# Nudiviruses in free-living and parasitic arthropods: evolutionary taxonomy

Jirka Manuel Petersen, Amy L Burgess, Monique M van Oers, Elisabeth A Herniou, Jamie Bojko

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Jirka Manuel Petersen, Amy L Burgess, Monique M van Oers, Elisabeth A Herniou, Jamie Bojko. Nudiviruses in free-living and parasitic arthropods: evolutionary taxonomy. Trends in Parasitology, 2024, 40 (8), pp.744-762. 10.1016/j.pt.2024.06.009. hal-04747493

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Submitted on 22 Oct 2024

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Review

Nudiviruses in free-living and parasitic
arthropods: evolutionary taxonomy

Jirka Manuel Petersen ID 1,2,*,@, Amy L. Burgess ID 3,4, Monique M. van Oers ID 1,
Elisabeth A. Herniou ID 2, and Jamie Bojko ID 3,4,*,@

The nudiviruses (family: *Nudiviridae*) are large double-stranded DNA (dsDNA) viruses that infect insects and crustaceans, and have most recently been identified from ectoparasitic members (fleas and lice). This virus family was created in 2014 and has since been expanded via the discovery of multiple novel viral candidates or accepted members, sparking the need for a new taxonomic and evolutionary overview. Using current information (including data from public databases), we construct a new comprehensive phylogeny, encompassing 49 different nudiviruses. We use this novel phylogeny to propose a new taxonomic structure of the *Nudiviridae* by suggesting two new viral genera (Zetanudivirus and Etanudivirus), from ectoparasitic lice. We detail novel emerging relationships between nudiviruses and their hosts, considering their evolutionary history and ecological role.

Taxonomic history of the family *Nudiviridae*

dsDNA viruses (see Glossary) are enormously diverse and are divided over separate taxonomic
realms [1]. One group of invertebrate-infecting dsDNA viruses sits in the class *Naldaviricetes* [2].
This includes the family *Nimaviridae* [3] and the order *Lefavirales*, housing the *Baculoviridae*,
*Hytrosaviridae*, recently proposed Filamentoviridae [4], and the *Nudiviridae* [5,6]. The *Nudiviridae*
comprise bacilliform viruses that infect hosts across the Pancrustacea [7], including insects (flies,
moths, beetles, crickets, craneflies) and crustaceans (lobsters, shrimp, crabs, amphipods), with
varying degrees of virulence and ecological impact [6].

**Pathological** and morphological data originally pointed towards the presence of baculoviruses in insect and crustacean hosts [8–10]. However, a baculovirus termed ‘Baculo-A’, derived from the swimming crab *Callinectes sapidus*, was described in the 1980s, but later revealed to be a nudivirus [11]. Similarly, the insect nudiviruses Oryctes rhinoceros nudivirus (OrNV), Heliothis zea nudivirus (HzNV-1), and Gryllus bimaculatus nudivirus were all originally described as baculoviruses [12–14]. Subtle morphological and pathological differences, including more obvious traits (i.e., lacking occlusion bodies leading to the term ‘non-occluded baculoviruses’; Box 1), supported the split from the other baculoviruses, leading to the suggestion of a baculoviral subfamily (‘Nudivirinae’) [15], which eventually became the family *Nudiviridae* [5]. Further pathological support was derived from the host specificity and tissue tropism of nudiviruses, such as those that infect the hepatopancreas of crustacean hosts [16,17], and those that manifest across the gut, fat-bodies and reproductive tissues of insects [6]. The localized nature of nudivirus infections within an infected individual stands as another primary distinction from baculoviruses, which typically induce systemic infections [18].

The *Nudiviridae* currently comprise four officially recognized genera: *Alphanudivirus* (including *endogenous* nudiviral elements), *Betanudivirus*, *Gammanudivirus*, and *Deltanudivirus* [5]. A fifth genus has been suggested for a group of divergent crustacean-infecting nudiviruses ('Epsilonnudivirus') [19] and two unofficial subfamilies have been considered, which would

Highlights

An updated phylogenomic tree of the *Nudiviridae* is presented, including 49 species, proposing two genera associated with ectoparasitic insects.

Evolution of the family *Nudiviridae* is estimated to have occurred in the mid-Devonian, matching the timeline of the insect's evolution.

All the crustacean-infecting nudiviruses have likely emerged from a single common ancestor (~143 million years ago), perhaps transmitted to Jurassic crustaceans by an insect host.

The *Nudiviridae* have a proclivity for ecto-
parasitic arthropods (i.e., lice and fleas),
linked to multiple diversification events
in the Triassic (early Betanudivirinae
split), Jurassic (involving ectoparasitic
hosts of alphanudiviruses), and
Cretaceous periods (Zeta/Etanudivirus
diversification).

Nudiviruses inhabit various geographical
and climatic zones, including members
from subpolar habitats.

<footnote n="1">¹Laboratory of Virology, Wageningen University and Research, 6708, PB, Wageningen, The Netherlands</footnote>

<footnote n="2">²Institut de Recherche sur la Biologie de l'Insecte, UMR7261 CNRS - Université de Tours, 37200 Tours, France</footnote>

<footnote n="3">³School of Health and Life Sciences, Teesside University, Middlesbrough, TS1 3BX, UK</footnote>

<footnote n="4">⁴National Horizons Centre, Teesside University, Darlington, DL1 1HG, UK</footnote>

<footnote>*Correspondence:
jirkamanuel.petersen@wur.nl
(J.M. Petersen) and
J.Bojko@tees.ac.uk (J. Bojko).
©X: @Jamiebojko (J. Bojko) and
@JirkaMp (J.M. Petersen).</footnote>
house the *Alphanudivirus* ('Alphanudivirinae') and the *Betanudivirus*, *Gammanudivirus*, and *Deltanudivirus* genera ('Betanudivirinae') [20]. An additional genus has been proposed for inclusion in the family *Nudiviridae* – the *Bracoviriform* (formerly 'Bracovirus') genus, which consists of endogenous nuviruses (Box 2) from parasitic wasps, and which are currently classified as *Polydnaviriformidae* (formerly 'Polydnaviridae') (Figure 1) [21,22].

Transcriptomic and metagenomic studies in invertebrate hosts have hinted at the existence of yet uncharacterized nuviruses in other arthropods – for example, nudivirus-like transcripts were found in two additional species in the order Decapoda: *Eriocheir sinensis* and a *Charybdis* sp. [23,24]. The bounty of next-generation sequencing data from arthropods (593 792 public datasets – 9 November 2023) in publicly available Sequence Read Archives (SRAs) [25] represents a major resource for discovering more nuviruses in order to address questions regarding taxonomy and evolutionary timeline.

Nudivirus genomes consist of a single circular dsDNA molecule, ranging in size from approximately 96 to 232 kilobase pairs (kbps), encoding between 87 and 154 protein-coding genes (PCGs) [6]. All lefavirals encode homologues of genes involved in viral (late) gene transcription, including the four subunits of the DNA-directed RNA polymerase (*lef-4*, *lef-5*, *lef-8*, and *lef-9*). In nuviruses, these four genes belong within a set of 28 'core' genes that can be found in all **exogenous** nudivirus genomes, whereas endogenous nuviruses can have an incomplete repertoire of core genes [26,27]. Nudiviral core genes fulfil crucial functions in virus replication, infectivity, virion assembly and morphology, but several have unresolved functions [6].

## Underexplored nudiviral diversity

Numerous unclassified viral agents with nudivirus-like pathology and morphology exist. Two studies conducted in the 1970s identified nuclear viruses with nonoccluded, rod-shaped virions in *Solenopsis* sp. (Hymenoptera: Formicidae) and *Gyrinus natator* (Coleoptera: Gyrinidae). These viruses exhibited nudivirus-like symptoms [28,29], but molecular data are missing. Another example involves the Tipula paludosa nucleopolyhedrovirus (TpNPV) [30], that is, based on single gene homology, more closely related to Tipula oleracea nudivirus (ToNV) than to baculoviruses [31], emphasizing the possibility that unclassified viruses (some with occlusion bodies; Box 1) could be nuviruses.

For the crustaceans, pathological studies have revealed nudivirus-like discoveries from (i) crabs: *Cancer pagurus* [16]; *Carcinus mediterraneus* (Tau-virus) [32]; *Paralithodes platypus* [33]; *Scylla serrata* [34]; and *Pinnotheres pisum* [35]; (ii) crayfish: *Astacus astacus* [36]; *Cherax quadricarinatus* [37]; *Pacifastacus leniusculus* [38]; *Cherax destructor* [39]; and *Astropotamobius*

### Box 1. Not all nuviruses are 'nudists': nudiviral occlusion bodies

Occlusion bodies (OBs) are crystalline structures assembled from viral proteins that are known for enwrapping the virions of certain insect viruses [105]. In baculoviruses, these dense protein structures protect the viral progeny from environmental stresses until they are ingested by their insect host. Despite their endurance towards many abiotic factors, baculovirus OBs easily dissolve under alkaline conditions to which they are exposed in the midgut of their hosts, leading to the release of virions responsible for primary infection [18].

