Differential Host Handling Behavior between Feeding

Journal of Insect Behavior https://doi.org/10.1007/s10905-018-9699-4

Differential Host Handling Behavior between Feeding and Oviposition in the Parasitic Wasp Haplogonatopus hernandezae Floria M.K. Uy 1,2

& Ana

Mercedes Espinoza 3,4

Received: 30 November 2017 / Revised: 2 October 2018 / Accepted: 5 October 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Lifetime fitness is directly influenced by the decision to use resources for either current or future reproduction. Thus, females should weigh the costs and benefits of each reproductive opportunity and allocate resources accordingly. Here, we explore decision-making and the time spent handling hosts of different instars in the parasitoid Haplogonatopus hernandezae, which uses a single planthopper host to either oviposit (i.e., current reproduction) or feed (i.e., future reproduction). Our results indicate that manipulation time in attacks that led to either oviposition and feeding increased with host instar and size. Consequently, attacks were less successful on older host instars than younger ones. Similarly, attack and handling time during oviposition was greater when manipulating fifth instar nymphs compared to younger ones, but oviposition time was similar. Surprisingly, host grasping by the chelate forelegs differed between oviposition and feeding events, and the specific chelate foreleg morphology of H. hernandezae facilitates the specific grasp of the clypeus and gena of the host. We also link this previously undescribed host grasping and differential handling behavior in this species to the final decision to oviposit or feed. Given the differences in handling time and effort among different host instars, we found that older hosts were more often chosen for consumption than younger hosts, and younger hosts were chosen more often for oviposition. Our study suggests that the tradeoff between current and future benefits is influenced by the investment in handling hosts of different instars, and the assessment of host suitability for successful offspring survival. Keywords Decision-making . Dryinidae . Haplogonatopus hernandezae . host-feeding .

host-handling . oviposition . parasitoid . Tagosodes orizicolus

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10905-0189699-4) contains supplementary material, which is available to authorized users.

* Floria M.K. Uy [email protected] Extended author information available on the last page of the article

Journal of Insect Behavior

Introduction Fitness strongly depends on the decision to invest either in current or future reproduction. This tradeoff is known as the cost of reproduction, which assumes that females make decisions to maximize reproductive success over their lifetime (Desouhant et al. 2005; Williams 1966). To maximize their reproductive success, females must weigh the costs and benefits of current reproduction opportunities against finding food to secure future reproduction (Rosenheim 1999; Sæther et al. 1993). Studies in many taxa, including moose, sticklebacks, sea birds, rodents, and burying beetles, suggest that these decisions to invest in current and future reproduction is influenced by female age, environmental constraints and resource availability (Ericsson et al. 2001; Poizat et al. 1999; Sæther et al. 1993; Scott and Traniello 1990; Weil et al. 2006). For example, if current reproduction is costly, females may choose to reproduce at a later time (Creighton et al. 2009). In addition, the decision to invest in current or future reproduction may be influenced not only by quantity and quality of available resources, but also by the energetic investment required to acquire these resources (Creighton et al. 2009; Heimpel and Rosenheim 1995; Yurewicz and Wilbur 2004). Therefore, understanding the tradeoff between current and future reproduction is essential for elucidating the evolution of adaptive decision making, and reproductive investment. Balancing the tradeoffs between current and future reproduction is especially important in organisms where current and future reproduction depend on a single resource (Desouhant et al. 2005; Rivero and West 2005). For example, carnivorous plants face a direct tradeoff between utilizing arthropods as prey or instead for pollination services, known as the pollinator-prey conflict (Jürgens et al. 2012; Youngsteadt et al. 2018; Zamora 1999). Similarly, one-piece nesting termites live inside wood that serves as both their nesting site and nourishment, rarely leaving to exploit new shelter (Korb and Hartfelder 2008). Because the colony dies after exhausting its food source, colony members must allocate enough resources for colony maintenance but leave enough for brood provision (Korb and Hartfelder 2008). Finally, in parasitoids that exhibit hostfeeding, hosts represent the single resource for either feeding or offspring development (Godfray 1994). This decision to either feed or oviposit, in turn, is influenced by intrinsic factors such as egg load and nutritional status of the parasitoid, and extrinsic factors such as the abundance, age, size, quality and the developmental stage of the specific host (Barzman and Daane 2001; Jervis et al. 1996; Jervis and Kidd 1986; King 1998; Kitamura 1988; Nishimura 1997). For example, old parasitoids with access to low quality hosts lay fewer eggs and feed more frequently than well-fed females (Lebreton et al. 2009). Some species of parasitoids may avoid early instars if the host is too small for survival of offspring (Jervis et al. 2008). They may also avoid late host instars as it may be harder to penetrate through their cuticle (Gross 1993), and/or hosts previously parasitized by conspecifics (Yamada and Kitashiro 2002). Previous research also shows that after this initial assessment, hosts of low quality (Lebreton et al. 2009), or those that are difficult to access due to defense mechanisms (Gross 1993), may be less preferred for oviposition and instead are used for feeding (Lebreton et al. 2009). Given the high diversity of host-parasitoid systems, we focus on synovigenic parasitoids because they provide an excellent a model system for exploring decisionmaking. These parasitoids can mature eggs throughout their adult stage (Jervis et al. 2008). Consequently, when encountering a host, the tradeoff between current and future


