J Behav (2010) 23:165–179 DOI 10.1007/s10905-010-9201-4

The Foraging Behavior of Lysiphlebus fabarum (Marshall), a Thelytokous Parasitoid of the in Iran

Arash Rasekh & J. P. Michaud & Hossein Allahyari & Qodratollah Sabahi

Revised: 19 November 2009 /Accepted: 14 January 2010 / Published online: 8 February 2010 # Springer Science+Business Media, LLC 2010

Abstract A series of laboratory experiments examined the foraging behavior of a thelytokous strain of Lysiphlebus fabarum (Marshall), a strongly proovigenic parasitoid of Aphis fabae Scopoli, in Iran. Females use chemical camoflage to forage undisturbed in ant-tended aphid colonies and solicit honeydew from aphids in the manner of ants. Rates of oviposition are very low (∼ 1.2 eggs / h) despite many aphid encounters and persistent ovipositor probing which appears to prime aphids for subsequent honeydew solicitation. Starved females spent 3.6 times longer in host patches (leaf disks with 15 2nd–3rd instar A. fabae) than did females sated on honey, and 40% of this time was spent soliciting honeydew. Five d-old females spent longer in patches than did one d-old females, and parasitized three times as many aphids. A 24 h pre-trial foraging experience did not reduce mean egg load significantly compared to a one h experience, but was sufficient to reduce patch residence time and number of aphids parasitized. reared under short day conditions (L:D=10:14) were more pessimistic foragers (remained longer in patches and parasitized more aphids) than females raised under long days (L:D=16:8). Wasps that encountered aphids previously parasitized by conspecifics began to superparasitize and remained longer in patches than females that encountered only unparasitized aphids. Encounters with other females had no effect on foraging behavior, possibly because cuticular camoflage interferes with conspecific recognition. The exceptionally low oviposition rate of this may reflect a life history in which individual fitness has evolved to be strongly dependent on continued ant attendance throughout the period of progeny development.

A. Rasekh Department of Plant Protection, Shahid Chamran University of Ahvaz, Ahvaz, Iran

J. P. Michaud (*) Kansas State University, Agricultural Research Center—Hays, 1232 240th Ave, Hays, KS 67601, USA e-mail: [email protected]

H. Allahyari : Q. Sabahi Department of Plant Protection, College of Agriculture, University of Tehran, Karaj, Iran 166 J Insect Behav (2010) 23:165–179

Keywords Aphis fabae . ants . day length . honeydew . oviposition . patch residence time

Introduction

Foraging parasitoids often face uncertainty with respect to food and host availability and adjust their behavior according to the conditions they encounter. For example, a female’s propensity to search for hosts, or accept them after they are encountered, can be influenced by intrinsic factors such as age, egg load, nutritional status, mating status, etc. These have been referred to as ‘physiological state variables’ (Collins and Dixon 1986;Roitbergetal.1993; Sirot et al. 1997). Parasitoids may also learn from experience (Godfray and Waage 1988; Vet et al. 1990; Chow and Mackauer 1992) and environmental cues can guide adaptive responses to hosts and host patches, provided these are reliable indicators of current or future foraging conditions (e.g., host availability, relative host quality, intensity of competition, etc.). Furthermore, external cues may interact with internal states to modulate pivotal decisions in foraging behavior such as host acceptance and patch leaving (Lucchetta et al. 2007). Lysiphlebus fabarum (Marshall) (: : ) is a ubiquitous parasitoid of the black bean aphid, Aphis fabae Scopoli, in many agroecosystems (Starý 1986; Nuessly et al. 2004). This wasp has mainly thelytokous reproduction in central Europe (Nemec and Starý 1985; Starý 1986; Kavallieratos et al. 2008) where it has been recorded from 44 different aphid hosts (Kavallieratos et al. 2004; Tomanović et al. 2009) and frequently parasitizes A. fabae on both crops and weeds (Hildebrand et al. 1997; Völkl and Stechmann 1998). Many species of the genus Lysiphlebus are remarkable for cuticular lipids that closely mimic those of their aphid hosts (Liepert and Dettner 1993). These myrmecophilous species are able to forage with impunity in ant-tended aphid colonies and so garner the protection of ants for their progeny while they are in vulnerable immature stages within their hosts (Völkl 2000). We observed that our particular strain of L. fabarum also engaged in apparent ant mimicry behavior—the use of antennation to solicit honeydew directly from aphids (Rasekh et al. 2010). Despite a median longevity of only 4.5 d, this wasp is able to utilize a combination of chemical and behavioral mimicry to obtain resources and services from members of trophic levels both above and below it. Host-feeding on hemolymph is more common in synovigenic parasitoids that require a source of protein to mature their eggs (Godfray 1994). However, L. fabarum is largely proovigenic, emerging with several hundred mature eggs in its ovaries (Belshaw and Quicke 2003). Since honeydew is primarily a source of sugar, honeydew solicitation by L. fabarum likely substitutes for nectar-feeding in providing the energy that fuels foraging behavior, even though recent studies suggest that honeydew may be a generally inferior sugar source (Wackers et al. 2008; Wyckhuys et al. 2008). Patch residence time (PRT) is often a focal point of behavioral analysis for parasitoid species that exploit hosts with clumped distributions (Waage 1979; J Insect Behav (2010) 23:165–179 167