While it was originally assumed that nuviruses were non-occluded baculoviruses [106], cases of OB formations in members of the *Nudiviridae* have emerged, challenging the derivation of their name from the Latin term 'nudus' (= naked or uncovered) [5]. Occluded nuviruses are present in both insect (ToNV, Tipula oleracea nudivirus) and crustacean (PvNPV, Penaeus vannamei nucleopolyhedrovirus; PmNV, Penaeus monodon nudivirus) hosts. The OBs of ToNV and PmNV are irregularly shaped [31,93], while PvNPV occludes its virions into pyramid-shaped OBs [9] (Figure 1). Furthermore, OBs of insect- and crustacean-infecting nuviruses seem to dissolve under different conditions. Congruent to baculoviruses in insects, the OBs of ToNV dissolved under alkaline treatment [107], while PmNV OBs showed resistance to high pH values, but dissociated when exposed to acidic conditions [93]. The dependency of OBs on per os transmission signifies the importance of coevolutionary adaptation, whereby nudiviral OBs might have been tailored to match their host's digestive environments.

## Glossary

**Ectoparasite:** a unicellular or multicellular organism that parasitizes its host externally.

**Endogenous:** in virology, refers to foreign genetic material that has integrated into the genome of a given organism (i.e., endogenous viral elements).

**Exogenous:** refers to viruses whose genetic material has not been integrated into the genome of its host but is packaged in infectious, replication-competent particles.

**Gene synteny:** the linear arrangement of genes across the genomic architecture of a given organism.

**Haematophagous:** the dietary trait of consuming blood.

**Molecular dating:** the process of analytically predicting the evolutionary timeline of a series of organisms using molecular data.

**Paleovirological:** descriptive term relating to paleovirology, which is the study of ancient viruses and how they have coevolved with their hosts, including the evolutionary inference of modern-day exogenous viruses.

**Pathological:** descriptive term relating to pathology, which is the study of diseases, their causes, processes, development, and consequences.

**Phylogeny:** involves the construction of phylogenetic trees to illustrate the evolutionary relationship and common ancestry of different species.

**Virulence:** describes the degree of disease a pathogen causes in its host.

**Viruses:** infectious mobile genetic elements that package their nucleic acid core in virions, assembled from at least one virus-encoded component.
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Trends in Parasitology

<caption>**Figure 1.** Histological and electron microscopic images of nudiviral ultrastructure and development.

(A) Multiple hepatopancreatic tubules (HPs) display infected hepatopancreatocytes with hypertrophic nuclei (dotted box), including a growing viroplasm (image obtained from *Faxonius rusticus*; [60]). (B) Inset image showing a higher magnified image of an infected nucleus (black arrow) (image obtained from *F. rusticus*; [60]). (C) A transmission electron micrograph of Menippe mercenaria nudivirus (MmNV), in the nucleus ('N') of an infected hepatopancreatic epithelial cell, including a virions-containing vesicle (black arrow) (image reproduced from [49]). (D) An enlarged view of the MmNV virions in the host nucleus (black arrow) (image reproduced from [49]). (E) An example of a nudivirus from *Penaeus* sp., which produces occlusion bodies (OBs) in pyramidal structures, containing virions (black arrows) (image reproduced from [9]). (F) Negatively stained rod-shaped virions of Heliothis zea nudivirus 1 (HzNV-1) visualized via electron microscopy (purified from infected HZ-AM1 cells via sucrose cushion).</caption>

*pallipes* [40]; (iii) shrimp: *Farfantapenaeus* (= *Pandalus*) *montagui* [41]; *Penaeus japonicus* [42]; *Penaeus duorarum* [43]; *Penaeus plebejus* [44]; and (iv) various amphipods [45–48]. To date, all sequenced ‘baculovirus-like’ isolates in crustaceans belong to the family *Nudiviridae* [49].

Evidence of nuviruses hiding in cyberspace

In total, 2110 SRA datasets from arthropods were searched for nudivirus-like sequences (Files S1 and S2 in the supplemental information online). Ten SRA submissions included large, circular
Box 2. Endogenous nudiviruses among insect hosts

Whole-genome sequencing techniques have revealed the presence of nudivirus-derived endogenous viral elements (EVEs) in various arthropods [108]. Although the circumstantial conditions for nudiviral genome integration is scarcely studied, it can be assumed that such integration is driven by two possible scenarios: an active integration mechanism employed by nudiviruses to establish persistent infections, as it was observed for HzNV-1 [109]; or indirect integration as a consequence of induced DNA damage and repair during replication in the host cell nucleus [110]. Evolutionarily, the integration of nudiviral genetic material into host genomes has resulted in the emergence of several endogenous and 'functional' nudiviruses – that is, the bracoviruses. These endogenized viruses serve as invaluable 'fossil records', offering insights into the evolutionary history of ancient viruses and their hosts [22]. Despite their ostensible frequency, nudiviral integration events produce only a few examples of functional endogenizations, and among those functional cases there are still varying degrees of benefit to the host. For instance, Fopius arisanus endogenous nudivirus (FaENV) and Venturia canescens endogenous nudivirus (VcENV) form virus-like particles (VLPs) in their parasitoid hosts (Fopius arisanus and Venturia canescens) that are injected together with the wasp eggs during parasitism-mediated oviposition. The particles of VcENV are considered to benefit the success of parasitism in its hymenopteran host (for instance via immune-protective coating of wasp eggs, carrying wasp-derived virulence proteins, and manipulation of parasitized host physiology), while FaENV particles carry DNA-empty nucleocapsids with unknown benefit to its parasitoid wasp. VLPs of VcENV and FaENV both possess viral envelopes with incorporated nudiviral proteins, but without a nucleic acid core. This stands in contrast to virions formed by bracoviruses, which are enveloped, and carry circular dsDNA molecules inside their nucleocapsids [6].

Next to these functional models of endogenous nudivirus-derived agents, there are scenarios where nudivirus integration
events have led to nonfunctionality. The endogenous nudiviral agents of three hemipteran insects (Nilaparvata lugens:
NIENV; Melanaphis sacchari: MsENV; Aphis glycines: AgENV), for instance, are unable to produce particles or at least have
no evident function for the insect host [6,26,27]. Furthermore, a study by Cheng and coauthors [108] found a total of 359
nudivirus-like sequences in the genomes of various arthropods, the majority being present in Hemiptera and Hymenop-
tera, but with no confirmed functionalities.

dsDNA molecules derived from the nudivirus genomes (from insects: *Franciscoloa pallida* (Y. Dong, Y. PhD thesis, University of the Sunshine Coast, Queensland, 2022),

*Franciscoloa roseicapillae* (Y. Dong, Y. PhD thesis, University of the Sunshine Coast, Queensland, 2022), *Myrsidea ptilorhynchii* (Y. Dong, Y. PhD thesis, University of the Sunshine Coast, Queensland, 2022), *Heterodoxus spiniger*, *Cuculoecus africanus*, *Echinophilopterus claytoni*, *Lagopoecus perplexus* [50], three datasets from *Ctenocephalides orientis* [51]); and eight included partial/ complete core gene sets (from insects: *Culex pipiens* [52], *Goniodes lagopi* [50]; from crustaceans: *Gammarus pulex*, *Gammarus fossarum*, *Echinogammarus berilloni*, *Astacus leptodactylus* [53], *Eriocheir sinensis*, *Charybdis* sp. [23]). Additionally, putative nudiviral sequences for which fragmented/incomplete core gene sequences were obtained from five species (insects: *Aleuroclava psidii* [54], *Dasineura brassicae* [55], *Phlebotomus chinensis* [56], *Trichocera saltator* [57,58]; crustacean: *Niphargus hrabei* [59]) (Files S1, S2, S3, and S4 in the supplemental information online).

Datasets from the oriental cat flea (*Ctenocephalides orientis*: Siphonaptera) led to the assembly of three distinct full nudivirus genomes that we refer to as *Ctenocephalides orientis* nudivirus isolates 1–3, CoNV1, CoNV2, and CoNV3, respectively (Genome range: 90 491–90 736 bp; PCGs: 86–87). Eight novel nudiviruses are from either the Amblycera or Ischnocera superfamily of ecto-parasitic lice (Psocodea) (File S2 in the supplemental information online). Genomic data disclosed seven complete louse nudiviral genomes, reflecting *Franciscoloa pallida* nudivirus (FrpNV); *Franciscoloa roseicapillae* nudivirus (FrrNV); *Myrsidea ptilorhynchii* nudivirus (MpNV); *Heterodoxus spiniger* nudivirus (HxsNV); *Cuculoecus africanus* nudivirus (CafNV); *Echinophilopterus claytoni* nudivirus (EcNV); and *Lagopoecus perplexus* nudivirus (LpNV). *Goniodes lagopi* nudivirus (GINV) presented as two contiguous sequences, but encoded homologues of all expected virus genes. Genome size ranges: 83 884–97 269 bp; PCGs: 88–104 proteins.