reproduction may be strongly influenced by various intrinsic and extrinsic factors (Rivero and West 2005). For example, in the synovigenic parasitoid Itoplectis naranyae, females are capable of maturing eggs without feeding on hosts (Ueno and Ueno 2007). However, females that previously fed on hosts had higher fecundity than those who did not feed (Ueno and Ueno 2007). Thus, the condition of the parasitoid should influence their decision to feed or oviposit. In addition, host quality has been shown to effect a parasitoids decision to either invest in immediate reproduction or to choose to survive and increase future reproduction (Rivero and West 2005; Siekmann et al. 2001; Sirot and Bernstein 1996). Finally, hosts are often not passive, and successful oviposition or feeding attempts by parasitoids may be reduced by defensive behaviors such as vigorous squirming and jerking movements, kicking and jumping (Gross 1993; Slansky 1986; Stadler et al. 1994; Villagra et al. 2002). Thus, handling time, which comprises the interval between the first contact with the host and the completion of the oviposition or host feeding event (Gross 1993), may vary depending on the specific chosen host. For example, smaller hosts may require less handling time and represent less injury risk for the parasitoid, because behavioral defenses are frequently more effective in older host developmental stages (Gross 1993). However, a small host may not offer enough nourishment and time for the offspring larvae to develop (Mora-Kepfer and Espinoza 2009). In contrast, a larger host may represent a higher energetic cost in handling, but also have a higher probability of successful development of the offspring. However, in some cases, larger hosts can overcome the attack by subsequently encapsulating the parasitoid eggs or larvae (Blumberg 1997), or may be too advanced in their life cycle to provide enough time for the parasitoid to develop (Vinson and Iwantsch 1980). The parasitoid must therefore balance the tradeoff between investment in host-handling and the appropriate host instar towards current or future fitness. Although the final outcome of either oviposition or host feeding in synovigenic and koinobiont parasitoids, and the defenses of its hosts have been documented (Harvey and Strand 2002; Jervis et al. 2001), little is known about decision-making and host-handling strategies depending specifically on the developmental stage of the host. In this study, we use the parasitoid wasp Haplogonatopus hernandezae (Dryinidae: Hymenoptera) to study host decision for either oviposition or feeding. A unique characteristic in the Dryinidae, and present in this species, is the presence of chelate forelegs (Guglielmino 2002; Olmi 1995; Tribull 2015), which they use to grasp and handle the host. The female wasp uses her chelae and mandibles to seize the host and consequently sting the ventral side of the thorax to paralyze the host (Hernandez and Belloti 1984; Mora-Kepfer and Espinoza 2009; Yamada and Imai 2000). Previous studies within Dryinidae suggest that the relationship between capturing the host and the host’s reaction to the attack may be a driving force in the evolution of this family of parasitoids (Guglielmino 2002; Olmi 1994). The particular morphology of the chelae in each species also serves as a key characteristic for the identification of adult females (Olmi 1994; Tribull 2015). However, the functional significance of chelate foreleg variation found even among closely related species is still poorly understood (Olmi 1984). Of particular interest, the potential relationship between chelae morphology and the specific attacked host has not been studied. Haplogonatopus hernandezae is a solitary and specialist endoparasitoid of the nymphs of the planthopper Tagosodes orizicolus in Colombia and Costa Rica


(Hernandez and Belloti 1984; Mora-Kepfer and Espinoza 2009; Olmi 1984; Olmi and Virla 2014). This species is strongly synovigenic, which is characteristic of apterous Dryinidae, and can therefore mature eggs throughout their reproductive life as an adult (Jervis et al. 2008). Females of H. hernandezae are also koinobiont parasitoids that temporarily paralyze their hosts, and have a high ovigeny index, small egg size, and high fecundity (Jervis et al. 2001, 2008; Jervis and Kidd 1986). Our study population reproduces exclusively by parthenogenesis, avoiding constraints associated with mating (Mora-Kepfer and Espinoza 2009). When finding a potential host, the female may presumably assess host quality (Mora-Kepfer and Espinoza 2009), and consequently choose to either feed or oviposit on this host (Hernandez and Belloti 1984). Females paralyze the host and proceed to probe its abdomen with their ovipositor, similarly to Haplogonatopus atratus, which has been described as an infanticide strategy to detect any eggs previously deposited by another female (Yamada and Kitashiro 2002). Our previous work in H. hernandezae did not find an effect of experience, as the proportion of hosts that were parasitized, predated upon or not attacked was similar in experienced and unexperienced females (Mora-Kepfer and Espinoza 2009). In addition, the proportion of male and female hosts being parasitized remained similar in females of different ages (Mora-Kepfer and Espinoza 2009). However, the effects of host instar in decision-making has not been previously explored in this parasitoid species. We predict that because planthopper nymphs actively try to escape, older nymphs may represent a higher handling investment than earlier instars. We also predict that host-handling will be different for feeding versus oviposition, because manipulation will influence the survival of both the host and parasitoid offspring. Thus, given the knowledge of the reproductive physiology of Dryinidae and our previous results for parasitism and predation in H. hernandezae, the aims of this study were threefold. First, we investigated the effect of inspecting the host to either oviposit or feed, and the decision to invest in feeding or ovipositing depending on host instar as a proxy for quality. Second, we described differential host-handling behavior between these two strategies and predict that the time spent in handling different host instars. Finally, because planthopper nymphs actively try to escape from the attack, we explored how the chelate forelegs are used in the initial grasp and subsequent handling in both feeding and oviposition attacks for different host instars.

