Baculovirus entry into the central nervous system of Spodoptera exigua caterpillars is independent of the viral protein tyrosine phosphatase

Abstract

Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.

Keywords: Autographa californica multiple nucleopolyhedrovirus; Spodoptera exigua; central nervous system; neuroparasitology; parasite-induced behavioural manipulation; protein tyrosine phosphatase.

Figure 1.

Schematic overview of the central nervous system of a mid-third instar S. exigua caterpillar, consisting of the supraoesophagal ganglion (brain), followed by the suboesophagal ganglion (SOG), three thoracic ganglia (TG1 to TG3) and seven abdominal ganglia (AG1 to AG7–8; abdominal ganglia 7 and 8 are fused). Each ganglion is connected to the adjacent ganglion by two connectives (not fully depicted). The much smaller frontal ganglion (FG) is connected to the brain which it is placed acutely caudally to. Scale bar (top left) represents 50 µm.

Figure 2.

Overview of the modified parts of the recombinant AcMNPV viruses used in this study, including AcMNPV WT-eGFP (WT), AcMNPV Δptp-eGFP (Δptp) where ptp was replaced by a Zeocin resistance gene (zeo^r), AcMNPV with ptp inserted back into the genome after deletion (repair) and AcMNPV encoding for a catalytic inactive PTP (catmut with the Cys-119 residue replaced with an alanine, C119A)). For all viruses, the polyhedrin (polh) gene and the fused egfp-vp39 genes were inserted between the left and right insertion sites (Tn7L and Tn7R) present in the bacmids. For AcMNPV repair and AcMNPV catmut, the ptp gene was inserted with the upstream hr1 repeat region. Antibiotic resistance genes inserted during the different cloning steps [44] include the gentamicin (gm^r), spectinomycin (spect^r) and kanamycin (kan^r) resistance genes.

Figure 3.

Accumulated percentage of S. exigua larvae infected with AcMNPV WT-eGFP (WT) showing external fluorescence. Larvae were examined daily from 1 to 7 days post infection (dpi) under a stereomicroscope with UV-lamp (Leica; Wild M3Z and HBO lamp), using 69 early second instar AcMNPV-infected (AcL2; dashed black line) and 56 early third instar AcMNPV-infected (AcL3; solid grey line) larvae. Percentages of fluorescence was the same during the course of infection for both treatments (Kaplan–Meier analysis, p = 0.69). For a few individuals the eGFP expression faded out after liquefaction and death. None of the (100) mock-infected individuals (ML2 and ML3) expressed eGFP at any dpi.

Figure 7.

Confocal laser scanning microscope images of parts of the central nervous systems of liquefied S. exigua larvae (6 and 7 days post infection (dpi)) upon AcMNPV WT-eGFP infection. (a) Brain of a mock-infected larvae for comparison with (b) brain and (c) SOG and first thoracic ganglion (TG1) of 6 dpi liquefied larvae (both from larvae infected as early second instar) and (d,e,e′) brain of 7 dpi liquefied larvae (from larvae infected as early third instar). Open arrow heads in (b,c,d) indicate OBs released from cells post lysis, whereas the open arrow in (c,d) indicates OBs still enclosed in cells. Filled arrows in (e,e′) indicate lysed cells with fragmented nuclei (TO-PRO-3, magenta channel). (e′) is a magnification of the square area indicated in (e). All panels in the figure represent a stack on the surface of the ganglia, except for (d), where the internal infection is visualized from a stack in the midline of the brain. A white signal indicates an overlap and high intensity of the green (AcMNPV WT-eGFP) and magenta channel. Scale bars represent 50 µm.

Figure 4.

Percentage of AcMNPV WT-eGFP-infected larvae (infected as second and third instars) expressing eGFP only in trachea (‘tracheal'; yellow line), or also superficially in the ganglia (‘superficial'; pink line), or also internally in ganglia (‘internal’; blue-green line) for all the analysed ganglia (brains to first abdominal ganglia) at different days post infection. This figure visualizes input data from 413 individual observations of infection levels in different ganglia (n = 32 for 1 dpi, 53 for 2 dpi, 69 for 3 dpi, 90 for 4 dpi, 77 for 5 dpi, 51 on 6 dpi and 41 for 7 dpi) from a total of 105 AcMNPV WT-eGFP-infected larvae, and does not include the data from the mock-infected larvae (n = 68) as none of the scans of 286 ganglia showed any eGFP expression.

Figure 5.

Percentage of AcMNPV WT-eGFP-infected larvae (infected as second and third instars) expressing no eGFP (‘No eGFP'; grey bars), eGFP only in trachea (‘tracheal'; yellow bars), or also superficially in the ganglia (‘superficial'; pink bars), or also internally in ganglia (‘internal'; blue-green bars) for the brain, SOG and first to third thoracic ganglia (TG1, TG2 and TG3) at different days post infection. The data represent the most advanced level of infection observed (see §2.7 scoring of infection). This figure shows data from 402 individual scorings of ganglia (data on AG1 are not represented in this figure).

Figure 6.

Confocal laser scanning microscope images of AcMNPV WT infection in the brain, SOG, first, second and third thoracic ganglia (TG1–TG3), and the first abdominal ganglia (AG1; for 3 days post infection (dpi) only) at 1–6 days dpi. All images represented here are from S. exigua larvae infected as early third instars with a 10⁸ OBs/ml concentration. Magenta channel for the TO-PRO-3 nuclei/dsDNA staining, green channel for AcMNPV WT-eGFP. White signal indicates an overlap and high intensity of the green and magenta channel. Scale bars represent 50 µm.

Figure 8.

(a) Schematic overview of AcMNPV WT-eGFP localization in the CNS (from brain to first abdominal ganglia (AG1)) of S. exigua, with (b–h) representative images of confocal laser scanning microscope (CLSM) imaging for each of the analysed ganglia. Based on CLSM images, the major localizations of AcMNPV WT-eGFP were visualized with green dots in (a), and trachea are visualized as appendages to the ganglia with red-brown lines (viral infections in the trachea are not represented in the schematic overview). (b) Brain with filled arrow indicating infected cell bodies surrounding the calyx and open arrow head indicating infected cell body in the centre of the superior neuropil; (c) frontal ganglion (FG); (d) SOG; (e) first thoracic ganglion (TG1); (f) second thoracic ganglion (TG2); (g) third thoracic ganglion (TG3); (h) first abdominal ganglion. (b–h) are from 3 to 5 dpi; representative images were picked to illustrate the overall trends of viral progression and localization. Magenta signal for the TO-PRO-3 nuclei/dsDNA staining, green channel for AcMNPV WT-eGFP. Scale bars represent 50 µm.

Figure 9.

Confocal laser scanning microscope images of the same brain area, showing the calices (all) and/or midline (mock, repair, catmut) of brains dissected at 4 days post infection from larvae infected as early third instar with a mock solution (Mock), or with one of the recombinant AcMNPV viruses expressing eGFP (AcMNPV WT-eGFP (WT), AcMNPV Δptp-eGFP (Δptp) lacking ptp, AcMNPV with ptp repaired back after deletion (repair) and AcMNPV encoding for a catalytic inactive PTP (catmut). White signal for TO-PRO-3 nuclei/dsDNA staining, green channel for eGFP signal from the recombinant viruses described. Scale bars represent 50 µm.