The common house mosquito (*Culex pipiens*: Culicidae) in the study by Feng *et al*. [52] provided 102 predicted nudivirus genes through transcriptomic data, which is considered to represent the
<table><thead><tr><th></th><th>Members</th><th>Genome</th><th>Hosts<br>(Orders)</th><th>Biology</th></tr></thead><tbody><tr><td>Alphanudivirus</td><td>DiNV<br>ENV<br>GbNV<br>KV<br>MNV<br>OrNV<br>TNV<br>PsNV<br>AmNV<br>OcNV<br>DuhNV</td><td>Endogenous:<br>Size (kbp):<br>~97 to 183<br>PCGs:<br>87 to 139</td><td>Coleoptera<br>Diptera<br>Hemiptera<br>Hymenoptera<br>Orthoptera</td><td>Transmission:<br>Ingestion of excretions or cadavers contaminated with non-occluded virions.<br><br>Pathology:<br>Virus accumulates mostly in midgut and fat body tissue. Infections can manifest in lethargy and malformations as well as decreased fertility, growth and longevity.</td></tr><tr><td>Betanudivirus</td><td>HzNV-1<br>HzNV-2</td><td>Size (kbp):<br>~228 to 232<br>PCGs:<br>113 to 154</td><td>Lepidoptera</td><td>Transmission:<br>Sexually during mating.<br><br>Pathology:<br>Malformations in reproductive organs. "Waxy plug" filled with high quantities of non-occluded virions forms at genital opening.</td></tr><tr><td>Gammanudivirus</td><td>PmNV<br>HgNV<br>CmNV<br>ApNV<br>CsNV<br>MrNV<br>MmNV<br>*PvNPV</td><td>Size (kbp):<br>~99 to 119<br>PCGs:<br>83 to 115</td><td>Decapoda</td><td>Transmission:<br>Presumably via ingestion of non-occluded virions, or occlusion bodies in few cases (i.a PmNV and PvNPV).<br><br>Pathology:<br>Virus accumulates in hepatopancreas.</td></tr><tr><td>Epsilonnudivirus</td><td>DhNV<br>FpNV<br>FrNV<br>FvNV</td><td>Size (kbp):<br>~119 to 147<br>PCGs:<br>97 to 129</td><td>Amphipoda</td><td>Transmission:<br>Non-occluded virions are presumably ingested via contaminated urine.<br><br>Pathology:<br>Virus accumulates in hepatopancreas.</td></tr><tr><td>Deltanudivirus</td><td>ToNV</td><td>Size (kbp):<br>~145<br>PCGs:<br>131</td><td>Diptera</td><td>Transmission:<br>Oral ingestion of occluded virions.<br><br>Pathology:<br>Larvae display epidermal discoloration and increased mortality.</td></tr><tr><td>Bracoviriform</td><td>CeBV<br>CiBV<br>MdBV<br>(in total 31 species recognized by ICTV)</td><td>Partial conservation of nidu viral core genes (varies among species).</td><td>Hymenoptera</td><td>Endogenous viral agents that form replication-incompetent particles in the ovaries of parasitoid wasps. Particles are injected with wasp eggs during oviposition to ensure parasitism success.</td></tr></tbody></table>

Trends in Parasitology

Figure 1. Overview of the family *Nudiviridae* covering official (*Alphanudivirus*, *Betanudivirus*, *Gammanudivirus*, *Deltanudivirus*) and unofficial (*Epsilonnudivirus*) genera as well as the related genus *Bracoviriform* (*Polydnaviriformidae*). Virus names in bold indicate official recognition by the International Committee on Taxonomy of Viruses (ICTV) with respective virus species names: DiNV, *Alphanudivirus droinnubilae*; ENV, *Alphanudivirus tertidromelanogasteris*; GbNV, *Alphanudivirus grybimaculati*; KV (*Alphanudivirus dromelanogasteris*); MNV (*Alphanudivirus quartudromelanogasteris*); OrNV (*Alphanudivirus oryrhinocerotis*); TNV (*Alphanudivirus alterdromelanogasteris*); HzNV-1/HzNV-2 (*Betanudivirus hezeae*); CmNV (*Gammanudivirus cameanadis*); CcNV (*Gammanudivirus cracrangonis*) is officially a gammanudivirus, but phylogenetic analyses support its assignment to *Epsilonnudivirus* (blue arrow); HgNV (*Gammanudivirus hogammari*); PmNV (*Gammanudivirus pemonodonis*); ToNV (*Deltanudivirus tipoleraceae*). (*) Penaeus vannamei nucleopolyhedrovirus (PvNPV) is currently assigned to the *Baculoviridae*, but shows clear phylogenetic relationship to the *Nudiviridae*. References for the respective virus taxa can be found in File S2 in the supplemental information online. Abbreviation: PCG, protein-coding gene.
entire collection of expressed genes for Culex pipiens nudivirus (CpNV). Transcriptomic data
from three amphipods and three decapods also revealed new nudivirus-like sequences (File
S2). We name these respectively, as: Gammarus pulex nudivirus (GpNV); Gammarus
fossarum nudivirus (GfNV); Echinogammarus berilloni nudivirus (EbNV); Astacus
leptodactylus nudivirus (AINV); Eriocheir sinensis nudivirus (EsNV) [24] and Charybdis
nudivirus (CharyNV). These encode 62–96 proteins.

Extensive nudivirus phylogeny leads to new evolutionary perspectives

Complete virus genomes are necessary for formal taxonomic inclusion by the International Com-
mittee on Taxonomy of Viruses (ICTV); however, most studies apply just the conserved genes in
their phylogenetic analyses [6,49]. By incorporating the core genes of incomplete viral genomes
(or transcripts), we can achieve identical high-level taxonomic accuracy (Figure 2). We included
16 novel nudivirus isolates in our phylogenomic analysis, providing the first phylogeny of nearly
50 nudiviruses.

Our phylogeny supports the current genus demarcation (*Alphanudivirus* to *Deltanudivirus*) and empha-
sizes the proposed Epsilonnudivirus genus as a monophyletic group through the addition of GpNV,
GfNV, and EbNV, as well as the most recently characterized crayfish nudiviruses Faxonius rusticus
nudivirus (FrNV), Faxonius propinquus nudivirus (FpNV), and Faxonius virilis nudivirus (FvNV) [60]
(Figure 2). The new decapod nudiviruses (AINV, EsNV, and CharyNV) cluster with the other decapod
nudiviruses in the genus *Gammanudivirus*. The fundamental monophyletic groups of the nudivirus
phylogeny remain stable and well supported, suggesting that the existing branches of the *Nudiviridae*
family are resiliently defined using core proteins [5].

The CoNV isolates and CpNV group in the genus *Alphanudivirus*, which adds fleas and mosquitos to the already broad insect host range of this genus (Figures 1 and 2). The louse-infecting nudiviruses share a most recent common ancestor (MRCA) with the beta-, gamma-, delta- and epsilonudiviruses, but branch off as a distinct clade that could be considered to form a novel genus in the proposed sub-family Betanudivirinae. The nudiviruses from amblyceran lice (FrpNV, FrrNV, MpNV) form their own monophyletic group, except for HxsNV, which clusters as a related group with four nudiviruses isolated from ischnoceran lice (CafNV, EcNV, GINV, LpNV).

The core **gene synteny** of alphanudiviruses is generally poorly conserved, with the exception of certain monophyletic groups, such as the *Drosophila*-infecting nudiviruses Esparto virus (ENV), Kallithea virus (KV), Drosophila innubila nudivirus (DiNV), and Mauternbach virus (MNV) (Figure 2). Nudiviral taxonomy must become united to determine whether gene synteny should be considered an appropriate taxonomic criterion, where in this case, the large genus *Alphanudivirus* may be broken into multiple additional genera. Accordingly, the core gene organisation within other genera was more congruent, with less drastic rearrangements, except for the expanded group of Epsilonnudivirus members, which shows ~five gene rearrangements (Figure 2). A similar observation could be made for the two monophyletic groups of louse-infecting nudiviruses (Figures 2 and 3). The group comprised of HxsNV, CafNV, EcNV, GINV, and LpNV shows nearly identical core gene synteny, though a slight gene shift can be observed in HxsNV due to multiple *pit-5* duplications. FrpNV and FrrNV share highly similar core gene synteny, while MpNV is most distinct in its gene organisation, relative to the other louse-infecting nudiviruses, perhaps indicating that MpNV may belong to a third clade of louse nudiviruses. Due to their monophyletic lineages and major differences in gene synteny (Figure 3B, C), the groups housing the louse nudiviruses might be considered as two novel genera for ICTV consideration, following the Greek alphabet – Zetanudivirus (FrpNV, FrrNV, MpNV) and Etanudivirus (HxsNV, CafNV, EcNV, GINV, LpNV).
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Phylogenetic tree with 49 nudiviruses, three bracoviruses, and the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as outgroup, coupled with intragenus core gene synteny compared among nudiviruses with complete genomes. The amino acid sequences of 17 nudiviral core genes were aligned with the MAFFT alignment tool (v7.490) of Geneious (v2023.2.1). Best-fit substitution models were determined separately for each core gene based on the Bayesian information criterion (BIC) generated in IQ-Tree [111] (File S2 in the supplemental information online). The multi-protein alignment file, containing 17 906 total sites, and the model information were subjected to maximum-likelihood (ML) phylogenetic analysis with 1000 bootstrap replicates. The inferred treefile was visualized with the online tool iTOL [112]. Percentage values of bootstrap supports are indicated as coloured circles. On the right, linear synteny plots generated with the R packages ggplot2 (v3.4.4) and gggenes (v0.5.1)</figcaption></figure>