Materials and Methods Insect Collection and Rearing We collected individuals of H. hernandezae and its host, the planthopper Tagosodes orizicolus, throughout the province of Guanacaste in the Pacific North of Costa Rica, during 2001 and 2002. All the insects were collected exclusively in inundated rice fields. We isolated nymphs and adults planthoppers that showed visible signs of parasitism, such as a dark cyst on their thorax or abdomen. We reared them individually in small cages designed for Dryinidae (Chandra 1980) to obtain parasitoid larvae that emerged from the host and pupated on the rice leaves. In addition, we established experimental populations of planthoppers that were reared in cages with rice plants placed on a flat tray filled with water to exclude ants, in an insectary under controlled


humidity (70–80%) and temperature (25–30 °C). We selected parasitoid females of two to four days of age, as our previous study in this species found that the proportion of parasitized and predated hosts does not vary during these first days of a parasitoid’s life (Mora-Kepfer and Espinoza 2009). During the first day that a female emerged as an adult, it was kept individually (Chandra 1980), and provided with ten third instar nymphs of T. orizicolus to assure it was not starving before the trial period. We specifically chose to feed wasps on their day of emergence as adults because starved parasitoids face a higher risk of sudden mortality (Harvey et al. 2001). Moreover, starved synovigenic parasitoids may choose not to oviposit and instead exclusively feed on hosts, or have low fecundity (Ueno and Ueno 2007). While establishing our parasitoid population in insectary conditions, we observed higher mortality in females that were not fed the first day compared to the ones that did feed from hosts, which could have compromised our experimental design for H. hernandezae (Uy, FMK unpublished data). Behavioral Assays To observe behavior, we placed each female in an 8.5 cm × 1.8 cm deep clear Petri dish with rice leaves and ten host nymphs of the same instar. We did not use instars 1–2 in the behavioral assays, because previous studies show that this species usually only feeds on nymphs of instars 1 and 2, as they are too small for oviposition and offspring survival (Hernandez and Belloti 1984; Mora-Kepfer and Espinoza 2009). Thus, we specifically selected instars 3 and 4 because they are frequently chosen for either oviposition or predation by Haplogonatopus species (Hernandez and Belloti 1984; Yamada and Kawamura 1999). Instar 5 was used to check whether manipulation became more difficult because of the host size. The nymphal instars were selected by using the morphological criteria described by Mora et al. (2001). We ran the experiment following our previously established protocol for this species (Mora-Kepfer and Espinoza 2009), for a total of seven days from 8 am to 12 pm, following the natural highest availability of hosts in the natural environment of inundated rice fields in Costa Rica. The experimental protocol is as follows: each day, a female parasitoid was introduced in a Petri dish for four hours with ten nymphs of one instar. After this observation period, the female was then housed individually until the next day. On the following day, she was transferred at 8 am to another container with ten new nymphs of a different instars for a new observation period. This procedure was repeated for seven consecutive days (Mora-Kepfer and Espinoza 2009). We rotated the order in which the nymphs of instars 3, 4 and 5 were presented to the parasitoid, to control for any experience bias. For each attack, we recorded handling behavior and the final outcome of either oviposition or predation behavior using a Sanyo Color CCD Camera Model VCC-3912 coupled to the ocular of a dissecting microscope. We recorded each interaction between the female wasp and the nymphs, and then transferred each attacked nymph to an individual container. Therefore, each female parasitoid encountered the opportunity to host-feed or oviposit each individual planthopper only during one observation period. The outcome of each attacked host was classified as successful (the female was able to paralyze the nymph and subsequently fed on it or oviposited) or unsuccessful (the parasitoid grasped but was not able to dominate and paralyze the host). To compare the paralyzation time of nymphs in


different instars and their manipulation in successful oviposition and feeding attacks, we established behavior categories for both the host and the parasitoid. Total and mean durations (seconds ± SE) of the total handling time and each of the behavior categories of the parasitoids were calculated. We recorded the duration and frequency of each of these behavior categories with the program JWatcher (Blumstein et al. 2000). We also determined a size ratio between the host and the parasitoid in each attack by measuring the head width of the dryinid (Yamada and Miyamoto 1998) and the total body length of the host nymph in the video images. We utilized our established method in this species to determine host instar and size as an indication of host quality (Mora-Kepfer and Espinoza 2009). Lastly, we froze several specimens during the specific moment of either oviposition or feeding attacks using liquid nitrogen. These frozen specimens were placed in a − 20 °C freezer, then transferred still frozen to the −20 °C absolute ethanol. After being fixed for more than one week, they were brought to room temperature, and then later dehydrated and sputter coated with gold. Finally, we took photographs of these specimens in attack mode to test for differences in host-handling between oviposition and feeding events with a Scanning Electron Microscope. Parasitoid Handling Behavior We described and divided the handling time of the parasitoid into two steps: 1) paralyzation as the period of seizing the host until stinging the host and paralyzing it, and 2) manipulation as the period of further handling until the achievement of ovipositing or host-feeding. During the second step of manipulation that led to oviposition attacks, we observed the following behaviors: a) probing, as when the parasitoid moved her sting several times along membranes between the abdominal segments of the nymph to inspect if a host was previously parasitized (Yamada and Kitashiro 2002), until she pressed against a point in the intersegmental membranes, b) drilling, as the period from pressing against this specific point to exposing her valvifers, c) oviposition, as the time between exposing the valvifers, retracting her sting into the third valvulae, pushing out her valvifers to the greatest extent possible and inserting the egg (Yamada and Imai 2000; Yamada and Kawamura 1999). In contrast, during the second step of manipulation that led to host feeding, we observed two consecutive behaviors: a) where the female grasped the host and directed her mandibles to press against the host body and b) feeding time, when the wasp fed from the nymph masticating with her mandibles into the thorax or abdomen tissue of the nymph. We divided use of the chelae into two categories. The first category of “set” occurred when a female attacked and then used its chelae synchronously to set the paralyzed host on the rice leaf so the planthopper nymph had its legs on the substrate, and would not fall off the rice leaf before recovering from the attack. After placing the nymph back on the substrate, the wasp released its grasp simultaneously with both chelae, the nymph recovered and walked off. The second category of “dropped” occurred when a female released the host after feeding, in asynchronous motion of its chelae and without placing the nymph’s legs back on the rice leaf. When dropped, the paralyzed host often fell lopsidedly or with its dorsal side towards the substrate, often falling off the rice leaf. We also recorded the host body parts that the wasp grabbed with its chelae.