McNamara and Houston 1985; Godfray 1994). Charnov’s(1976) marginal value theorem can be extended from foraging for prey to foraging for patches; a rate- maximizing forager should select a patch residence time so that the marginal rate of fitness gain at the time of leaving equals the long term average rate of fitness gain in the habitat. However, parasitoids typically have little information about overall habitat quality, and this may be limited to a few recent experiences. Some behaviors typically occur outside the host patch (e.g., habitat-seeking, mate-seeking, or food- seeking) whereas others occur only within it (e.g., host attack, oviposition, decision to leave). However, thelytokous parasitoids avoid the time expenditure associated with seeking a mate (Price 1980), and also the cost of producing sons (Maynard Smith 1978). Parasitoids such as L. fabarum that feed directly from their hosts save even more time and energy by avoiding the need to search for food outside host patches (Jervis and Kidd 1986). Here it becomes more difficult to assign a discrete function to behaviors associated with host encounters, i.e., are they directed at acquiring food or parasitizing hosts? We made direct observations of L. fabarum within artificially constructed patches of its host, the black bean aphid, to explore its time allocation to behaviors associated with feeding and parasitism, while manipulating intrinsic and extrinsic factors that are predicted to impact these behaviors. Specifically, we hypothesized that females in a low-energy state (created by a period of starvation) would increase the proportion of time they allocated to soliciting honeydew at the expense of parasitism rate. We hypothesized that younger females would leave patches earlier and parasitize fewer aphids than older ones, and that long versus short pre-trial foraging experiences would produce the same effect. In addition, we hypothesized that females exposed to short day lengths, aphids previously parasitized by other females, or conspecific females, would remain longer in patches and parasitize more aphids relative to females receiving control experiences, since these signals are indicative of unfavorable reproductive conditions.

Materials and Methods

Insect Colonies

A thelytokous colony of L. fabarum was established from mummies collected from black bean aphids infesting broad bean in a field in Zanjan Province, Iran, in June 2007. A stock colony of A. fabae was maintained on potted broad bean, V. faba var. Sarakhsi, grown in pots filled with fertilized sawdust in growth chambers at 20±1°C, 65–75% RH, and a 16:8 (L:D) photoperiod. The parasitoid was reared on A. fabae fed on broad bean under the same conditions. All aphids used in experiments were 4 d (± 6 h) old at 20°C (late 2nd–early 3rd instar nymphs). Synchronous cohorts of wasps were produced by exposing 2nd instar A. fabae to 3 d-old female wasps in a 5:1 ratio in ventilated plastic cylinders (8.0 cm diameter x 20.0 cm) for a period of 6 h and then transferring the aphids to potted bean plants in a growth chamber until mummies formed. Mummies were carefully removed from plants by scraping them gently off the leaves and then isolated in gelatin capsules 168 J Insect Behav (2010) 23:165–179

(vol.=0.95 cm3) until emergence, whereupon each adult female was released into her own ventilated plastic cylinder (3.5 cm diameter×7.0 cm) and provisioned with diluted honey (as droplets on a strip of wax paper) and water (on a cotton roll). The water was refreshed daily and the diluted honey every second day. Unless otherwise noted, all females had continuous access to food prior to testing andwereusedinexperimentswhentheywere72±4hofagewithoutprior exposure to aphids. All experiments were carried out on a laboratory bench under fluorescent light and an ambient temperature of 20°C within a climate-controlled, walk-in growth chamber.