(Figure legend continued at the bottom of the next page.)
Molecular dating tree – nudiviral paleovirology

Using the available molecular information, we performed a **molecular dating** analysis with conserved nudiviral core genes from 49 nuviruses, three bracoviruses, seven baculoviruses, and the hytrosavirus Musca domestica Salivary Gland Hypertrophy Virus (MdSGHV), to estimate the ages of viral evolutionary events from a **paleovirological** perspective. A timedated tree was generated via the RelTime Branch-Lengths method implemented in MEGA11 [61]. The RelTime method is a non-Bayesian approach that operates on a relative rate framework (RRF), computing branch-specific relative rates directly from the branch lengths of inferred non-clock trees, while relaxing the molecular clock throughout the tree [62]. In contrast to Bayesian methods, RelTime does not require a tree prior and a clock model as parameters, and calculates confidence intervals (CIs) of divergence times using the Tao *et al.* method [63]. Next to the non-clock tree, a single calibration constraint of 103.38 ± 4.41 million years ago (Mya) (red-circled Node 15; Figure 4A and Table 1) was used as input under a log-normal distribution, representing the MRCA of Chelonus inanitus bracovirus (CiBV) and Cotesia congregata bracovirus (CcBV) [64]. We accounted for the possibility of strongly divergent evolutionary rates of the endogenized bracoviruses compared with the exogenous nuviruses by performing an evolutionary rate analysis with HyPhy (v2.5.61) [65] under the Fixed Effects Likelihood (FEL) model [66]. The analysis of eight nudiviral core genes conserved among four bracoviruses and 32 exogenous nuviruses resulted in ratios of non-synonymous to synonymous substitutions (dN/dS) <1, which indicates a strong negative selection across all nudiviral core genes (File S2 in supplemental information online). The bracoviral genes encoding virion components displayed higher evolutionary rates (dN/dS: 0.133–0.315) than those of exogenous nuviruses (dN/dS: 0.048–0.118), which is congruent with findings from a previous study [67], while the transcription-associated genes evolve at more similar rates in both bracoviruses and nuviruses. Despite their endogenization, the nudiviral core genes of bracoviruses appear to have a more relaxed mutation rate than free-living nuviruses and a possible underestimation of the age estimates in the molecular dating analysis needs to be considered.

Our molecular dating analysis provided estimates of shared evolutionary events among members of the *Nudiviridae* and *Baculoviridae*. The MRCA of all nuviruses, including the bracoviruses (~280 Mya; the Paleozoic Era, Permian) traces further back than the MRCA of baculoviruses (~222 Mya; the Mesozoic Era, Triassic), which is coherent with an earlier

<footnote>show core gene (coloured arrows) rearrangements compared among nuviruses of the same genus in phylogenetic order. A scale bar (base-pairs) is presented at the top and bottom of the tree. Coloured lines between arrows connect homologous core genes of phylogenetic neighbors. Each dual synteny plot starts with the DNA polymerase of the respective virus. Virus species with superscript letters were not included in the gene synteny plot, either because they originate from transcriptomic data (T), are missing accurate genomes (M), or are endogenous (E). The original phylogenetic tree with node supports and additional references can be found in File S2, and accession numbers of genes used from each virus are included in File S1 in the supplemental information online. Abbreviations of virus names: AINV, Astacus leptodactylus nudivirus; AmNV, Apis mellifera nudivirus; ApNV, Aratus pisonii nudivirus; CafNV, Cuculoecus africanus nudivirus; CcBV, Cotesia congregata bracovirus; CcNV, Crangon crangon nudivirus; CharyNV, Charybdis nudivirus; CiBV, Chelonus inanitus bracovirus; CmNV, Carcinus maenas nudivirus; CoNV, Ctenocephalides orientis nudivirus; CsNV, Callinectes sapidus nudivirus; DhNV, Dikerogammarus haemobaphes nudivirus; DINV, Drosophila innubila nudivirus; EbNV, Echinogammarus berilloni nudivirus; EcNV, Echinophilopterus claytoni nudivirus; ENV, Esparto virus; EsNV, Eriocheir sinensis nudivirus; FaENV, Fopius arisanus endogenous nudivirus; FpNV, Faxonius propinquus nudivirus; FrNV, Faxonius rusticus nudivirus; FrpNV, Franciscoloa pallida nudivirus; FrrNV, Franciscoloa roseicapillae nudivirus; FvNV, Faxonius virilis nudivirus; GbNV, Gryllus bimaculatus nudivirus; GfNV, Gammarus fossarum nudivirus; GINV, Goniodes lagopi nudivirus; GpNV, Gammarus pulex nudivirus; HgNV, Homarus gammarus nudivirus; HxsNV, Heterodoxus spiniger nudivirus; HzNV1, Heliothis zea nudivirus 1; HzNV2, Helicoverpa zea nudivirus 2; KV, Kallithea virus; LpNV, Lagopoecus perplexus nudivirus; MdBV, Microplitis demolitor bracovirus; MmNV, Menippe mercenaria nudivirus; MNV, Mauternbach virus; MpNV, Myrsidea ptilorhynchii nudivirus; MrNV, Macrobrachium rosenbergii nudivirus; NIENV, Nilaparvata lugens endogenous nudivirus; OrNV, Oryctes rhinoceros nudivirus; PmNV, Penaeus monodon nudivirus; PsNV, Phenacoccus solenopsis nudivirus; PvNPV, Penaeus vannamei nucleopolyhedrovirus; TNV, Tomelloso virus; ToNV, Tipula oleracea nudivirus; VcENV, Venturia canescens endogenous nudivirus.</footnote>
<figure><img src="image_4.png" /></figure>

Trends in Parasitology

Figure 3. Inter-genus synteny of nudiviral core genes among representatives of nudivirus genera. (A) Circular plot showing the core gene synteny of nudivirus representatives from the genera *Alphanudivirus* (α), *Betanudivirus* (β), *Gammanudivirus* (γ), *Deltanudivirus* (δ), *Epsilonnudivirus* (ε), and *Etanudivirus* (η) compared with *Zetanudivirus* (ζ). (B) Comparative core gene synteny of Zetanudivirus [Franciscoloa pallida nudivirus (FrpNV)] and Etanudivirus [Cuculoecus africanus nudivirus (CafNV)]. (C) Comparative core gene synteny of Zetanudivirus [Myrsidea ptilorhynchi nudivirus (MpNV)] and Etanudivirus (CafNV). The curved bars each represent the genome of the indicated nudivirus species, and are colored according to the genus they belong to. Sites with indented triangles represent the start of the genome (start codon of DNA polymerase). Gene directions are visualized as golden (positive strand) and silver (negative strand) rectangles. Ribbons represent genome connections between core gene homologs of the respectively colored nudiviruses. Distances between ticks represent 5 kb. The circular plots were generated with Circa (www.omgenomics.com/circa/).
<figure><img src="image_5.png" /></figure>

Trends in Parasitology

Figure 4. Molecular dating analysis of nudiviruses, baculoviruses, and bracoviruses in relation to evolutionary timeline of crustacean and insect host clades. (A) Molecular dating tree from double-stranded (ds)DNA viruses. Phylogenetic virus tree with optimized branch lengths served as input to compute the molecular dating tree with the MEGA11 (v11.0.10) software by using the RelTime-Branch Lengths option [61]. A single calibration constraint of 103.38 ± 4.41 million years ago (Mya) (red-circled Node 15) was specified with log-normal distribution, representing the most recent common ancestor (MRCA) of Chelonus inanitus bracovirus (CiBV) and Cotesia congregata bracovirus (CcBV) [64]. The age estimates and 95% confidence intervals (CIs) of the numbered nodes can be found in Table 1 in the main text. The original molecular dating tree from MEGA11, including error bars and additional details on the methodology, can be found in File S2 in the supplemental information online. (B) Timeline of insect and crustacean hosts. The web-based TimeTree of Life (TToL5) resource [66] was used to build the host tree by providing a text file with relevant species names. The TToL5 resource is a publicly accessible database that infers divergence times of taxa based on the median value of existing evolutionary studies. (C) Bar chart showing the non-synonymous to synonymous substitutions (dN/dS) values across eight nudiviral core genes from four bracoviruses and 32 exogenous nudiviruses. The dN/dS values were inferred from the codon-aligned nucleotide sequences with HyPhy (v2.5.61) under the Fixed Effects Likelihood (FEL) model (File S2).