Host Behavior We divided host behavior into three categories: 1) resistance, as the period in which the nymph offered resistance such as kicking, jumping and jerking after being caught by the parasitoid, 2) paralyzed, as the period in which the nymph showed no movement after being stung, and 3) recovery, as the period from which the female wasp left the nymph after oviposition until the host was able to move again and walk. In contrast to oviposition, feeding events resulted in the host failing to recover and instead dying from the wound inflicted by the parasitoid. Statistical Analyses We performed a Spearman correlation to determine if there was a relation between the host instar and its size. To analyze whether the proportion of oviposition, feeding and unsuccessful attacks varied according to nymph developmental stage, we used a Chisquare test and its standardized residuals. To compare the duration of each behavior category of the nymphs according to their developmental stages and avoid pseudoreplication constraints, we performed Repeated Measures ANOVAS. We used this same test to explore differences in the handling time of the parasitoid and the time of occurrence of each category of oviposition and host feeding behavior among the different nymphal stages of the host. Differences between the final manipulation strategies of the hosts after the predation and oviposition events were analyzed by using a Chi-square test. To determine if the number stinging events varied according to the nymphal instars, we employed a Repeated Measures ANOVA. We analyzed the use of the chelae to grasp the host in oviposition and predating events by using a Chi-square test. Finally, we also explored differences in the grasping with the chelae among nymphal instars using a Chi-square test.

Results Host and Parasitoid Behavior during Attacks Out of a total of 199 attacks, 58.3% were successful oviposition events, 21.6% were successful predation events, and 20.1% were unsuccessful, either because the planthoppers escaped from the grasp of the wasp (14%) or the planthopper was seized but then rejected (6.1%). We found a positive relation between the host age and size (R = 0.06, T (n-2) = 10.28, N = 165, P = 0.001). The percentage of unsuccessful attacks due to the nymphs escaping was higher for fifth instar nymphs compared to fourth instars and third instars (χ2 = 13.31, df = 4, P = 0.010, N = 161, Fig. 1). The handling time prior to oviposition was also higher as the nymphal instar increased (H = 20.60, P = 0.00, Fig. 2). In contrast, the handling time prior to feeding did not change in relation to the host stage (H = 0.94, P = 0.62, Fig. 2). The time that nymphs resisted an attack varied depending on whether the parasitoid fed or instead oviposited on a host. Resistance time was longer in older nymphs for attacks that led to oviposition (H = 23.44, P < 0.001, Fig. S1A). However, the resistance


Fig. 1 Effect of host instar on success in oviposition and feeding attacks by females of Haplogonatopus hernandezae. Events in which the hosts escaped the grasp of the parasitoid were categorized as unsuccessful attacks

time did not differ for different host instars for attacks that ended in feeding (H = 0.32, P = 0.85, Fig. S1A). The same tendency was found for the paralyzation time: older hosts took longer to be paralyzed in attacks that led to oviposition (H = 20.40, P < 0.001, Fig. S2B). The time to paralyze the hosts did not differ among the nymphal instars in attacks that led to feeding (H = 0.24, P = 0.88, Fig. S2B). Finally, the recovery time was shorter for 5th instar nymphs following oviposition (H = 12.06, P = 0.02, Fig. S2C). The time to catch and paralyze a host was influenced by host age and the decision to either feed or oviposit. The time to paralyze a host was greater with older host instars in attacks that led to oviposition (H = 8.95, P = 0.01), but this trend was absent in attacks that led to feeding (H = 0.14, P = 0.93). Handling time was also higher for 5th instar hosts in those attacks that ended in oviposition (H = 20.97, P < 0.001), but not in those that led to feeding (H = 20.98, P = 0.421).

Fig. 2 Time of occurrence (seconds) of each behavior category of the parasitoid to either feed or oviposit in hosts of different instars. LSD Posthoct Test. Significant differences among attacks towards host instars are indicated with different letters (P < 0.05)