The Experimental Arena

Experiments were performed by releasing individual L. fabarum females into glass Petri dishes (3.5 cm diameter×1 cm) containing a leaf disk of broad bean on which 15 2nd instar A. fabae had been permitted to settle and feed for 2–3h.Oncea female encountered the first aphid, the lid of the arena was removed to create an open patch and a stopwatch was started. Each female was observed continuously under a stereomicroscope while she remained in the patch and the time of onset and duration of all distinguishable behavioral events, as well as defensive behaviors of the aphids such as kicking and producing droplets of cornicle secretions, were recorded using an MP3 voice recorder. The audio recordings were subsequently transcribed and used to tally female behaviors, time allocation and aphid defensive responses. Patch time was tallied for each female as the time from her first encounter with an aphid until the time she walked over the lip of the dish and left the patch. Various female behaviors were categorized as follows: resting (wasp in a state of immobility), searching (walking across the leaf with periodic antennation of the surface), host antennation, abdominal bending (forward orientation of the abdomen beneath the thorax in preparation for attack; usually associated with visual examination of the host at a fixed distance), and probing (insertion of the ovipositor). Note that tallies of ‘encounters’, ‘probes’ etc. refer to total events and thus include numerous multiple bouts of behavior with the same aphids. In order to estimate rates of parasitism, a subset of the aphids attacked by each female in an experimental arena was removed for rearing. Since many attacks were brief and repeated, only aphids receiving ovipositor insertions ≥ 25 s in duration were removed. Previous work (Rasekh et al. 2010) indicated that only 7.5% of parasitized aphids received ovipositor insertions < 25 s. Each such cohort of aphids attacked by a female was placed on an excised bean shoot in a mini-cage on a small container of water. After 4 days in a growth chamber, all attacked aphids from each replicate were dissected and the parasitoid larvae within them counted.

Starvation

Since L. fabarum females mimic ants in order to obtain honeydew directly from aphids (Rasekh et al. 2010) we reasoned that a female’s level of satiation could influence her proportional time allocation to various behaviors associated with aphid exploitation. To test this, we starved a series of females (n=20) for a period of 5±1 h J Insect Behav (2010) 23:165–179 169 prior to testing in the experimental arena, while providing another series (n=20) with continuous access to water and diluted honey.

Age

To test for effects of female age on foraging behavior, we introduced females (n=36) into experimental arenas at either 24±4 h of age or at 120±4 h of age.

Foraging Experience and Egg Load

A series of two d-old females were divided into two groups. Females of one group (n=15) were each isolated for 24 h with 50 s instar A. fabae on a bean shoot (long foraging experience). Females of the other group (n=15) were allowed to forage for 1 h under the same conditions (short foraging experience) on the day of the experiment. Each female was rested for 1.5–3.0 h (confined without aphids) before beingintroducedintotheexperimentalarenaonherthirddayoflife.Sincethe long foraging experience could potentially result in a reduction of egg load relative to the short foraging experience, we dissected females following termination of each replicate. The ovaries of each female were dissected out in a drop of saline solution on a microscope slide and then squashed under a cover slip to release the eggs. Initial egg loads were obtained by adding the number of eggs laid by the wasp in the experiment to the number of eggs remaining in the ovaries following dissection.

Day Length

This experiment was designed to determine if female foraging behavior would be affected by day length, an indicator of season, and whether day length was assessed by during the larval stage, the adult stage, or both. One group of parasitoids (n=36) were reared in aphids under long day conditions corresponding to mid- summer (L:D=16:8) and then divided into two further groups once they emerged as adults, one exposed to long days, the other to short days corresponding to late autumn (L:D=10:14). A second group of parasitoids (n=36) were reared under short day conditions and then divided into two groups upon emergence to receive the two different day length treatments as adults. Females were then tested in experimental arenas on their third day of adult life.

