Novel partiti-like viruses are conditional mutualistic symbionts in their normal lepidopteran host, African armyworm, but parasitic in a novel host, Fall armyworm

Abstract

Recent advances in next generation sequencing (NGS) (e.g. metagenomic and transcriptomic sequencing) have facilitated the discovery of a large number of new insect viruses, but the characterization of these viruses is still in its infancy. Here, we report the discovery, using RNA-seq, of three new partiti-like viruses from African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), which are all vertically-transmitted transovarially from mother to offspring with high efficiency. Experimental studies show that the viruses reduce their host's growth rate and reproduction, but enhance their resistance to a nucleopolyhedrovirus (NPV). Via microinjection, these partiti-like viruses were transinfected into a novel host, a newly-invasive crop pest in sub-Saharan Africa (SSA), the Fall armyworm, S. frugiperda. This revealed that in this new host, these viruses appear to be deleterious without any detectable benefit; reducing their new host's reproductive rate and increasing their susceptibility to NPV. Thus, the partiti-like viruses appear to be conditional mutualistic symbionts in their normal host, S. exempta, but parasitic in the novel host, S. frugiperda. Transcriptome analysis of S. exempta and S. frugiperda infected, or not, with the partiti-like viruses indicates that the viruses may regulate pathways related to immunity and reproduction. These findings suggest a possible pest management strategy via the artificial host-shift of novel viruses discovered by NGS.

Fig 1. Phylogenetic analysis of partiti-like viruses found in Spodoptera exempta.

Maximum likelihood tree constructed with IQ-TREE 1.6.6 using amino acid sequences of the conserved RdRp domain of SEIV1, SEIV2, SEIV3, thirteen other partiti-like viruses from invertebrates, and 22 members from the family Partitiviridae. Alphapartitivirus, Betapartitivirus, Gammapartitivirus, Deltapartitivirus and Cryspovirus are the five genera within the family Partitiviridae, the members of which were reported to infect plant, fungi and protists. The bootstrap values (5000 pseudoreplicates) > 50% are indicated on the nodes.

Fig 2. Distribution of the partiti-like viruses in S. exempta.

(a,b) Relative quantification of SEIV1 (a) and SEIV2 (b) using β-actin and GAPDH as reference genes at different stages (n = 4) (ANOVA: SEIV1: F = 10.111, d.f. = 8,27, P < 0.0001; SEIV2: F = 100.73, d.f. = 8,27, P < 0.0001). (c,d) Relative quantification of SEIV1 (c) and SEIV2 (d) in different tissues of larvae (for hemolymph, n = 3; others, n = 6) (SEIV1: F = 26.827, d.f. = 3,17, P < 0.0001; SEIV2: F = 13.819, d.f. = 3,17, P < 0.0001). (e,f) Relative quantification of SEIV1 (e) and SEIV2 (f) in different tissues of female (n = 3) (SEIV1: F = 10.875, d.f. = 3,8, P = 0.0034; SEIV2: F = 6.681, d.f. = 3,8, P = 0.0143). (g,h) Relative quantification of SEIV1 (g) and SEIV2 (h) in different tissues of male (n = 3) (SEIV1: F = 11.114, d.f. = 3,8, P = 0.0032; SEIV2: F = 14.539, d.f. = 3,8, P = 0.0013). 1^st = first instar-stage larvae, 2^nd = second instar-stage larvae, 3^rd = third instar-stage larvae, 4^th = fourth instar-stage larvae, 5^th = fifth instar-stage larvae, 6^th = sixth instar-stage larvae, p = pupae, F = Female, M = Male, Hem = Hemolymph, Fa = Fat body, Gu = Gut, Cu = Cuticle, Le = leg, He = Head, Th = Thorax, Ab = Abdomen. Means ± SD. Different letters indicate statistically significance differences.

Fig 3. Life-history parameters of the partiti-like viruses-positive and -negative individuals of S. exempta and S. frugiperda.

