicates of
 chemical trails and four replicates of control samples were collected for
 the queenless ant nests, while ten replicates of chemical trails and five
 replicates of control samples were collected for the field ant
 nests. <i>Chemical
 analysis</i> Chemical trail and control
 extracts were concentrated to ~10 μL under gentle helium flow, and 2 μL
 were analyzed by GC/MS. All injections were performed on a Clarus 600
 GC/MS apparatus (Perkin Elmer Inc., Wellesley, PA) equipped with a 30 m x
 0.25 x 0.25 fused-silica capillary column (Zebron ZB-5MS, Phenomenex Inc.,
 Torrance, CA). Extracts were injected in splitless mode with the oven
 programmed at 100 ºC for 1 min, raised at 10 ºC/min up to 180 ºC,
 maintained for 1 min, and then 5 ºC/min up to 280 ºC with 20-min hold.
 Injector temperature was set at 250 ºC and helium at 1 mL/min was used as
 carrier gas. The detection was performed in EI mode at 70 eV with
 ionization source and transfer line set at 180 ºC and 250 ºC,
 respectively. Scan mode was employed (<i>m/z</i> 35-500) and
 tentative identification was based on retention indices according alkane
 standards and diagnostic ions reported in literature, as there is no
 commercial sources for these cuticular hydrocarbons [38–42].
 Statistical analysis
 We compared the
 total time spent in the colony using ANOVA. The normality assumption was
 assessed using Shapiro’s test, and the homoscedasticity assumption was
 assessed with Levene’s test. The total time spent on the filtered paper
 was not normally distributed and was analyzed with a Wilcoxon test.
 Proportional and count data were analyzed with generalized linear models
 (GLMs). Initially, we assumed a Poisson error variance for count data
 (number of times the wasp left the colony, and number of stings per
 colony) and a binomial error variance for proportional data (colony
 detection; proportion of colonies with at least one host stung by the
 parasitic wasp; proportion of filter papers detected by the parasitic
 wasp). We assessed the assumed error structures by a heterogeneity factor
 equal to the residual deviance divided by the residual degrees of freedom.
 If we detected an over- or underdispersion, we re-evaluated the
 significance of the explanatory variables using an <i>F</i>
 test after rescaling the statistical model by a Pearson’s Chi-square
 divided by the residual degrees of freedom [43]. We present the means of
 untransformed proportion and count data (in preference to less intuitive
 statistics such as the back-transformed means of logit-transformed
 data). Principal component analysis (PCA) was performed
 to visualize, through score and loading plots, differences in the
 chromatographic peak areas of all compounds in the four experimental cases
 (laboratory assay: queenless ant nests and controls; field assays: field
 queenright ant nests and controls). The chromatographic peak areas of all
 compounds were integrated for each replicate. The resulting data were
 arranged in a matrix of 24 rows (replicates) and 14 columns (chemical
 compounds as variables). In this data set, the zero value was assigned to
 those compounds not detected in a given experimental case. The minimum
 value of peak area was around 10<sup>4 </sup>units, the median
 was ∼10<sup>5 </sup>units, and the maximum was
 ∼10<sup>6</sup> units. For compounds detected at trace levels,
 below the integration threshold, we used 10<sup>3</sup> units
 as area value, which is 1 log-unit below the minimum integrated area.
 Then, to normalize the data distribution, area values were transformed by
 applying the quadratic root transformation. The <i>prcomp</i>
 function was employed to perform the PCA and the number of principal
 components to be considered was determined by examining their eigenvalues
 (l) and proportion of variances by using the
 <i>get_eigenvalue</i> function in the factoextra package. The
 <i>ggplot</i> function in the package ggplot2 was employed to
 visualize the scores. All data analyses were performed with the R v.3.6.3
 statistical package [44].