evolutionary study [22]. Node 1 indicates the time at which the MRCA of both nudiviruses and baculoviruses is estimated at 378 Mya (Devonian period of the Paleozoic Era), while the divergence of holometabolous insects from polyneopteran insects is estimated at ~383 Mya
<table><caption>Table 1. Sixteen notable nodes from the molecular dating tree (see Figure 4 in the main text) together with their estimated divergence times and 95% CIs</caption><thead><tr><th>Node</th><th>Time, Mya</th><th>95% CI, Mya</th><th>MRCA of<sup>a</sup></th></tr></thead><tbody><tr><td>1</td><td>378</td><td>344–415</td><td>Baculo-, braco-, and nudiviruses</td></tr><tr><td>2</td><td>280</td><td>257–304</td><td>Braco- and nudiviruses</td></tr><tr><td>3</td><td>248</td><td>223–275</td><td>β-, γ-, δ-, ε-, ζ-, η-NVs, and bracoviruses</td></tr><tr><td>4</td><td>222</td><td>196–253</td><td>β-, γ-, δ-, ε-NVs, and bracoviruses</td></tr><tr><td>5</td><td>222</td><td>197–249</td><td>Baculoviruses</td></tr><tr><td>6</td><td>205</td><td>176–238</td><td>β-, γ-, and ε-NVs</td></tr><tr><td>7</td><td>195</td><td>167–228</td><td>δ-NVs and bracoviruses</td></tr><tr><td>8</td><td>173</td><td>148–203</td><td>α-, β-, and γ-BcVs</td></tr><tr><td>9</td><td>171</td><td>150–195</td><td>α-NVs</td></tr><tr><td>10</td><td>145</td><td>123–173</td><td>CoNV(1–3) and α-NVs excluding PsNV</td></tr><tr><td>11</td><td>143</td><td>118–172</td><td>γ- and ε-NVs</td></tr><tr><td>12</td><td>122</td><td>100–149</td><td>ε-NVs</td></tr><tr><td>13</td><td>120</td><td>104–138</td><td>ζ- and η-NVs</td></tr><tr><td>14</td><td>106</td><td>87–129</td><td>α- and β-BcVs</td></tr><tr><td>15</td><td>102</td><td>99–105</td><td>Bracoviruses</td></tr><tr><td>16</td><td>96</td><td>78–118</td><td>γ-NVs</td></tr></tbody></table>

according to TimeTree [68]. Hence, the divergence of these viral families likely happened early in insect evolution.

Another Paleozoic hallmark involves the MRCA of all nudiviruses (~280 Mya), during the Permian, where the unofficial subfamilies Alphanudivirinae (including all alphanudiviruses) and Betanudivirinae (comprising all other nudiviruses, including the lice nudiviruses) split [20] (Figure 4A). In the evolutionary history of eumetabolous insects, hemipteran and psocodean in-sects diverged approximately 9 million years before the diversification of holometabolous insects (as inferred from Figure 4B). The emergence of the two nudiviral subfamilies could be linked to the diversification of these respective insect groups, with the theory that ancestral hemipteran and psocodean insects were hosts to the ancestors of the Alphanudivirinae and Betanudivirinae, re-spectively. This assumption is supported by the observation that the hemipteran *Alphanudivirus*, *Phenacoccus solenopsis* nudivirus (PsNV), and the psocodean nudiviruses (lice-infecting) branch off at the deepest nodes of their respective subfamilial clades (Figure 4A). This is further supported by the lack of hemipteran nudiviruses outside of the Alphanudivirinae, and no psocodean nudiviruses outside of the betanudivirinae, to date. Furthermore, the mosquito-infecting CpNV shares an MRCA with the other dipteran nudiviruses (e.g., ENV) and coleopteran nudiviruses (OrNV, Oryctes rhinoceros nudivirus; DuhNV, Diabrotica undecimpunctata howardi nudivirus), suggesting that the earlier divergence of CpNV does not follow the evolutionary lineage of the dip-teran and coleopteran hosts. We do not yet have enough data on the diversity of beta- and deltanudiviruses in holometabolous insects to explore the emergence of these lineages further – one plausible option may be that these hosts (*Tipula* spp. and *Heliothis* spp.) attained these vi-ruses from other, yet unidentified, host groups.

The MRCA between the CoNV isolates (*Alphanudivirus*) and the rest of the alphanudiviruses (excluding PsNV) was estimated at 145 Mya (Node 10; 95% CI: CI lower = 123 Mya, CI upper =

<footnote><sup>a</sup>Abbreviations: α-NV, *Alphanudivirus*; β-NV, *Betanudivirus*; γ-NV, *Gammanudivirus*; δ-NV, *Deltanudivirus*; ε-NV, *Epsilonnudivirus*; ζ-NV, *Zetanudivirus*; η-NV, *Etanudivirus*; α-BcV, *Alphabaculovirus*; β-BcV, *Betabaculovirus*; γ-BcV, *Gammabaculovirus*; δ-BcV, *Deltabaculovirus*.</footnote>
173 Mya), hinting that one of the oldest ancestors of alphanudiviruses might have been associated with an ancestral flea. Amniotes emerged ~325 Mya, during their colonization of terrestrial environments [69]. This coincides with the emergence of the Siphonaptera, which emerged ~300 Mya (Figure 4B) and have been termed 'dinosaur fleas', which are thought to have switched to mammals [70]. The late Jurassic and early Cretaceous sit within the 123–173 Mya period, a time when early mammals (e.g., marsupials) evolved alongside the diversification of boreid fleas [71]. The diversification of these flea nudiviruses, within the *Alphanudivirus* genus, could plausibly have occurred alongside the evolution of mammalian flea hosts.

The MRCA of all viruses in the Betanudivirinae clade can be traced back as the third deepest node among all observed events (Node 3; 248 Mya; Figure 4A), primarily including the early branching of the lice-infecting zeta- and etanudiviruses from the rest of the Betanudivirinae (Node 3). The diversification of the amblyceran zetanudiviruses and the ischnoceran etanudiviruses (HxsNV being the amblyceran exception) from one another is estimated between 104 and 138 Mya (Node 13; 120 Mya; Figure 4A). The radiation of those two nudivirus lineages is coherent with the estimated origin of the clade Phthiraptera (115 Mya) [72] and the MRCA of its subclades Amblycera and Ischnocera between 105 and 110 Mya [73]. Suggestively, the evolutionary split of zeta- and etanudiviruses may have been driven by the diversification of the Phthiraptera clade into amblyceran and ischnoceran lice during the early-mid Cretaceous Epoch. Given the time period (early-mid Cretaceous), and the age of bird-like dinosaur evolution and diversification events (90–170 Mya) [74], this virological event could have taken place on the back of a feathered dinosaur, mimicking this same ecological system we now see in modern day birds, infested with lice, and hyperparasitized by nudiviruses.

Within the Betanudivirinae, the divergence of gammanudiviruses represents a relatively recent event in nudivirus evolutionary history. Since the crustacean diversification occurred in the Ordo-vician (~500 Mya) [68], it seems that this group may have acquired nudiviruses via a zoonotic event, possibly from an insect. Given that all crustacean nudiviruses identified to date (gamma- and epsilonnudiviruses) have a single ancestor, estimated at ~140 Mya (Node 11; late Jurassic – early Cretaceous), it seems possible that a single host-switching event resulted in the crustacean infection capability that we see today. Perhaps the tissue tropism of crustacean-infecting compared with insect-infecting nudiviruses can explain this phenomenon. Crustacean nudiviruses target the gut, as do baculoviruses, but baculoviruses and other insect-infecting nudiviruses are also able to infect other tissue types – a trait missing in the *Gammanudivirus* and *Epsilonnudivirus* members. *Gammanudivirus* and *Epsilonnudivirus* appear to be a younger lineage of the *Nudiviridae*, beginning to parasitize the ancient arthropod group, Crustacea, during the late Jurassic.