Encounters with Parasitized Aphids

Parasitized aphids were produced by exposing 2nd instar A. fabae to female wasps in a ventilated plastic cylinder (8.0 cm diameter×20.0 cm) in a 5:1 ratio for a period of 24 h. Three day-old L. fabarum females (n=36) were divided randomly into two treatment groups. Individuals of one group were each placed in a Petri dish with 20 previously parasitized aphids for 20 min, whereas those of the other group each received 20 unparasitized 2nd instar A. fabae. Following this exposure, females of both treatment groups were then isolated in empty dishes for 60–70 min prior to testing in experimental arenas. 170 J Insect Behav (2010) 23:165–179

Conspecific Encounters

To test for effects of conspecific encounters on foraging behavior, females (n=26) were held either singly or in pairs in gelatin capsules (as above) for 90–120 min prior to testing in the open patch protocol.

Statistical Analysis

Since the type of data generated by these types of experiments is either not normally distributed or is otherwise unsuited to parametric analysis, we used the nonpara- metric Mann Whitney U-test to compare treatment effects (SPSS 1998). Although medians and quartiles are conventionally reported for nonparametric comparisons, they lack descriptive value for low frequency events (e.g., the important variable ‘no. aphids parasitized’). Consequently, we chose to report means and standard errors in tables. We used Spearman’s correlation coefficient to analyze the relationships between time spent antennating aphids and number of honeydew droplets obtained, and between female egg load and PRT.

Results

Starvation

Starved females remained more than three times as long in experimental arenas as did sated females and therefore yielded higher scores for the incidence and duration of most foraging behaviors (Table 1), although it was difficult to distinguish whether aphids were being sought for food or for hosts. Hungry females began searching

Table 1 Behavioral Data (mean±SE) for 3 Day-Old Lysiphlebus fabarum Females That Either had Continuous Access to Diluted Honey (Sated, n=20), or were Deprived of Food for 5 h (Starved, n=20) Prior to Testing in an Arena Containing 15 Second Instar Aphis fabae on a Bean Leaf Disk

Variable Starved Sated UP

Patch residence time (min) 72.1±5.6 20.1±2.8 7.0 < 0.001 Resting time (min) 7.3±1.7 0.3±0.2 39.5 < 0.001 Search time (min) 8.1±0.9 4.5±0.5 83.0 0.001 Antennation time (min) 27.8±4.5 1.5±0.5 4.0 < 0.001 Abdominal bending time (min) 13.9±2.0 7.2±1.2 101.5 0.007 Probing time (min) 7.7±1.1 4.1±0.7 114.5 0.020 No. aphid encounters 39.9±4.6 18.3±4.7 72.5 < 0.001 No. aphids escaping probes 0.4±0.2 1.7±0.4 121.0 0.033 No. aphids secreting honeydew 2.3±0.43 0.1±0.01 49.0 < 0.001 No. honeydew droplets consumed 1.9±0.36 0.05±0.05 54.5 < 0.001 No. aphids probed 7.9±1.0 3.3±0.8 60.0 < 0.001 No. aphids parasitized 0.6±0.23 0.2±0.12 158.5 0.134 J Insect Behav (2010) 23:165–179 171 more quickly than sated females upon introduction into a patch and encountered their first aphid in a significantly shorter period (U=111.0, P=0.015). Because of the large effect of treatment on PRT, the incidence and duration of various behaviors was expressed as a fraction of PRT and then re-analyzed. Females from both treatments encountered a similar number of aphids per unit of PRT (U=151.0, P=0.185). However, starved females spent proportionally ten-fold more time resting (U=69.0, P<0.001), almost five-fold more time antennating aphids (U=24.0, P<0.001) than sated females. This coincided with less than half the proportional amount of time searching (U=89.5, P=0.002), abdominal bending (U=1107.5, P=0.014) and probing aphids (U=124.5, P=0.040). A significantly smaller proportion of aphids kicked following encounters with hungry females than following encounters with sated fed females (U=62.5, P<0.001). When all data were pooled, there was a significant correlation between the time wasps spent antennating aphids and the number of honeydew droplets secreted by aphids (rs=0.626, P2-tailed<0.001).

Age

Five d-old females began foraging more quickly than did one d-old females and encountered their first aphid earlier (U=94.5, P=0.031). Older females also remained more than three times as long in arenas and scored higher values for the incidence and duration of almost all behaviors (Table 2). However, when values were expressed as proportions of total patch time there were no significant differences (P>0.15 in all cases) between young and old females, suggesting that age had no effect on proportional time allocation to various behaviors during patch exploitation.

Foraging Experience and Egg Load

Females in the two treatment groups began the experiment with a mean difference in initial egg load of 22 eggs, which was not statistically significant (Table 3).