For trials that ended in oviposition, the handling time of hosts after paralysis was also longer in 5th instar hosts compared to 3rd and 4th instar hosts, both for the probing period and anchorage time to lay an egg (Table S1). However, once anchoring had occurred, the time required for oviposition did not vary among host instars (Table S1). Handling time prior to feeding was not different among the host stages, nor did the duration of feeding itself (Table S2). For trials where the planthopper hosts were later parasitized, the number of 4th and 5th instar nymphs that were stung twice by the parasitoid was significantly greater than for younger hosts (G = 30.47, df = 2, P = 0.00, N = 101). In feeding attacks, the most frequent behavior in all host stages was stinging continuously while the dryinid fed on the nymphs (G = 7.17, df = 2, P = 0.127, N = 41). Host Manipulation after Feeding and Oviposition Events Close-up video images revealed that the final manipulation of each host after being attacked differed between trials that let to oviposition or feeding. If the female decided to feed on a nymph, she paralyzed the host and fed on the dorsal side of the thorax or abdomen by masticating with her mandibles. With a few exceptions in fifth instar nymphs, we observed that the wound caused the death of the host, confirming the observations of our previous study (Mora-Kepfer and Espinoza 2009). The most frequent behavior of the parasitoid after feeding was to quickly drop the paralyzed nymph (Fig. 3a.), which fell with its lateral or dorsal side on the substrate or rice leaf. In some cases, the parasitoid stepped on the nymph it had just fed on as it started to forage after the attack (N = 10), or released the grip on the nymph and started foraging while still dragging the nymph with the other chela for a few seconds (N = 8). In contrast, after the female finished ovipositing on a host, she slowly set the paralyzed host with its ventral side oriented towards the substrate (χ2 = 69.13, df = 2, P = 0.00, Fig. 3a). The wasp released the nymph simultaneously with both chelae, and after the nymph recovered from the attack, it walked or jumped when it became active again. The video images also indicated that the wasp’s chelate forelegs are used for grasping specific body parts of the host and that this gripping differs in oviposition and feeding attacks. The female grasped the nymph either by its clypeus (holding the clypeus of the nymph with one chela and legs or abdomen with the other chela) or other body structures (holding the host with both chelae the side of the thorax or abdomen or legs, rather than the clypeus). The clypeus was grasped most frequently in all nymphal stages after paralysis and until the dryinid either dropped or gently set it on the substrate, (χ2 = 28.97, df = 1, P < 0.001, Fig. 3a). We also found that the clypeus of the host is used for grasping with the chelae in attacks that led to oviposition. The clypeus was gripped significantly more frequently than other host body structures after the host was paralyzed (χ2 = 49.58, df = 1, P < 0.001, Figs. 3b and 4). However, in attacks that led to feeding, the wasp did not grasp specific body structures of the host.

Discussion Our findings provide novel evidence for differences in host-handling behavior between oviposition and feeding attacks, which provide evidence for the role of host instar in the


Fig. 3 a Final manipulation of each host after either an oviposition or a predation event. b Differential chela grasp of host body structures by the female parasitoid during feeding and oviposition attacks

choice of investing in current reproduction, or instead feed from a host presumably to secure resources for future reproduction. In H. hernandezae, females chose hosts for either oviposition or feeding based on host size, which is similar to other parasitoids (Gross 1993; Wang and Messing 2004). Although we did not measure host quality directly, our results are consistent with the hypothesis that size may be correlated to the nutrient content of the host (Vinson and Iwantsch 1980) and/or the ability of hosts to evade parasitism and predation (Gerling et al. 1990). That is, our data indicate that even though a dryinid is capable of handling and attacking nymphs of all instars, manipulation time increased with host age and size. Consequently, the number of successful attacks that ended in oviposition or feeding were strongly influenced by the nymphal instar. In addition, older hosts were chosen for consumption more frequently than younger hosts, while younger hosts were parasitized more often than older hosts. This result is consistent with other studies suggesting that hosts that are of lower quality and


Fig. 4 Open chela (ch) of the parasitoid grasping the host face: clypeus (cp) and gena (g). The parasitoid grasped the host face and anchored its chelated forelegs, and consequently begun to paralyze the host to initiate oviposition. Scale = 400 μm

less suitable for oviposition and/or are more difficult to handle are used for feeding, which ultimately provides the necessary energy for future reproductive efforts when finding more suitable hosts (Lebreton et al. 2009). Host Defense and Handling Time As in studies of other parasitoids, we observed that older host nymphs defended themselves more vigorously by shaking and kicking when held by a wasp causing the resistance time to increase (Bokononganta et al. 1995; Rivero 2000). Hence, the time invested in handling an older, larger host increases in oviposition attacks. Previous work suggests that oviposition cost (including searching and handling the host) is related to the time and/or energy expenditure to oviposit an egg (Stephens and Krebs 1986). If laying eggs in different host stages results in different oviposition costs, a tradeoff exists between offspring rearing conditions and difficulty of oviposition and host suitability for the offspring (Nishimura 1997). Handling time prior to oviposition increased with host instar for several reasons. First, resistance time is significantly greater in older nymphs. That is, a parasitoid may be able to successfully approach a small host to oviposit, while being less exposed to the range of the defensive leg kicking, but, in larger hosts, a closer approach may be needed. This forces the parasitoid to move within the “leg distance” of the host (Gerling et al. 1990), making it more difficult to sting the host to paralyze it. Our observations show that older nymphs actively kick the ventral side of the wasp body as it stings the thorax to paralyze the nymph. Further, body shaking and jumping were more efficient in older hosts as a strategy to escape than in younger hosts. Second, after capture, fifth instar nymphs required more investment to be paralyzed for longer periods compared to younger instars. Searching for a site on the host body to parasitize also took longer in this instar. Previous studies show that a female of Haplogonatopus atratus examines the membranes between the host abdominal terga with her ovipositor before ovipositing to detect if the host has been previously parasitized (Yamada and Kitashiro 2002; Yamada and Ikawa 2005; Yamada and