## Global distribution of nudiviruses and their hosts

When all nudiviruses are considered, their distribution extends across all continents (Figure 5, Key figure). Marine, freshwater, and terrestrial hosts harbor nudiviruses – it seems that if insects or crustaceans occupy an area, nudiviruses likely occupy it as well. For insects, their environmental and ecological relationships are primarily associated with terrestrial systems, to a lesser extent freshwater systems, since we did not identify any nudiviruses from marine mammal-infesting insects. However, a handful of nudiviruses have been isolated from insects with a freshwater life stage (i.e., mosquitos), as well as a putative virus with nudivirus-like pathology from a water beetle (*G. natator*) [29]. Regarding terrestrial climatic zones, our findings complement the presence of nudiviruses across known biotopes (e.g., tropical: EcNV, FrpNV, FrrNV, MpNV; subequatorial: CpNV, HxsNV; equatorial: CafNV, CoNV), but also expand their habitat to the yet unrecognized sub-polar zones, with LpNV and GINV from Alaska.
Key figure

World map of confirmed and putative locations where nudivirus have been identified

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Coordinates (latitude and longitude) of nudivirus-related find spots were used as input for the R packages maps (v3.4.2) and ggplot2 (v3.4.4) with support by the packages dplyr (v1.1.4) and ggpubr (v0.6.0). (A) Differently coloured shapes represent already identified nuviruses with fully sequenced genomes, or putative yet uncharacterized nuviruses based on pathological (P) or transcriptomic implications (T) from literature and personal data. (B) Newly characterized nuviruses found in mosquito and ectoparasitic insects (flea and lice). Nudivirus names are shown at the top with their respectively coloured insect hosts below (separated by broken lines). For the lice and flea, the warm-blooded animals that those ectoparasitic insects infest are displayed at the bottom (left to right): Eolophus roseicapilla, Centropus phasianinus, Ptilonorhynchus violaceus, Canis lupus, Chrysococcyx cupreus, Northiella haematogaster, Lagopus lagopus/muta, Tympanuchus phasianellus, Canis familiaris. The list of nuviruses and their coordinates can be found in File S1 in the supplemental information online. Abbreviations: CafNV, Cuculoecus africanus nudivirus; CoNV, Ctenocephalides orientis nudivirus; CpNV, Culex pipiens nudivirus; EcNV, Echinophilopterus claytoni nudivirus; GINV, Goniodes lagopi nudivirus; FrpNV, Franciscoloa pallida nudivirus; FrrNV2, Franciscoloa roseicapillae nudivirus 2; LpNV, Lagopoecus perplexus nudivirus; MpNV, Myrsidea ptilorhynchii nudivirus; HxsNV, Heterodoxus spiniger nudivirus.</figcaption></figure>

Insect hosts of nuviruses from across Asia include fleas (C. orientis) from infested dogs in Perak (Malaysia) [51]; the hemipteran, *A. psidii*, which infested guava trees (*Psidium guajava*) in Xinyang, China [54]; *H. spiniger* lice from wolves in India; and *C. pipiens* and *P. chinensis* from China [52,56]. Siphonaptera, Hemiptera, and Diptera samples had been collected across both Asia and Europe, while the lice hosts (*Psocodea*) cover a broader continental range. Most lice samples
were gathered from bird hosts in New South Wales and Queensland (Australia; *F. pallida*, *F. roseicapillae*, *M. ptilorhynchi*, *E. claytoni*), while *L. perplexus* and *G. lagopi* were sampled in Alaska (North America), and another (*C. africanus*) in Ghana (Africa; Figure 5B). Given the ability of **ectoparasites** to traverse diverse environments by hitchhiking on the animals on which they feed (including birds), the widespread distribution of nuviruses could be explained by migratory birds through evolutionary history.

In addition to the geographical dispersal, the observed nuvirial host diversity highlights further layers of ecological complexity (Figures 5 and 6). Next to their importance as pathogens of commercially important arthropods, and as biocontrol agents against certain insect pests (i.e., OrNV [75]; bracoviruses [76]), their presence in **haematophagous** (blood-feeding) insects emphasizes a yet undescribed ecological niche this virus family fills. The eight psocodean species, the mosquitoes *C. orientis* and *C. pipiens*, as well as the sandfly *P. chinensis* all feed on the host tissue or blood of endothermic animals, including humans. However, *C. pipiens* and *P. chinensis* can

<figure><img src="image_7.png" /></figure>

Trends in Parasitology

Figure 6. A Sankey diagram, elaborating on the host diversity, environmental diversity, host diet, ecological role, and potential impact associated with nuviruses and their hosts (Crustacea and Hexapoda). The numbers indicate how many nuviruses are associated with the respective trait. The noted haematophagous lice (Amblycera) also consume host skin and feathers (mallophagy), in contrast to the exclusively mallophagous ischnoceran lice. The Sankey diagram was generated with the web-based online tool (sankeymatic.com), supported by File S1 in the supplemental information online.
complete their life cycles without the need of hematophagy [77,78], while obligate ectoparasites such as lice and fleas spend most of their lives on the hosts from which they feed [79,80]. The proximate interaction between ectoparasites and their hosts are usually accompanied by a long history of coevolutionary adaptations [81,82]. The discovery of these large dsDNA viruses in lice and fleas introduces a yet undescribed trophic level to these ectoparasite-host systems. Since viruses are intracellular parasites [83], one may consider this tripartite relationship a form of hyperparasitism [84–86] or, at least, showcase another parasitic strategy that members of the *Nudiviridae* have converged upon [87]. Another tripartite model is known from parasitoid wasps and their endogenized bracoviruses, whose nudiviral ancestor integrated into an ancestral wasp genome ~100 Mya and coevolved with the wasp to become a domesticated mutualist [88]. Our findings emphasize an expanding societal relevance of nudiviruses that might eventually reach beyond applications in biocontrol and arthropod farming, to the sectors of healthcare and veterinary medicine (Figure 6). Where phage therapy is used to control bacterial infection, perhaps ‘nuditherapy’ might help animals to get rid of lice and flea pests in the future, providing that empirical data are obtained one day to show these newly discovered nudiviruses are pathogenic to their insect hosts.

Crustacean-infecting nudiviruses were found across marine and freshwater systems of China, Australia, Europe, inland USA, the Atlantic and Pacific coasts of Canada and the USA, as well as the Gulf of Mexico (Figure 5). The host diversity of these groups includes Decapoda and Amphipoda; however, all these hosts are omnivores (primarily detritivorous) in their freshwater, euryhaline, or marine habitat (Figure 6). Five crustacean hosts of nudiviruses are commonly present in fisheries, and three are involved in aquaculture efforts, highlighting the importance of understanding the interactions between crustacean and virus, including their economic impact (Figure 6). Examples include *C. sapidus* ('blue crab fishery') [89,90]; *Homarus gammarus*, where HgNV has only been identified from hatcheries to date [91]; and the more intensive *Penaeus monodon* aquaculture systems across Asia [92], where PmNV causes 'wasting disease', lowering yield via a reduction in juvenile shrimp growth [93]. The remaining ten nudiviruses have been sampled from wild environments, where some are native components of co-evolved ecosystems [45,94], while other nudiviruses infect invasive species [17,19].

Ecologically, the ‘wild’ crustaceans that host nudiviruses all form part of the aquatic benthos. For example, amphipods are considered important nutrient recyclers [95] and the mangrove crab, *Aratus pisonii*, is an essential part of mangrove ecosystems as a detritivorous and semi-terrestrial species [96]. The four invasive non-native species that harbor nudiviruses include: the globally invasive *Carcinus maenas* (aka. invasive green crab), *Dikerogammarus haemobaphes* (aka. the demon shrimp), and the invasive crayfish *Faxonius propinquus* and *Faxonius rusticus* [60,97–99]. Some of the fishery and aquaculture species are also considered invasive species in certain locations, such as *C. sapidus* in Europe [100]; however, nudiviruses have not been screened-for in these invasive populations. The nudiviruses in invasive crustacean populations have the potential to increase in prevalence and they may to some extent modify host behavior [98,101]; however, there remains a need for broader ecological studies to untangle how these viruses might influence biological invasions – that is, by potential transmission of nudiviruses to native crustacean populations, comparable with what happened when gray squirrels introduced squirrelpox to red squirrel populations [102]. Finally, there is one putative crustacean host with a parasitic lifestyle. The pea crab, *P. pisum*, parasitizes mussels, and specimens with a histologically demonstrated nudivirus infection were collected from the south coast of the UK [35]. However, no genomic data have been collected to confirm the relationship with the *Nudiviridae*.

Overall, a reasonable diversity of known nudiviruses are associated with facultative or obligate ectoparasitic/parasitoid hosts – fleas, lice, wasps, mosquito, pea crabs. Most terrestrial
nuviruses have been detected in insect hosts involved with agricultural systems, but their occurrence is likely much broader, whereas several crustacean hosts are associated with fisheries and aquaculture. For the majority, these viruses sit within insect/crustacean taxa with an herbivorous or omnivorous lifestyle. The ecological role of the latter group is often prey species, or have a role in nutrient recycling.

