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Host behavioural manipulation has been reported for many parasite-host combinations. An intriguing example and the main topic of study in this thesis, is host behavioural manipulation by baculoviruses, in which host insects serve as a tool to enhance the transmission rate, and hence fitness, of these viruses.
Chapter 2 of this thesis reviews the documented changes of insect behaviour upon virus infection and summarizes the limited knowledge available so far about the underlying mechanisms. Special attention is given to baculovirus-induced behavioural changes in caterpillars. The ecological and evolutionary consequences of virus-induced insect behavioural changes are also discussed.
Baculoviruses induce pre-death climbing behaviour in their lepidopteran hosts. Death at elevated positions is thought to disperse progeny virus over larger areas of plant foliage. Hoover et al. (Science, 2011) found that the egt gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was involved in inducing tree-top disease in L. dispar larvae. Our previous work revealed that Spodoptera exigua (Se)MNPV induced tree-top disease in S. exigua larvae, which is light-dependent. Chapter 3 aimed to determine whether the egt gene from SeMNPV is also involved in SeMNPV-induced tree-top disease. The results in Chapter 3 showed that S. exigua larvae infected with SeMNPV lacking the egt gene died at low positions and larvae infected with a mutant virus lacking the egt gene died earlier than larvae infected with wild type (WT) virus. Larvae infected with the mutant virus died before the onset of the climbing behaviour seen in wild-type infected larvae. Therefore, we concluded that SeMNPV EGT enzyme facilitates tree-top disease in infected S. exigua larvae by extending the survival time of its host.
Besides viral and host genes, environmental conditions may also affect the outcomes of behavioural manipulation. In Chapter 4, the importance of the time point at which light is provided (relative to the moment of infection) in the induction of tree-top disease was investigated. The results showed that light from above was needed between 43 and 50 hours post infection (hpi) to induce tree-top disease. This time interval is prior to the actual climbing, which occurred in the dark between 57 and 67 hpi. When infected larvae were not exposed to light between 43 and 50 hpi, they eventually died at low positions.
The second type of behavioural manipulation induced by baculoviruses in their hosts is enhanced locomotion behaviour (or hyperactivity). Previous work demonstrated the involvement of the ptp gene from Autographa californica (Ac)MNPV in triggering hyperactivity in S. exigua larvae. The phosphatase activity of the encoded PTP protein was also needed for this process. SeMNPV also carries a protein tyrosine phosphatase, called ptp2, which is phylogenetically unrelated to ptp. Ptp2 is more similar to the protein tyrosine phosphatase gene (ptph2) from Microplitis demolitor bracovirus, that induced apoptosis in host immune cells. The idea is that this induction of apoptosis reduces the number of immune cells and that as a consequence the virus can evade from immune responses, leading to increased virus propagation. In Chapter 5, we investigated whether the SeMNPV PTP2 protein also has a pro-apoptotic effect on cultured insect cells and on S. exigua immune cells. The results showed that PTP2 induced mild apoptosis in cultured insect cells (Sf21 cells) and that the catalytic site of PTP2 was needed for this process. Moreover, the SeMNPV ptp2 gene was also involved in inducing apoptosis in host hemocytes. Larvae infected with WT virus produced more viral occlusion bodies (OBs) than larvae infected with an SeMNPV mutant lacking the ptp2 gene. We hypothesized that SeMNPV suppresses the host immune system by inducing apoptosis in hemocytes and that this immune suppression finally leads to higher OB yields.
Parasites may achieve behavioural manipulation by invading or affecting the host central nervous system (CNS). However, little information was available on the morphology and function of the CNS of S. exigua larvae, and of caterpillars in general. In Chapter 6, we first constructed a 3D model of the S. exigua larval brain and described different brain areas, by immunolabelling serotonergic cells in the larval brain and gnathal ganglion (GNG, or subesophageal ganglion,SEG). Furthermore, we described the distribution of serotonergic neuron cells in the brain and GNG. Individual cell clusters were mapped and the number of serotonergic cells in each cluster in the brain and the GNG were estimated. As a next step, we studied virus localization and temporal invasion patterns in the larval brain and GNG, using green fluorescent protein-labelled AcMNPV virus particles (GFP-AcMNPV). The results showed that AcMNPV started to invade the host CNS from the second day post infection (dpi) at the lateral sides, and further spread to specific cells throughout the brain at three dpi (when hyperactivity is observed). The data in this chapter provided more information on the larval CNS and has pointed out several interesting directions for future research.
On the molecular level, AcMNPV PTP (AcPTP) must interact with a host or viral protein that leads to the induction of hyperactivity. To identify host and viral proteins that interact with AcPTP, a PTP substrate analysis was performed using a mass spectrometry approach. The results in Chapter 7 describe host and viral proteins that co-purified with AcPTP. The results indicate that AcPTP is potentially involved in many different steps of the virus replication cycle, including nucleocapsid assembly, nucleocapsid transport, ODV envelopment and/or virus dissemination. The results suggest that AcPTP functions both as a structural protein and as a phosphatase enzyme. Some of the interactions potentially have an important role in cellular signalling pathways and potentially in brain invasion.
Chapter 8 discusses the main findings of this thesis and gives implications regarding to future research on baculovirus-induced behavioural manipulation. Overall, the results of this thesis showed that the viral egt gene and the timing of light both play an important role in baculovirus-induced tree-top disease. Moreover, the ptp2 gene plays a significant function in host immune system modulation. We studied the morphology of the S. exigua larval brain and the serotonergic neuron distribution in the host CNS and explored how baculoviruses invade the host CNS. Lastly, potential interaction partners of the PTP protein, involved in the induction of hyperactivity, were described.
|Qualification||Doctor of Philosophy|
|Award date||31 Aug 2018|
|Place of Publication||Wageningen|
|Publication status||Published - 2018|