Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions (‘tree-top’ disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.
Animals experience continuous invasions by parasites and are abused for replication and spread of the invaders. To enhance transmission, many parasites alter host behaviour. Notable examples include altered mosquito-feeding behaviour by malaria parasites  and suicidal behaviour of crickets infected with hairworms . These responses often entail a differential response to environmental cues (see [3,4] for reviews).
Insect pathogenic baculoviruses are natural disease agents that are adapted to both their hosts and the pertinent ecosystem. The ecology of baculoviruses is therefore complex and needs to be better understood in order to gain the maximum benefit of the use of these viruses as biocontrol agents . Baculoviruses alter the behaviour of their caterpillar hosts by inducing hyperactivity [6,7] and by causing infected caterpillars to migrate to the top of the plant prior to death [8–10]. This behaviour is thought to be adaptive for the virus, as it ensures optimal dissemination of progeny virus onto lower foliage  and enhances visibility for birds, spreading the virus over longer distance . However, the mechanisms underlying this adaptive behavioural manipulation have remained largely obscure and the host response triggered by the virus to cause this behaviour is unknown.
Here, we tested the hypothesis that ‘tree-top’ disease results from a positive phototactic response, i.e. attraction to light. This hypothesis was experimentally tested by investigating the effect of light on the movement induced by the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in its host S. exigua. We conclude that ‘tree-top’ disease of the infected caterpillars is caused by a strong attraction to light prior to death.
2. Material and methods
(a) Insect larvae and virus
Spodoptera exigua larvae were reared on artificial diet at 27°C with 50% relative humidity as described before  and a 14 L : 10 D photoperiod (7.00 lights on, 21.00 lights off). Wild-type (WT) SeMNPV viral occlusion bodies (OBs)  were amplified once in S. exigua third instars to obtain more virus. OBs were purified from infected larvae as described earlier .
(b) Behavioural assays under normal light/dark conditions (14 L : 10 D)
Newly moulted third instar S. exigua larvae (starved overnight for 16 h) were infected with a 90% lethal virus concentration (LC90) dose (105 OBs ml−1) of virus using droplet feeding as described earlier . Subsequently, virus-infected larvae were placed individually in glass jars (120 mm tall × 71 mm wide), which were closed with a metal lid containing small holes to allow ventilation. Jars were lined with mesh wire to facilitate climbing and contained a piece of artificial diet (approx. 3.5 cm3) at the bottom. Jars were incubated at 27°C with 50% relative humidity. A 14 L : 10 D (7.00 lights on, 21.00 lights off, as during rearing) regime was applied using three luminescent tubes of 18 W each, with a 30 cm distance between the luminescent tubes and jar lids. The vertical position of the larvae was monitored twice per day, starting from one day post-infection (dpi) until all larvae were either dead or had pupated. Larvae that did not die owing to virus infection (died of other causes or survived despite being droplet fed with virus) were excluded from analyses. Death owing to baculovirus infection was assayed by screening for liquefaction of the host larva after death, which is a clear indication of baculovirus infection. The assay was performed as two independent replicates.
(c) Behavioural assays under dark conditions or using light from a single direction
For behavioural assays under dark conditions, a 0 L : 24 D photoperiod was applied. In assays in which light was applied only from above, a 14 L : 10 D photoperiod was applied as described above, but jar walls were protected from light using aluminium foil. Jars were covered with a piece of transparent plastic Saran wrap containing three holes for ventilation. The jars were then placed in a black box to block any light from other directions than above. In assays in which light was applied only from below, a 14 L : 10 D photoperiod was applied as described above, with 30 cm distance between the luminescent tubes and the jar bottoms. Jar walls were protected from light using aluminium foil, and jars were closed with metal lids containing small holes for ventilation. A black box was placed over the jars to block any light from other directions than below.
For those three assays (dark, light from above and light from below), the height at death was recorded at 4 dpi. Larvae that did not die owing to virus infection (died of other causes or survived despite being droplet fed with virus) were excluded from the analyses. Death owing to baculovirus infection was assayed by screening for liquefaction of the host larva after death, which is a clear indication of baculovirus infection. Each of these three behavioural assays was performed as two independent replicate experiments.
(d) Behavioural assays using uninfected larvae
Newly moulted third instar S. exigua larvae (starved overnight for 16 h) were droplet-fed a virus-free solution as previously described . Larvae were placed in glass jars as described above and incubated under either normal light/dark conditions (14 L : 10 D) or completely dark conditions (0 L : 24 D). The vertical position of the larvae was monitored twice per day until all larvae had pupated. To avoid light exposure to larvae kept under dark conditions, a 20 W red light bulb was used during the measurements of the position of the larvae in the jar.
(e) Statistical analysis
Significant differences in the height at death of virus-infected larvae between treatments were analysed using a Kruskal–Wallis non-parametric tests (SPSS). Significant differences between the height of larvae at 76 and 95 h post-infection were analysed using a Wilcoxon signed-rank test. For significant correlations between % surviving larvae and the average height of the larvae, a non-parametric Kendall's τ correlational analysis was performed.