## Concluding remarks

Histology and electron microscopy identified the first nuviruses, and now genomic data provide us with a vast resource of nudivirus diversity across invertebrate hosts. The complete genomes of eight nuviruses (CafNV, CoNV, EcNV, FrpNV, FrrNV, HxsNV, LpNV, MpNV) were hidden in cyberspace from pre-published studies, while an additional eight nuviruses were identified as partial gene sets or from transcriptomic data (AINV, CharyNV, CpNV, EbNV, EsNV, GINV, GpNV, GfNV). Five other resources hint at the presence of putative nuviruses, but with insufficient assembly recovery (File S2). These new nuviruses, coupled with the isolates sequenced between 2007 and 2023 [11,17,19,20,31,49,60,91,93,94,103], have allowed for a new taxonomic perspective, suggesting two new genera ('Etanuvirus' and 'Zetanuvirus') to house nuviruses from ectoparasitic lice. Nuviruses first emerged around 280 Mya – crustacean nudivirus infections may have once been considered the older lineage; however, they appear to have the youngest evolutionary history. Insect-infecting nuviruses appear to be the first to emerge, including their potential evolution and diversification in lice on early birds in the Cretaceous period (see Outstanding questions). Future studies are required to address questions regarding the morphology and pathology of these new nuviruses, which may in turn reveal their ecological importance and new prospects for biocontrol of deleterious mammalian and avian ectoparasites, where chemical resistance is common [104] (see Outstanding questions). Our findings merely scratch the surface, but we are hopeful that our study will act as a steppingstone for forthcoming exploration into nudiviral taxonomic reasoning to develop delineation criteria and explore further nudivirus diversity across the Arthropoda.

## Acknowledgments

The writing of this article has received funding from the European Union's Horizon 2020 research and innovation program INSECT DOCTORS under the Marie Skłodowska-Curie grant agreement No. 859850. J.B. and A.L.B. would like to thank Teesside University for PhD funding to support A.L.B. We would like to thank *The Journal of Structural Biology* and *The Journal of Virology* for their approval to reproduce pre-published images. J.M.P. would like to thank the Wageningen Electron Microscopy Centre for the help in obtaining the negatively stained micrograph of HzNV-1.

## Declaration of interests

All authors declare no conflict of interests.

## Supplemental information

Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.pt.2024.06.009.

## References

1. Koonin, E.V. et al. (2020) Global organization and proposed megataonomy of the virus world. *Microbiol. Mol. Biol. Rev.* 84, e00061-19

2. Van Oers, M.M. et al. (2023) Correction to: developments in the classification and nomenclature of arthropod-infecting large DNA viruses that contain pi genes. *Arch. Virol.* 168, 255

3. Wang, H.-C. et al. (2019) ICTV virus taxonomy profile: Nimaviridae. *J. Gen. Virol.* 100, 1053-1054

4. Guinet, B. et al. (2024) A novel and diverse family of filamentous DNA viruses associated with parasitic wasps. *Virus Evol.* 10, veae022

5. Harrison, R.L. et al. (2020) ICTV virus taxonomy profile: Nudiviridae. *J. Gen. Virol.* 101, 3-4

6. Petersen, J.M. et al. (2022) The naked truth: an updated review on nuviruses and their relationship to bracoviruses and baculoviruses. *J. Invertebr. Pathol.* 189, 1077-1100

7. Hegna, T.A. et al. (2020) The fossil record of the Pancrustacea. *Nat. History Crustacea* 8, 21-52

8. Anderson, I. and Prior, H. (1992) Baculovirus infections in the mud crab, *Scylla serrata*, and a freshwater crayfish, *Cherax quadricarinatus*, from Australia. *J. Invertebr. Pathol.* 60, 265-273

## Outstanding questions

Several examples of 'baculoviruses' have been pathologically defined in arthropods, but will these cases become identifiable as nuviruses once genomic sequencing is conducted?

The ectoparasitic hosts of nuviruses have chewing mouthparts (lice) or piercing mouthparts (flea) to feed on their warm-blooded hosts. Is it possible that nuviruses of ectoparasites are orally transmitted like the majority of known nuviruses, or might they be sexually transmitted like the unconventional betanuviruses, or follow a yet undescribed transmission route, for example, by circulating in blood-streams from which they can be taken up again by other ectoparasites?

If the nuviruses of fleas and lice are as detrimental to their ectoparasitic hosts as other members of *Nudiviridae*, what potential value could they hold for ecological associations and for biological control of ectoparasitic infestations?

Our phylogenomic review highlights that novel taxonomic rankings may be needed to better explore diversity in the *Nudiviridae*. Does the presence of these new taxa indicate novel features and patterns that could promote feasible delineation criteria for (re)defining nudivirus species?

Are the evolutionary pressures guiding nudivirus evolution greater governed by biotic (e.g., trophic, host biology) or abiotic (e.g., environmental) factors?

We identify that the Crustacea appear likely to have acquired nuviruses from insect hosts. Is it possible that other insectivorous arthropods (i.e., the Arachnida) might also house nuviruses, which have jumped across host phyla?
9. Bonami, J.-R. et al. (1997) The polyhedra of the occluded baculoviruses of marine decapod crustacea: a unique structure, crystal organization, and proposed model. *J. Struct. Biol.* 120, 134–145

10. Groff, J. et al. (1993) Detection of a nonoccluded baculovirus in the freshwater crayfish *Cherax quadricarinatus* in North America. *J. Aquat. Anim. Health* 5, 275–279

11. Bojko, J. et al. (2022) Rediscovering 'Baculovirus-A' (Johnson, 1976): the complete genome of 'Callinectes sapidus' nudivirus'. *J. Invertebr. Pathol.* 194, 107822

12. Boucias, D. et al. (1989) Characterization of a non-occluded baculovirus (subgroup C) from the field cricket, *Gryllus rubens*. *Arch. Virol.* 106, 93–102

13. Crawford, A.M. and Sheehan, C. (1985) Replication of *Oryctes* baculovirus in cell culture: viral morphogenesis, infectivity and protein synthesis. *J. Gen. Virol.* 66, 529–539

14. Kelly, D. et al. (1981) Induction of a nonoccluded baculovirus persistently infecting *Heliothis zea* cells by *Heliothis armigera* and *Trichoplusia ni* nuclear polyhedrosis viruses. *Virology* 112, 174–189

15. Wang, Y. and Jehle, J.A. (2009) Nudiviruses and other large, double-stranded circular DNA viruses of invertebrates: new insights on an old topic. *J. Invertebr. Pathol.* 101, 187–193

16. Bateman, K. and Stentiford, G. (2008) Cancer pagurus bacilliform virus (CpBV) infecting juvenile European edible crabs *C. pagurus* from UK waters. *Dis. Aquat. Org.* 79, 147–151

17. Bateman, K.S. et al. (2021) Identification and full characterization of two novel crustacean infecting members of the family Nudiviridae provides support for two subfamilies. *Viruses* 13, 1694

18. Ros, V.I.D. (2020) Baculoviruses: general features (*Baculoviridae*). In Reference Module in Life Sciences (Zuckerman, M. and Bamford, D.H., eds), pp. 739–746, Elsevier

19. Allain, T.W. et al. (2020) A novel nudivirus infecting the invasive demon shrimp *Dikerogammarus haemobaphes* (Amphipoda). *Sci. Rep.* 10, 14816

20. Liu, S. et al. (2021) Nudivirus sequences identified from the Southern and Western corn rootworms (Coleoptera: Chrysomelidae). *Viruses* 13, 269

21. Bézier, A. et al. (2009) Polydnavirus hidden face: the genes producing virus particles of parasitic wasps. *J. Invertebr. Pathol.* 101, 194–203

22. Thézé, J. et al. (2011) Paleozoic origin of insect large dsDNA viruses. *Proc. Natl. Acad. Sci. U. S. A.* 108, 15931–15935

23. Porter, A.F. et al. (2019) Diversity and evolution of novel invertebrate DNA viruses revealed by meta-transcriptomics. *Viruses* 11, 1092

24. Shen, Z. et al. (2021) Metatranscriptomic analysis reveals an imbalance of hepatopancreatic flora of Chinese mitten crab *Eriocheir sinensis* with hepatopancreatic necrosis disease. *Biology* 10, 462

25. Leinonen, R. et al. (2010) The sequence read archive. *Nucleic Acids Res.* 39, D19–D21

26. Cheng, R.-L. et al. (2014) Brown planthopper nudivirus DNA integrated in its host genome. *J. Virol.* 88, 5310–5318

27. Liu, S. et al. (2020) Endogenous viral elements integrated into the genome of the soybean aphid, *Aphis glycines*. *Insect Biochem. Mol. Biol.* 123, 103405

28. Avery, S. et al. (1977) Virus-like particles in a fire ant, *Solenopsis* sp., (Hymenoptera: Formicidae) from Brazil. *Florida Ent.* 60, 17–20

29. Gouranton, J. (1972) Development of an intranuclear nonoccluded rod-shaped virus in some midgut cells of an adult insect, *Gyrinus natator* L. (Coleoptera). *J. Ultrastruct. Res.* 39, 281–294

30. Smith, K.M. and Xeros, N. (1954) An unusual virus disease of a dipterous larva. *Nature* 173, 866–867

31. Bézier, A. et al. (2015) The genome of the nucleopolyhedrosis-causing virus from *Tipula oleracea* sheds new light on the Nudiviridae family. *J. Virol.* 89, 3008–3025