Kawamura 1999; Yamada and Sugaura 2003). If a female dryinid located an egg from a previous oviposition event of another female, the egg was stung to kill this offspring. This pattern suggests that longer handling and inspecting time with a larger and older host serves to make sure it has not been previously parasitized (Ito and Yamada 2007; Ito and Yamada 2006). Thus, older hosts were frequently stung twice, prior to finding a spot to oviposit. The resistance time for these hosts was longer and more than one period of paralyzation was necessary to perform a successful oviposition in them majority of the 5th instar planthopper hosts. Finally, after paralyzation, anchoring time (during which drilling took place) was also longer for older nymphs, suggesting a greater difficulty in penetrating the host cuticle. This pattern suggests that the ovipositor sheaths possibly serve as an anchor during the initial phases of the process (Vilhelmsen 2003). This results coincides with observations for other parasitoids, in which a strong correlation was found between the cuticle thickness of the host and oviposition success (van Lenteren et al. 1998). As such, last instar nymphs become more costly in energy and timeconsuming to handle. Overall, our results suggest that increased handling time for older hosts may favor parasitoids in using them for feeding. Alternatively, because the hosts may develop into adults soon, parasitoids may favor younger hosts to assure that the parasitoid offspring will have enough time to develop and leave the host to pupate. That is, choosing the appropriate host instar is essential for the survival of the developing parasitoid larva due to differences in growth rates and time limitations (Roitberg and Bernhard 2008). A female adult of T. orizicolus lives on average 31 days and males live less at an average of 14.6 days (Gomez Sousa and Kamara 1980), and H. hernandezae larva can take up to 27 days to develop inside the host (Mora-Kepfer and Espinoza 2009). In a previous study, we found that H. hernandezae chooses female nymph hosts for oviposition and males hosts are fed upon, suggesting ovipositing in female nymphs will allow enough time for the parasitoid larva to develop and result in successful current fitness (MoraKepfer and Espinoza 2009). Host-Handling Behavior Host-handling behavior likewise varies between oviposition and feeding attacks, specifically in the grasping of the host by the chelae and the final manipulation after the attack. The clypeus was the body structure of the host most frequently used for grasping in oviposition attacks. The particular chela morphology, having an enlarged claw with subapical tooth and six lamellae, and segment 5 of the front tarsus having two rows of nearly eight lamellae (Olmi 1984), facilitates efficient grasping of the host. This suggests the unexpected observation that the function of the particular chela morphology is to specifically grasp the clypeus and gena of T. orizicolus. When a wasp decided to feed on a nymph, stinging, which was used both to grasp and paralyze was continuous, caused damage to the host body. The clypeus and other body parts were used frequently for grasping since the feeding did not need to be in a specific site. Instead, the parasitoid fed from anywhere on the thorax or the abdomen. The host was subsequently killed by destructive feeding and dropped. As predation events are single encounters, the host age or the manipulation that a nymph receives has no further consequences on the future reproduction of the parasitoid.


In contrast, when a female decides to lay an egg, she must manipulate the host carefully to assure that her progeny will be able to survive and develop successfully. Our observations showed that in oviposition attacks the wasp grasped the host precisely by the clypeus and either the tip of the abdomen in young nymphs or a leg in older instars. After the dryinid oviposited, she set the nymph carefully with its ventral side towards the rice leaf by releasing the grip of the chelate forelegs simultaneously, which allowed the host to recover from being paralyzed and hold on to the rice leaf. Rice fields in Costa Rica are frequently flooded; therefore, this behavior might save the parasitized host from falling into the water while still paralyzed to assure the successful recovery of this nymph, and the survival of the parasitoid egg that will develop inside the host. Conclusions Our study shows an effect of host instar on the decision to either oviposit or feed. The subsequent handling behavior also differed between these two choices as hosts used for oviposition were handled more carefully after oviposition, suggesting that the host carrying the parasitoid’s egg will have a higher probability of recovering and surviving after being paralyzed in a flooded rice field. In contrast, hosts that were used for feeding were handled carelessly and did not recover from the attack. Although our results do not directly test future fitness, they provide novel evidence for costs and benefits of manipulation of hosts of different instars, and choice of current fitness versus feeding according to nymphal instar, as a proxy of host quality. Thus, the decision to feed or oviposit may be influenced by the assessment of available host instars, the investment in handling a host of a specific developmental instar, and the tradeoff between current and future reproductive fitness by the parasitoid. Our findings raise new questions about the mechanisms that influence decision-making and differential behavior in parasitoids. Future studies that include manipulations of the physiological state of the parasitoid and host density to explore its effect on offspring survival, would provide key insights into the mechanisms underlying the tradeoff between current and future reproduction. Acknowledgements We thank C. Barboza and R. Mora-Castro for their assistance in maintaining the planthopper and parasitoid colonies in the insectary of the Centro de Biología Celular y Molecular (CIBCM) of the University of Costa Rica. P. Hanson, W. Eberhard, A. Uy and W. Searcy, and members of the Uy and Searcy labs provided insightful comments on this manuscript. We are grateful to M. Vargas for Scanning Electron Microscope images. The filming equipment was provided by the Animal Behavior Laboratory at the Department of Biology, University of Costa Rica.

References Barzman MS, Daane KM (2001) Host-handling behaviours in parasitoids of the black scale: a case for antmediated evolution. J Anim Ecol 70:237–247 Blumberg D (1997) World crop pests. Elsevier, Amsterdam Blumstein D, Evans C, Daniel J (2000) JWatcher animal behaviour laboratory. Macqueri University, Sydney Bokononganta A, Neuenschwander P, Vanalphen J, Vos M (1995) Host stage selection and sex allocation by Anagyrus mangicola (Hymenoptera: Encyrtidae), a parasitoid of the mango mealybug, Rastrococcus invadens (Homoptera: Pseudococcidae). Biol Control 5:479–486