Table 2 Behavioral Data (mean±SE) for Lysiphlebus fabarum Females of Two Ages (n=18 for Each Age) That were Each Tested in an Open Arena Containing 15 Second Instar Aphis fabae on a Bean Leaf Disk

Variable One day old Five days old UP

Patch residence time (min) 10.1±1.8 33.2±4.2 39.0 < 0.001 Resting time (min) 0.02±0.02 0.29±0.15 108.5 0.091 Search time (min) 2.8±0.4 8.9±0.9 27.0 < 0.001 Antennation time (min) 0.28±0.12 2.00±0.85 121.0 0.203 Abdominal bending time (min) 3.7±0.8 11.1±1.9 65.0 0.002 Probing time (min) 2.9±0.8 10.6±1.6 55.0 < 0.001 No. aphid encounters 7.0±1.4 19.1±2.4 52.5 < 0.001 No. aphids kicking 0.44±0.15 1.61±0.39 92.0 0.027 No. aphids probed 3.1±0.8 10.4±1.5 51.0 < 0.001 No. aphids parasitized 0.5±0.17 1.6±0.33 88.0 0.019 172 J Insect Behav (2010) 23:165–179

Table 3 Behavioral Data (mean±SE) for 3 d-Old Lysiphlebus fabarum Females That Had Each Been Exposed to 50 s Instar Aphis fabae for Either One h (High Egg Load, n=15) or 24 h (Low Egg Load, n=15) andThenRested1.5–3.0 h Prior to Testing in an Open Arena with 15 Second Instar Aphis fabae on a Bean Leaf Disk

Variable Low egg load High egg load UP

Initial egg load 197.1±7.0 218.9±9.8 80.5 0.187 Patch residence time (min) 12.7±2.7 26.8±3.4 48.0 0.007 Search time (min) 3.9±0.8 5.8±0.5 74.5 0.116 Antennation time (min) 0.23±0.1 0.40±0.19 110.0 0.935 Abdominal bending time (min) 4.5±1.2 12.7±1.9 37.5 0.001 Probing time (min) 3.6±0.9 6.6±1.1 67.5 0.061 No. aphid encounters 7.3±1.7 15.0±1.5 46.5 0.005 No. aphids kicking 0.0 0.33±0.16 52.5 0.011 No. aphids probed 3.9±0.9 8.9±1.2 45.0 0.004 No. aphids parasitized 0.27±0.18 1.07±0.23 51.0 0.010

Nevertheless, females from the short host exposure treatment remained more than twice as long in arenas as those receiving the long host exposure treatment, spent more time in abdominal bending, and probed and parasitized more aphids. When proportional time allocation was analyzed, females from the short host exposure treatment spent proportionally less time searching than females from the long host exposure treatment (U=54.0, P=0.015) but proportionally similar time in abdominal bending (U=72.0, P=0.098). Females from both treatments encountered a similar number of aphids per unit of PRT (U=103.0, P=0.713) and spent a similar proportion of their time probing them (U=97.5, P=0.539), but females with short host exposures parasitized more aphids per unit of PRT (U=53.0, P=0.013). Despite the lack of a significant difference in egg load between treatments, there was a strong correlation between female PRT and initial egg load (rs=0.673, P2-tailed<0.001).

Day Length

Wasps that experienced only short day conditions spent more time in arenas than did wasps that experienced only long day conditions and had higher scores for the incidence and duration of all behaviors associated with parasitism (Table 4). Wasps that experienced only short day conditions encountered their first aphid sooner than did those that experienced only long day conditions (U=71.0, P=0.003), but there were no significant differences in proportional time allocation (P>0.20 in all cases). The ‘long day / short day’ and ‘short day / long day’ treatments (data not shown) were included to resolve the relative sensitivity of immatures and adults to day length. Consequently, these two treatments were compared only to the ‘long day / long day’ and ‘short day / short day’ treatments, respectively. Whereas an increase in day length across life stages had a significant effect on wasp foraging behavior, a decrease did not. When larvae developed under long days, a short day length adult experience had no significant effect on any measure of wasp behavior (P>0.38 in all cases). However, when larvae developed under short days, a long day adult J Insect Behav (2010) 23:165–179 173