Behavioural assays were performed to investigate the effect of light on tree-top disease induced by SeMNPV infection in S. exigua larvae. We first performed a behavioural assay to study the timing of SeMNPV-induced tree-top disease. To this end, third instar S. exigua larvae were infected with an LC90 dose of SeMNPV WT baculovirus and behavioural assays were performed under normal light/dark conditions (14 L : 10 D). Virus-infected larvae started to rapidly ascend from 76 hours post-infection (hpi) (35 mm at 76 hpi; 69 mm at 90 hpi; figure 1a) (Wilcoxon signed-rank test, Z =−3.392, p = 0.001). Shortly after these larvae ascended, the majority succumbed to virus infection (100% survival at 76 hpi; 20% survival at 90 hpi, figure 1a), and a negative significant correlation was found between the average height of the larvae and the % of surviving larvae (Kendall's τ coefficient = −0.757, p = 0.001). Thus, these larvae showed a strong climbing response in the last hours prior to death, resulting in death at elevated positions.
We hypothesized that light could be a critical parameter in inducing this climbing prior to death. To this end, the height at death of infected larvae exposed to different light conditions was measured. This showed that infected larvae exposed to complete darkness (0 L : 24 D) died at low positions (23 ± 3.6 mm) (figure 1b), indicating that light was required for death at elevated positions. We then investigated whether the presence of light sensu strictu was sufficient to evoke climbing behaviour or whether the infected larvae had become positive phototactic. To this end, infected larvae were exposed to light from a single direction. Larvae exposed to light only from above (14 L : 10 D) died at significantly higher positions (82 ± 6.5 mm) (figure 1b) than larvae kept under dark conditions (23 ± 3.6 mm) and larvae exposed to light only from below (14 L : 10 D) (2.3 ± 0.9 mm) (figure 1b) (Kruskall–Wallis test across all three groups K = 79.877; d.f. = 2; p < 0.0001). These data show that SeMNPV induces strong positive phototaxis in infected caterpillars.
To understand whether this response was specifically evoked by baculovirus infection, or whether larvae were also positive phototactic in the absence of virus infection, behavioural assays were performed using uninfected third instar larvae. These larvae showed several climbing peaks coinciding with larval moults during development from third instar to pupae (moult to fourth instar at 28 h; moult to fifth instar at 99 h; figure 2a), which is a common phenomenon in uninfected S. exigua larvae . Prior to pupation, larvae descended to the bottom to pupate in the diet plug (figure 2a).
In contrast to the climbing prior to death observed in baculovirus-infected caterpillars, the climbing peaks related to moulting were not light-dependent. Under completely dark conditions (0 L : 24 D), larvae also showed climbing peaks that coincide with moulting (moult to fourth instar at 24 h; moult to fifth instar at 73 h; figure 2b). This indicates that climbing related to moulting observed in uninfected caterpillars and the positive phototaxis prior to death in infected caterpillars are unrelated processes.
Although several studies have investigated baculovirus-induced hyperactivity and tree-top disease of infected caterpillars [6,7,10,16], the proximate mechanisms have remained largely elusive. Here, we demonstrate that SeMNPV-induced tree-top disease in infected caterpillars is the result of an altered behavioural response to light. Because the climbing behaviour of uninfected (third and fourth instar) larvae is light-independent, we conclude that this positive phototactic response is specifically triggered during virus infection. The observation that uninfected third and fourth instar larvae climb even in the absence of light may suggest that this moulting-related climbing behaviour is the result of a response related to geotaxis (movement in response to gravity).
In our study, experiments were carried out under a relatively extreme light regime (high-intensity light versus complete darkness). To which extent these conditions reflect the situation in nature is hard to predict, as this will depend on geographical position, time of year/day and the density of the plant foliage on which the larva resides. Future studies should be able to tell us the importance of light in caterpillar tree-top disease in a more ecologically relevant setting.
A recent study by Hoover et al.  identified a gene from the baculovirus Lymantria dispar MNPV that induces tree-top disease in L. dispar larvae. This gene encodes ecdysteroid uridine diphosphate-glucosyl transferase (EGT), which inactivates host moulting hormones and extends the larval lifespan . Whether EGT plays a similar role in tree-top disease in SeMNPV-infected larvae (e.g. by targeting a host pathway related to light perception or phototaxis) remains to be determined.
Manipulation of host climbing behaviour occurs in numerous parasite–host systems and several parasites are known to induce host phototactic responses (reviewed in [3,4]). For example, positive phototaxis is seen in gammarids infected with parasitic worms  and in crickets infected with Gordian worms . Future research is needed to understand whether mechanistic parallels underlying phototaxis exist between these systems and baculovirus-infected caterpillars.
Overall, we conclude that tree-top disease in SeMNPV-infected caterpillars is the result of positive phototaxis prior to death and that this response is specifically triggered during virus infection.
Data are available at doi:10.5061/dryad.f082m.
S.v.H., J.M.V., M.V.O. and V.I.R. conceived the study and designed experiments. S.v.H. and Y.H. performed the experiment and analysed the data. All authors have contributed to writing the manuscript.
S.v.H. and V.I.D.R were supported by the Program Strategic Alliances of the Royal Dutch Academy of Arts and Sciences (project 08-PSA-BD-01) and V.I.D.R. is supported by a VENI grant of the Netherlands Organisation for Scientific Research (project 863.11.017).
Conflict of interests
The authors declare no conflict of interests.
Amaya Serrano, University of Pamplona, Spain, is acknowledged for kindly providing the SeMNPV virus stock.
- Received August 27, 2014.
- Accepted December 4, 2014.
- © 2014 The Author(s) Published by the Royal Society. All rights reserved.