Journal of Insect Behavior Chandra G (1980) Dryinid parasitoids of rice leafhoppers and planthoppers in the Philippines II. Rearing techniques. Entomophaga 25:187–192 Creighton JC, Heflin ND, Belk MC (2009) Cost of reproduction, resource quality, and terminal investment in a burying beetle. Am Nat 174:673–684 Desouhant E, Driessen G, Amat I, Bernstein C (2005) Host and food searching in a parasitic wasp Venturia canescens: a trade-off between current and future reproduction? Anim Behav 70:145–152 Ericsson G, Wallin K, Ball JP, Broberg M (2001) Age-related reproductive effort and senescence in freeranging moose, Alces alces. Ecology 82:1613–1620 Gerling D, Roitberg BD, Mackauer M (1990) Instar-specific defense of the pea aphid, Acyrthosiphon pisum: influence on oviposition success of the parasite Aphelinus asychis (Hymenoptera: Aphelmidae). J Insect Behav 3:501–514 Godfray HCJ (1994) Parasitoids: behavioral and evolutionary ecology. Princeton University Press Gomez Sousa J, Kamara F (1980) Determination of some biological parameters of Sogatodes orizicola (Muir). Centro Agric 7:13–21 Gross P (1993) Insect behavioral and morphological defenses against parasitoids. Annu Rev Entomol 38:251– 273 Guglielmino A (2002) Dryinidae (Hymenoptera Chrysidoidea): an interesting group among the natural enemies of the Auchenorrhyncha (Hemiptera). Denisia 4:549–556 Harvey JA, Strand MR (2002) The developmental strategies of endoparasitoid wasps vary with host feeding ecology. Ecology 83:2439–2451 Harvey JA, Harvey IF, Thompson DJ (2001) Lifetime reproductive success in the solitary endoparasitoid, Venturia canescens. J Insect Behav 14:573–593 Heimpel GE, Rosenheim JA (1995) Dynamic host feeding by the parasitoid Aphytis melinus: the balance between current and future reproduction. J Anim Ecol 64:153–167 Hernandez MP, Belloti A (1984) Ciclos de vida y habitos de Haplogonatopus hernandezae Olmi (Hymenoptera: Dryinidae) controlador natural del saltahojas del arroz Sogatodes orizicola (Muir). Rev Colomb Entomol 10:3–8 Ito EMI, Yamada YY (2006) Seemingly maladaptive refraining from infanticidal probing at the third parasitism attack by the semi-solitary parasitoid Echthrodelphax fairchildii (Hymenoptera: Dryinidae). Insect Science 13:229–233 Ito E, Yamada YY (2007) Imperfect preference for singly parasitized hosts over doubly parasitized hosts in the semisolitary parasitoid Echthrodelphax fairchildii: implications for profitable self-superparasitism. Entomol Exp Appl 123:207–215 Jervis MA, Kidd NAC (1986) Host-feeding strategies in Hymenopteran parasitoids. B Biol Rev Camb Philos Soc 61:395–434 Jervis MA, Hawkins BA, Kidd NAC (1996) The usefulness of destructive host feeding parasitoids in classical biological control: theory and observation conflict. Ecol Entomol 21:41–46 Jervis MA, Heimpel GE, Ferns PN, Harvey JA, Kidd NA (2001) Life-history strategies in parasitoid wasps: a comparative analysis of ‘ovigeny. J Anim Ecol 70:442–458 Jervis MA, Ellers J, Harvey JA (2008) Resource acquisition, allocation, and utilization in parasitoid reproductive strategies. Annu Rev Entomol 53:361–385 Jürgens A, Sciligo A, Witt T, El-Sayed AM, Suckling DM (2012) Pollinator-prey conflict in carnivorous plants. Biol Rev Camb Philos Soc 87:602–615 King BH (1998) Host age response in the parasitoid wasp Spalangia cameroni (Hymenoptera: Pteromalidae). J Insect Behav 11:103–117 Kitamura K (1988) Comparative studies on the biology of Dryinid wasps in Japan : 5) development and reproductive capacity of hosts attacked by Haplogonatopus apicalis (Hymenoptera, Dryinidae) and the development of progenies of the parasites in their hosts. Japanese J Entomol 56:659–666 Korb J, Hartfelder K (2008) Life history and development - a framework for understanding developmental plasticity in lower termites. Biol Rev Camb Philos Soc 83:295–313 Lebreton S, Darrouzet E, Chevrier C (2009) Could hosts considered as low quality for egg-laying be considered as high quality for host-feeding? J Insect Physiol 55:694–699 Mora R, Retana A, Espinoza AM (2001) External morphology of Tagosodes orizicolus (Homoptera: Delphacidae) revealed by scanning Electron microscopy. Ann Entomol Soc Am 94:438–448 Mora-Kepfer F, Espinoza AM (2009) Parasitism and predation of the planthopper Tagosodes orizicolus (Homoptera: Delphacidae) by a dryinid parasitoid in Costa Rica. Rev Biol Trop 57:203–211 Nishimura K (1997) Host selection by virgin and inseminated females of the parasitic wasp, Dinarmus basalis (Pteromalidae, Hymenoptera). Funct Ecol 11:336–341 Olmi M (1984) A revision of the Dryinidae (Hymenoptera). Mem Am Entomol Inst 37:947–1913