Table 4 Behavioral Data (mean±SE) for 3 d-Old Lysiphlebus fabarum Females (n=18 per treatment) Experiencing Either Long or Short Days as Both Immatures and Adults Prior to Foraging in an Open Arena Containing 15 Second Instar Aphis fabae on a Bean Leaf Disk

Variable Day length UP

L:D=16:8 L:D=10:14

Patch residence time (min) 13.9±2.7 29.6±3.1 54.0 < 0.001 Search time (min) 3.4±0.6 7.7±1.0 59.0 0.001 Antennation time (min) 0.5±0.3 1.3±0.5 127.0 0.279 Abdominal bending time (min) 6.6±1.2 13.0±1.6 72.0 0.004 Probing time (min) 3.3±1.1 3.2±0.6 133.0 0.034 No. aphids encountered 10.4±2.0 17.6±1.9 91.0 0.024 No. aphids probed 3.9±1.0 7.0±1.0 95.0 0.034 No. aphids parasitized 0.17±0.09 0.67±0.16 96.0 0.037 experience decreased PRT (U=93.0, P=0.029), search time (U=100.0, P=0.051), abdominal bending time (U=379.0, P=0.002) and encounter rates with aphids (U=81.5, P=0.010) relative to a short day adult experience, although there was no effect on time spent probing aphids (U=117.0, P=0.161) or the number of aphids parasitized (U=152.0, P=0.767).

Encounters with Parasitized Aphids

Females that encountered aphids previously parasitized by a conspecific female prior to testing displayed longer PRTs than females encountering only healthy, unparasitized aphids and also scored higher levels of activity for all foraging behaviors (Table 5). Treatment females also probed more aphids per minute of patch time than did their control counterparts (U=58.0, P=0.001) and superparasitized a

Table 5 Behavioral Data (mean±SE) for 3 d-Old L. fabarum Females (n=18 per treatment) Encountering Either Healthy or Parasitized (24 h Previously) Aphids Prior to Foraging in an Open Arena Containing 15 Second Instar Aphis fabae on a Bean Leaf Disk

Variable Prior aphid encounters UP

Healthy Parasitized

Patch residence time (min) 20.6±3.7 36.2±4.0 70.5 0.003 Search time (min) 5.3±0.7 9.1±1.0 73.0 0.004 Abdominal bending time (min) 4.8±1.0 8.1±1.0 91.0 0.024 Antennation time (min) 2.4±1.5 5.4±1.7 94.1 0.031 Probing time (min) 6.6±1.4 12.2±1.3 76.0 0.006 No. aphids probed 6.2±1.2 14.3±1.3 46.0 < 0.001 No. aphids parasitized 0.7±0.2 1.9±0.3 67.0 0.002 174 J Insect Behav (2010) 23:165–179 total of five aphids, the first observation of multiple eggs per aphid in any experiment.

Conspecific Encounters

Females that encountered a conspecific prior to foraging behaved no differently than females that did not (Table 6).

Discussion

In a previous paper (Rasekh et al. 2010), we described in detail how this particular strain of L. fabarum effectively mimics ant solicitation behavior in order to obtain honeydew directly from aphids, thus rendering the aphid colony a source of both hosts and food. We hypothesized that the level of female satiation would be an important state variable affecting the proportion of time spent parasitizing hosts versus soliciting honeydew from them. Since starved females remained 3.6 times longer in patches than did sated females without parasitizing any more aphids, we can infer that their additional investment was directed toward obtaining food rather than hosts and conclude that the former is far more labor-intensive than the latter. The tendency of starved females to rest proportionately more than sated females likely reflects their initial energy deficit, and their greater proportional investment in antennation of aphids, their more intensive solicitation of honeydew. Most aphid antennation by L. fabarum is associated with honeydew solicitation (Rasekh et al. 2010) and the correlation between antennation time and actual honeydew secretion supports this interpretation. The lower rate at which starved females elicited kicking responses from aphids compared to sated females reflects the suppression of aphid defensive behavior during solicitation and is consistent with the inference that A. fabae respond much as they do to solicitation by ants. Starved females expended an average of almost 15 min in solicitation for every honeydew droplet obtained. Although this low rate of return may partially reflect imperfect mimicry, it is also possible that the aphids have been selected to withhold the reward to some extent because their benefits accrue in direct proportion to the