(a,b) S. exempta larval period (a) (females: t-test: t = 8.192, d.f. = 212, P < 0.0001; males: t = 6.550, d.f. = 176, P < 0.0001) and pupal period (b) (females: t = 3.729, d.f. = 212, P = 0.0002; males: t = -0.079, d.f. = 176, P = 0.94 (n: F+ = 114, F- = 100, M+ = 100, M- = 78). (c,d) Fertility (number of neonate larvae produced) of F+M+ and F-M- S. exempta pairs in all samples (c) (t = -5.031, d.f. = 74, P < 0.0001;n: F+M+ = 61, F-M- = 25) and after excluding pairs that produced no eggs (d) (t = -4.107, d.f. = 58, P = 0.00013; n: F+M+ = 43, F-M- = 24). (e) S. frugiperda pupal period (females: t = 4.82, d.f. = 225, P < 0.0001; males: t = 3.46, d.f. = 203, P <0.0001; n: females: F+ = 110, F- = 117; males: M+ = 123, M- = 82). (f,g) Fertility (number of neonate larvae produced) of F+M+ and F-M- S. frugiperda individuals in all samples (f) (t = -4.087, d.f. = 57, P = 0.00014; n: F+M+ = 30, F-M- = 29) or after excluding pairs that produced no eggs (g) (t = -0.978, d.f. = 50, P = 0.0031; n: F+M+ = 23, F-M- = 29). Means ± SD. *** = P<0.001, based on t-tests at each point.

Fig 4. Relationship between the baculoviruses (SpexNPV and SfMNPV) and the partiti-like viruses in larvae of S. exempta (a-c) and S. frugiperda (d-f).

(a,d) Effect of NPV dose (log₁₀-transformed number of occlusion bodies per larva) on larval survival to pupation in S. exempta (a) (logistic regression: χ² = 25.798, d.f. = 1, P < 0.0001) and S. frugiperda (d) (logistic regression: χ² = 9.447, d.f. = 1, P = 0.0022). The points are the raw means for each dose. The thick lines are the fitted values and the shaded zones are the standard errors around these fitted values; blue points, lines and shading are the three partiti-like viruses-negative larvae (V-); red points, lines and shading are the three partiti-like viruses-positive larvae (V+). The numbers of larvae at different concentrations for SpexNPV (0 (control), 1×10³, 5×10³, 2.5×10⁴, and 1×10⁵ OBs/larva) were 63, 64, 67, 82, 79 for virus-negative individuals and 96, 109, 134, 133, 127 for virus-negative individuals; for SfMNPV (0 (control), 8×10³, 4×10⁴, 2×10⁵, and 1×10⁶ OBs/larva) they were 92, 85, 104, 149, 87 for virus-negative individuals and 100, 95, 111, 106, 90 for virus-positive individuals. (b,e) The pupal period of survivors of NPV-challenge in S. exempta (b) (linear model: F = 12.263, d.f. = 1,265, P = 0.00054) and S. frugiperda (e) (linear model: F = 2.30, d.f. = 1,413, P = 0.0940). (c,f) NPV copy numbers (log₁₀-transformed) at 72 h post-challenge with SpexNPV (c) (F = 10.095, d.f. = 18, P = 0.0052) and SfMNPV (f) (F = 7.504, d.f. = 18, P = 0.0134). The concentrations of NPV were 2.5×10⁴ OBs/larva and 2×10⁵ OBs/larva for SpexNPV and SfMNPV respectively. V- = the three partiti-like viruses-negative larvae, V+ = the three partiti-like viruses-positive larvae. Means ± SD. *** = P<0.001, based on t-tests at each time-point.

Fig 5. Transcriptome analysis using the partiti-like virus-positive individuals compared to related virus-negative individuals in S. exempta and S. frugiperda.

(a,c) PCA analysis of global gene expression of DEGs in the comparison of partiti-like virus-positive groups and related virus-negative groups in S. exempta (a) and S. frugiperda (c). Blue stands for the partiti-like viruses-positive samples and red stands for the partiti-like viruses-negative samples. (b,d) Heatmaps of–log10 p-values of KEGG pathway representing the up- and down- regulated DEGs in S. exempta (b) and S. frugiperda (d). “*” indicate the significantly enriched pathways (p < 0.05). Red color shows up-regulation pathways, green color show down-regulation pathways, gray color shows no value, the redder/greener the color, the lower P-values.

Fig 6. The quantity of DEGs with log2(TPM) related to the expression of different pathways.

(a) the Jak-STAT signaling pathway of S. exempta larvae; (b) the Insect hormone biosynthesis pathway of S. exempta adult females; (c) the Steroid hormone biosynthesis pathway of S. exempta adult female; (d) the Toll and Imd signaling pathway of S. frugiperda larvae; and (e) the insulin signaling pathway expression of S. frugiperda adult females. Colors in log2(TPM) indicate the gene expression levels, the hotter (redder) the color, the higher the gene expression level. The original data for TPM are shown in S9 Data.