Getting on host’s nervesbaculovirus-associated changes in the neuronal system of spodoptera exigua

Resumen

Baculoviruses constitute a diverse group of entomopathogenic viruses characterised by their high specificity and their strong field incidence. These two characteristics have facilitated their usage as bioinsecticides for controlling lepidopteran crop pests. During the baculovirus infection in the larvae, the virus spreads through the insect body, infecting and replicating in many different tissues. As a consequence of this systemic infection, baculovirus produces behavioral changes in its host to promote its viral dispersion in the field, in a clear example of evolutionary advantage. These well-known behavioral phenotypes, whose main examples are the enhanced locomotion activity and the tree-top disease, have been connected with the presence in the baculovirus genome of genes previously acquired from the host. In addition, the existence of this parasite-induced phenotypes may also imply expression changes in host genes that would play a key role in controlling behavioral patterns. Thus, we hypothesize that the already described baculovirus-associated behavioral phenotypes and others that might be discovered, could also be connected to changes in the neural systems of the insect. The nervous system of the insect is composed of the central nervous system and the peripheral nervous system. Both of them participate in the control of the insect’s physiology and behavior, and also in the communication of the insect with its surrounding environment. Our hypothesis is supported by the fact that baculovirus presence in brain or antennas has been already observed. Considering this background, the main purpose of this doctoral thesis is to study the influence of the Spodoptera exigua Multiple Nucleopolyhedrovirus (SeMNPV) in the neuronal systems of the lepidopteran crop pest Spodoptera exigua. Within the central nervous system, we have centred our efforts in the study of the neuropeptidergic system, formed by neuropeptides that regulate the insect’s internal physiology. Within the peripheral nervous system, we have focused on the chemosensory-related genes, concretely in the odorant receptors, that are the centre piece of the insect’s olfaction. For analysing the SeMNPV influence in both systems,we have followed a common pipeline. We first have described the neuropeptide and chemosensory-related gene repertoires of S. exigua. Then, we have studied SeMNPV-associated changes in the expression of these gene repertoires. Finally, we have selected gene candidates for functional characterization to understand their role in the host-pathogen interaction. In the first chapter, the neuropeptidome of S. exigua is annotated. That supposes the identification of 63 neuropeptide unigenes from a transcriptome assembled with samples of larval heads, larval gut and adult brains. We also build phylogenetical trees with the S. exigua neuropeptides and those from other related species whose neuropeptidomes are already available. Other information as the description of brain-gut neuropeptides, a comparison of the neuropeptidome expression in different tissues and developmental stages, and how external factors as light or temperature can influence it, complete the description of the neuropeptidome of S. exigua. In the second chapter, we analyse how the baculovirus infection affects the expression of neuropeptide genes following different approaches. At first, we perform differential gene expression in RNA-Seq samples of SeMNPV- and mock-infected larval head samples. Some genes related with the ecdysis process appear to be up-regulated, connecting this with previously observed phenotypes produced by the baculovirus infection. More comprehensive results appear studying the SeMNPV influence in brain samples through RT-qPCR. One putative neuropeptide gene, proctolin-like, resembling the insect neuropeptide proctolin, is clearly down-regulated after the SeMNPV infection. For studying its role in the host-pathogen interaction, recombinant baculoviruses are generated expressing this gene, in a gain-of-function strategy. Proctolin-like overexpression resulted in a decrease of the larval locomotion activity and digestion, confirming its similar role to the neuropeptide proctolin. These results permit us to hypothesize about the proctolin-like regulation after the baculovirus infection and the consequences of its down-regulation, possiblymaking larvae to become more active and bigger and thus, increasing viral fitness by releasing more viral progeny to the environment. In the third chapter, the chemosensory-related gene repertoire of S. exigua is reannotated, improving the previous annotations and using a new nomenclature system that permits its comparison with that of other related species. This new description focuses on larval chemosensory genes, contrary to the previous annotations that used adult transcriptomes. We also analyse how the pre-exposition to specific odorants provokes changes in the expression of some chemosensory genes and we devise a new method for studying behaviourally active odorants against S. exigua larvae. In the fourth chapter, we analyse the SeMNPV influence in the expression of the chemosensory-related genes, mainly focusing on odorant receptors. Using RNA-Seq samples of larval head, some odorant receptors appear to be up-regulated after the virus infection. Two of them, SexiOR23 and SexiOR35 are selected for their functional characterisation using heterologous expression in Drosophila and electrophysiological techniques. SexiOR35 result in a broad-tuned receptor able to recognise many different plant odorants. Behavioral assays with baculovirus- and mock-infected samples reveal changes in the larval perception of odorants recognised by SexiOR35 after baculovirus infection. This supposes the first description of baculovirus-associated changes in the olfaction of lepidopteran larvae. The results obtained from this doctoral thesis discover baculovirus-associated changes in the neuronal systems of S. exigua. On one hand, the down-regulation of proctolin-like, would make the SeMNPV-infected larvae to become bigger and more active, increasing the viral fitness in the environment. On the other hand, the up-regulation of specific odorant receptors during the SeMNPV infection would produce strong changes in the odorant preferences of the larvae, producing behavioral alterations that could increase the viral incidence or unveiling a larval response to decrease the consequences of the viral infection. These results open new questionsthat aim to understand if the observed changes are directly caused by the virus or are collateral effects of the infection. They also allow to hypothesize about the biological significance of these phenotypes and to discover new potential targets for pest control of the lepidopteran pest S. exigua.