Table 6 Behavioral Data (mean±SE) for 3 Day-Old Lysiphlebus fabarum Females That Either Encountered a Conspecific Female for 1.5–2.0 h (Grouped, n=13), or were Held in Solitude (Solitary, n=13) Prior to Testing in an Open Arena Containing 15 Second Instar Aphis fabae on a Bean Leaf Disk

Variable Solitary Grouped UP

Patch residence time (min) 38.2±6.6 42.0±6.2 79.0 0.801 Search time (min) 10.9±1.6 14.5±2.7 64.0 0.311 Antennation time (min) 4.3±1.8 4.8±1.7 71.0 0.511 Abdominal bending time (min) 10.8±1.8 10.6±1.7 83.0 0.960 Probing time (min) 11.7±2.3 11.4±2.1 82.0 0.920 No. aphids probed 11.8±1.8 11.3±0.3 84.0 0.979 No. aphids parasitized 1.5±0.4 1.2±0.4 76.5 0.687 J Insect Behav (2010) 23:165–179 175 length of time ants remain in the colony. In comparison to sated females, the more intensive solicitation behavior of starved females appeared to diminish aphid defensive reactions such that fewer aphids escaped being probed with the ovipositor when these females reverted to attack behavior (Table 1). Typically, species of Lysiphlebus with cuticular camoflage are able to lay between 20 and 50 eggs per hour in the presence of ants due to suppression of aphid defensive responses by the ants (Völkl 2000). Thus, it is possible that we observed low rates of parasitism because of the absence of ants in our experiments. Furthermore, it is possible that the parasitoid may parasitize A. fabae at higher rates on other host plants, as rates of parasitism on broad bean can be significantly lower than on weedy species of Amaranthaceae and Compositae (Völkl and Stechmann 1998). Parasitism rates have been observed to vary according to host plant in other aphidiines, even among plants in the same genus (Kavallieratos et al. 2002). Nevertheless, the strain of L. fabarum studied by Völkl and Stechmann (1998) parasitized A. fabae at a rate of 0.6 to 0.7 aphids per minute on suitable host plants, more than an order of magnitude higher than the average rates we observed in these experiments. Females of L. fabarum can be described as quite strongly proovigenic; although they are likely able to mature additional eggs should their egg load become depleted, they emerge with more mature eggs than they could seemingly lay in their lifetime (Belshaw and Quicke 2003), suggesting their fitness function is more likely time- limited than egg-limited. Five d-old females remained in patches three times as long as one d-old females, spent more time in all behaviors associated with aphid parasitism, and parasitized three times as many aphids. Studies of other parasitoids consistently demonstrate longer PRT for older females (see references in Wajnberg 2006). Females of iteroparous species always face a trade-off between investing in current versus future reproductive effort (Stearns 1992; Bernstein and Jervis 2008). For parasitoids, this means that foraging time becomes increasingly limiting with advancing age, while eggs decline in value (Rosenheim 1996; van Baalen 2000). Thus, young females reserve more reproductive effort for future bouts than older females and leave patches earlier (Wajnberg et al. 2006). However, the proportional allocation of time to various behaviors did not differ between young and old females in our experiment, suggesting that the overall strategy for patch exploitation does not change with age, only the per-patch investment. A parasitoid’s egg load can influence her foraging decisions (Völkl and Mackauer 1990), even in species that are unlikely to become egg-limited (Michaud and Mackauer 1995). However, it can be difficult to distinguish effects of egg load from effects of prior host experience, given that proovigenic females must be provided large numbers of hosts to reduce their egg load (Rosenheim and Rosen 1991). Pre- trial exposure of females to aphids for a period of 24 h reduced their initial egg load by an average of only 22 eggs, not significantly different from a one h aphid exposure, but consistent with previous observations that L. fabarum females lay only about one egg per hour (Rasekh et al. 2010). Despite this, females with short pre- trial host exposures remained more than twice as long in arenas as females receiving long pre-trial host exposures and parasitized more than three times as many aphids. They also spent a larger proportion of their time examining aphids in the abdominal bending posture, and laid more eggs per unit of patch time, even though they encountered similar numbers of aphids. Thus, the extent of previous foraging 176 J Insect Behav (2010) 23:165–179 experience affected patch time allocation; longer pretrial host exposure may have led to greater female fatigue, or the larger number of aphid encounters obtained in that period may have led to more optimistic assessments of habitat quality. However, effects of egg load likely contributed, since initial female egg loads were positively correlated with their PRT. Foraging prospects for parasitoids can be expected to progressively deteriorate with the onset of autumn and shorter day length (Roitberg et al. 1992). Our results indicate that L. fabarum responds to day length, primarily in immature stages, with an asymmetric interaction between life stage experiences (immature vs imago). The optimism of females exposed to long days prior to emergence was not diminished by a short day experience after emergence, whereas the pessimism of females developing under short days was partially reversed when they experienced long days as adults. This result suggests that exposure to long days during immature stages has a lasting impact on adult foraging behavior, whereas the effect of immature exposure to short days is to some degree reversible, a conceivably adaptive response under spring conditions. Encounters with conspecific females prior to foraging had no effect on female PRT or any component of behavior within the patch, suggesting that females did not respond to conspecifics as potential competitors. Studies on other parasitoids have found either no effect on PRT (Visser et al. 1992), or an increase (Michaud and Mackauer 1995; Ohno 1999). Our result is quite possibly due to the cuticular camoflage; L. fabarum females may be no better than ants at distinguishing conspecifics from aphids, which leads to mutual ovipositional probing in group encounters (Rasekh et al. 2010). However, females did respond to encounters with previously parasitized aphids, remaining in patches longer and scoring higher for most types of foraging behavior. Previously parasitized aphids represent poor quality hosts and it has been previously shown that encounters with previously parasitized hosts (Roitberg et al. 1992; Michaud and Mackauer 1995) or even low quality hosts of a different species (Michaud 1996) cause females to become pessimistic and raise their reproductive investment in a subsequent patch of higher quality hosts. This was also the only treatment to result in superparasitized aphids, an indication that females began laying additional ‘insurance eggs’ to increase their probability of host capture in the face of imminent competition from conspecifics (Visser et al. 1992). Low rates of parasitism within host patches have been explained as a means of spreading risk when progeny survival is highly variable among patches (Ayal and Green 1993; Cronin and Strong 1993; Michaud 1994) or avoiding self- superparasitism (Rosenheim and Mangel 1994). However, neither seem a likely explanation for the low rate of parasitism observed in this strain of L. fabarum because neither are consistent with the large number of aphids attacked, i.e., females making as many as 45 to 50 probes per h and per oviposition. A risk-spreading strategy would dictate earlier oviposition, shorter PRT, and less time spent probing hosts without laying eggs. The extensive probing of aphids appears to serve an ancillary function in honeydew solicitation. Rasekh et al. (2010) showed that females obtained more than twice as many honeydew droplets from A. fabae nymphs that received ovipositor probes prior to solicitation than from those that had not. By probing large numbers of aphids, a female primes them for subsequent honeydew solicitation, yielding a benefit to herself and any attending ants. J Insect Behav (2010) 23:165–179 177