Journal of Insect Behavior Olmi M (1994) The Dryinidae and Embolemidae (Hymenoptera: Chrysidoidea) of Fennoscandia and Dennmark (vol. 30). Brill Academic Publishing, Netherlands Olmi M (1995) Dryinidae. The Hymenoptera of Costa Rica. Oxford University Press, Oxford Olmi M, Virla EG (2014) Dryinidae of the Neotropical region (Hymenoptera: Chrysidoidea). Zootaxa 3792:1– 534 Poizat G, Rosecchi E, Crivelli AJ (1999) Empirical evidence of a trade–off between reproductive effort and expectation of future reproduction in female three-spined sticklebacks. Proc R Soc B 266:1543–1548 Rivero A (2000) The relationship between host selection behaviour and offspring fitness in a koinobiont parasitoid. Ecol Entomol 25:467–472 Rivero A, West SA (2005) The costs and benefits of host feeding in parasitoids. Anim Behav 69:1293–1301 Roitberg B, Bernhard P (2008) Behavioral ecology of insect parasitoids: from theoretical approaches to field applications. John Wiley & Sons, Hoboken Rosenheim J (1999) Characterizing the cost of oviposition in insects: a dynamic model. Evol Ecol 13:141–165 Sæther B-E, Andersen R, Pedersen H (1993) Regulation of parental effort in a long-lived seabird an experimental manipulation of the cost of reproduction in the antarctic petrel, Thalassoica antarctica. Behav Ecol Sociobiol 33:147–150 Scott MP, Traniello JFA (1990) Behavioural and ecological correlates of male and female parental care and reproductive success in burying beetles (Nicrophorus spp.). Anim Behav 39:274–283 Siekmann G, Tenhumberg B, Keller MA (2001) Feeding and survival in parasitic wasps: sugar concentration and timing matter. Oikos 95:425–430 Sirot E, Bernstein C (1996) Time sharing between host searching and food searching in parasitoids: statedependent optimal strategies. Behav Ecol 7:189–194 Slansky F (1986) Nutritional ecology of endoparasitic insects and their hosts: an overview. J Insect Physiol 32: 255–261 Stadler B, Weisser WW, Houston AI (1994) Defence reactions in aphids: the influence of state and future reproductive success. J Anim Ecol 63:419–430 Stephens DW, Krebs JR (1986) Foraging theory. Princeton University Press, Princeton Tribull C (2015) Phylogenetic relationships among the subfamilies of Dryinidae (Hymenoptera, Chrysidoidea) as reconstructed by molecular sequencing. J Hymenopt Res 45:15–29 Ueno T, Ueno K (2007) The effects of host-feeding on synovigenic egg development in an endoparasitic wasp, Itoplectis naranyae. J Insect Sci 7:1–13 van Lenteren JC, Isidoro N, Bin F (1998) Functional anatomy of the ovipositor clip in the parasitoid Leptopilina heterotoma (Thompson) (Hymenoptera: Eucoilidae), a structure to grip escaping host larvae. Int J Insect Morphol Embryol 27:263–268 Vilhelmsen L (2003) Flexible ovipositor sheaths in parasitoid Hymenoptera (Insecta). Arthropod Struct Dev 32:277–287 Villagra CA, Ramı́rez CC, Niemeyer HM (2002) Antipredator responses of aphids to parasitoids change as a function of aphid physiological state. Anim Behav 64:677–683 Vinson SB, Iwantsch GF (1980) Host suitability for insect parasitoids. Annu Rev Entomol 25:397–419 Wang X-g, Messing RH (2004) The ectoparasitic pupal parasitoid, Pachycrepoideus vindemmiae (Hymenoptera: Pteromalidae), attacks other primary tephritid fruit fly parasitoids: host expansion and potential non-target impact. Biol Control 31:227–236 Weil ZM, Martin LB, Workman JL, Nelson RJ (2006) Immune challenge retards seasonal reproductive regression in rodents: evidence for terminal investment. Biol Lett 2:393–396 Williams GC (1966) Natural selection, the costs of reproduction, and a refinement of Lack's principle. Am Nat 100:687–690 Yamada YY, Ikawa K (2005) Superparasitism strategy in a semisolitary parasitoid with imperfect self/non-self recognition, Echthrodelphax fairchildii. Entomol Exp Appl 114:143–152 Yamada YY, Imai N (2000) Identification of the sex of eggs and the mating status of female adults in Echthrodelphax fairchildii (Hymenoptera: Dryinidae) based on oviposition behavior. Entomol Sci 3:579– 583 Yamada YY, Kawamura M (1999) Sex identification of eggs of a dryinid parasitoid, Haplogonatopus atratus, based on oviposition behaviour. Entomol Exp App 93:319–322 Yamada Y, Kitashiro S (2002) Infanticide in a Dryinid parasitoid, Haplogonatopus atratus J. Insect Behav 15: 415–427 Yamada YY, Miyamoto K (1998) Payoff from self and conspecific Superparasitism in a Dryinid parasitoid, Haplogonatopus atratus. Oikos 81:209–216 Yamada YY, Sugaura K (2003) Evidence for adaptive self-superparasitism in the dryinid parasitoid Haplogonatopus atratus when conspecifics are present. Oikos 103:175–181

Journal of Insect Behavior Youngsteadt E, Irwin RE, Fowler A, Bertone MA, Giacomini SJ, Kunz M, Suiter D, Sorenson CE (2018) Venus flytrap rarely traps its pollinators. Am Nat 191:539–546 Yurewicz KL, Wilbur HM (2004) Resource availability and costs of reproduction in the salamander Plethodon cinereus. Copeia 2004:28–36 Zamora R (1999) Conditional outcomes of interactions: the pollinator–prey conflict of an insectivorous plant. Ecology 80:786–795

Affiliations Floria M.K. Uy 1,2 & Ana Mercedes Espinoza 3,4

1

Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica

2

Department of Biology, University of Miami, Coral Gables, FL 33124, USA

3

Centro de Investigaciones en Biología Celular y Molecular, Universidad de Costa Rica, San Pedro, Costa Rica

4

Escuela de Agronomía, Facultad de Ciencias Agroalimentarias, Universidad de Costa Rica, San Pedro, Costa Rica