We suspect that the low rate of oviposition by L. fabarum reflects a high level of parasitoid coevolution with both ant-tended aphids and their formicine defenders. Although many parasitoids may need to parasitize large numbers of hosts in order to ensure the survival of only a few progeny, this is not so for L. fabarum. Thelytokous reproduction eliminates the need for males, reducing the host requirement by 50%, and ant attendance greatly improves the survival of daughters (Völkl 1992). Most aphid colonies begin as small clusters of individuals, probably quite analogous to our experimental patches in size, if not in age structure. Few colonies (or the larval parasitoids within them) may survive to reach the exponential growth stage when predator recruitment is timely, but those that do benefit not only the ants that tend the colony, but any parasitoid progeny they contain. Survival of immature parasitoids is likely to be higher within large aphid colonies, as these not only retain ant protection throughout parasitoid development, but also enjoy ‘selfish herd’ benefits in terms of reduced predation risk (sensu Hamilton 1971) and these scale with colony size. If female fitness is contingent on continued ant attendance, and is linked to colony survival for a period equal to, or greater than, the period required for progeny development, then the assumption of rate-maximization within patches may no longer hold and the optimal clutch size may be quite small.

Acknowledgements We thank P. Sloderbeck for reviewing the manuscript and G. Heimpel for useful suggestions. The comments of several anonymous reviewers further improved the manuscript. Voucher specimens no. 209 were deposited at the Kansas State University Museum of Entomological and Prairie Research. This is contribution No. 09-271-J of the Kansas State Agricultural Experiment Station.

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