32. Pappalardo, R. et al. (1986) T(tau) virus infection of *Carcinus mediterraneus*: histology, cytopathology, and experimental transmission of the disease. *J. Invertebr. Pathol.* 47, 361–368

33. Johnson, P.T. (1988) The rod-shaped nuclear viruses of crustaceans: gut-infecting species. *Dis. Aquat. Org.* 4, 123–141

34. Owens, L. et al. (2010) Intranuclear bacilliform virus and hepatopancreatic parvovirus (PmergDNV) in the mud crab *Scylla serrata* (Forskal) of Australia. *Aquaculture* 310, 47–51

35. Longshaw, M. et al. (2012) Parasites and pathogens of the endosymbiotic pea crab (*Pinnotheres pisum*) from blue mussels (*Mytilus edulis*) in England. *J. Invertebr. Pathol.* 109, 235–242

36. Edgerton, B.F. et al. (2004) Understanding the causes of disease in European freshwater crayfish. *Conserv. Biol.* 18, 1466–1474

37. Edgerton, B. and Owens, L. (1997) Age at first infection of *Cherax quadricarinatus* by *Cherax quadricarinatus* bacilliform virus and *Cherax giardiavirus*-like virus, and production of putative virus-free crayfish. *Aquaculture* 152, 1–12

38. Anderson, L.G. et al. (2021) Patterns of infection in a native and an invasive crayfish across the UK. *J. Invertebr. Pathol.* 184, 107595

39. Edgerton, B. et al. (1996) Description of a bacilliform virus from the freshwater crayfish, *Astacus astacus*. *J. Invertebr. Pathol.* 68, 187–190

40. Edgerton, B. et al. (2002) An intranuclear bacilliform virus associated with near extirpation of *Austropotamobius pallipes* Lereboulet from the Nant watershed in Ardèche, France. *J. Fish Dis.* 25, 523–531

41. Bateman, K. and Stentiford, G. (2017) A taxonomic review of viruses infecting crustaceans with an emphasis on wild hosts. *J. Invertebr. Pathol.* 147, 86–110

42. Takahashi, Y. et al. (1994) Electron microscopic evidence of bacilliform virus infection in kuruma shrimp (*Penaeus japonicus*). *Fish Pathol.* 29, 121–125

43. Couch, J.A. (1974) An enzootic nuclear polyhedrosis virus of pink shrimp: ultrastructure, prevalence, and enhancement. *J. Invertebr. Pathol.* 24, 311–331

44. Lester, R. et al. (1987) Light and electron microscope evidence of baculovirus infection in the prawn *Penaeus plebejus*. *Dis. Aquat. Org.* 3, 217–219

45. Bojko, J. and Ovcharenko, M. (2019) Pathogens and other symbionts of the Amphipoda: taxonomic diversity and pathological significance. *Dis. Aquat. Org.* 136, 3–36

46. Bojko, J. et al. (2013) Baseline histopathological survey of a recently invading island population of 'killer shrimp', *Dikerogammarus villosus*. *Dis. Aquat. Org.* 106, 241–253

47. Warren, D.A. et al. (2022) Histopathological survey for parasite groups in *Gammarus varsoviensis* (Amphipoda). *Dis. Aquat. Org.* 149, 47–51

48. Warren, D.A. et al. (2023) Histopathological screening of *Pontogammarus robustoides* (Amphipoda), an invader on route to the United Kingdom. *J. Invertebr. Pathol.* 200, 107970

49. Bojko, J. et al. (2023) A new member of the Nudiviridae from the Florida stone crab (*Menippe mercenaria*). *Virology* 588, 109910

50. Sweet, A.D. et al. (2020) Lousy grouse: comparing evolutionary patterns in Alaska galliform lice to understand host evolution and host-parasite interactions. *Ecol. Evol.* 10, 8379–8393

51. Beliavskaia, A. et al. (2023) Metagenomics of culture isolates and insect tissue illuminate the evolution of *Wolbachia*, *Rickettsia* and *Bartonella* symbionts in *Ctenocephalides* spp. fleas. *Microb. Genom.* 9, mgen001045

52. Feng, Y. et al. (2022) A time-series meta-transcriptomic analysis reveals the seasonal, host, and gender structure of mosquito viromes. *Virus Evol.* 8, vacc006

53. Tom, M. et al. (2013) Crustacean oxi-reductases protein sequences derived from a functional genomic project potentially involved in ecdysteroid hormones metabolism – a starting point for function examination. *Gen. Comp. Endocrinol.* 194, 71–80

54. Song, N. et al. (2022) The mitogenome of *Aleuroclava psidii* (Singh, 1931) (Hemiptera: Aleyrodidae) and increased number of mitochondrial gene rearrangements in whiteflies. *Front. Biosci. Landmark* 27, 154

55. Sana, S. et al. (2023) De novo transcriptome assemblies of five major European oilseed rape insect pests. *BMC Genom. Data* 24, 15

56. Wang, J. et al. (2022) Total RNA sequencing of *Phlebotomus chinensis* sandflies in China revealed viral, bacterial, and
eukaryotic microbes potentially pathogenic to humans. *Emerg. Microbes Infect.* 11, 2080–2092

57. Käfer, S. et al. (2019) Re-assessing the diversity of negative strand RNA viruses in insects. *PLoS Pathog.* 15, e1008224

58. Misof, B. et al. (2014) Phylogenomics resolves the timing and pattern of insect evolution. *Science* 346, 763–767

59. Pérez-Moreno, J.L. et al. (2018) Transcriptomic insights into the loss of vision in Molinár János Cave's crustaceans. *Integr. Comp. Biol.* 58, 452–464

60. Stratton, C.E. et al. (2024) North American crayfish harbour diverse members of the Nudiviridae. *SSRN*, Published online April 12, 2024. https://doi.org/10.2139/ssrn.4790364

61. Tamura, K. et al. (2021) MEGA11: molecular evolutionary genetics analysis version 11. *Mol. Biol. Evol.* 38, 3022–3027

62. Tamura, K. et al. (2018) Theoretical foundation of the RelTime method for estimating divergence times from variable evolutionary rates. *Mol. Biol. Evol.* 35, 1770–1782

63. Tao, Q. et al. (2020) Reliable confidence intervals for RelTime estimates of evolutionary divergence times. *Mol. Biol. Evol.* 37, 280–290

64. Murphy, N. et al. (2008) Phylogeny of the parasitic microgastroid subfamilies (Hymenoptera: Braconidae) based on sequence data from seven genes, with an improved time estimate of the origin of the lineage. *Mol. Phylogenet. Evol.* 47, 378–395

65. Kosakovsky Pond, S.L. et al. (2020) HyPhy 2.5 – a customizable platform for evolutionary hypothesis testing using phylogenies. *Mol. Biol. Evol.* 37, 295–299

66. Kosakovsky Pond, S.L. and Frost, S.D. (2005) Not so different after all: a comparison of methods for detecting amino acid sites under selection. *Mol. Biol. Evol.* 22, 1208–1222

67. Herniou, E.A. et al. (2013) When parasitic wasps hijacked viruses: genomic and functional evolution of polydnaviruses. *Philos. Trans. R. Soc. B, Biol. Sci.* 368, 20130051

68. Kumar, S. et al. (2022) TimeTree 5: an expanded resource for species divergence times. *Mol. Biol. Evol.* 39, msac174

69. Brocklehurst, N. and Benson, R.J. (2021) Multiple paths to morphological diversification during the origin of amniotes. *Nat. Ecol. Evol.* 5, 1243–1249

70. Watson, V. and Rothschild, B. (2021) Deep origin of parasitic disease in vertebrates. In *The Evolution and Fossil Record of Parasitism: Coevolution and Paleoparasitological Techniques* (De Baets, K. and Huntley, J.W., eds), pp. 317–358, Springer

71. Zhu, Q. et al. (2015) Fleas (Siphonaptera) are Cretaceous, and evolved with Theria. *Mol. Phylogenet. Evol.* 90, 129–139

72. De Moya, R.S. et al. (2021) Phylogenomics of parasitic and nonparasitic lice (Insecta: Psocodea): combining sequence data and exploring compositional bias solutions in next generation data sets. *Syst. Biol.* 70, 719–738

73. Zhang, Y. et al. (2024) Stem chewing lice on Cretaceous feathers preserved in amber. *Curr. Biol.* 34, P916–922 E1

74. Yu, Y. et al. (2021) Deep time diversity and the early radiations of birds. *Proc. Natl. Acad. Sci.* 118, e2019865118

75. Etebari, K. et al. (2020) Transcription profile and genomic variations of *Oryctes rhinoceros* nudivirus in coconut rhinoceros beetles. *J. Virol.* 94, e01097–20

76. Cusumano, A. and Volkoff, A.-N. (2021) Influence of parasitoid-associated viral symbionts on plant-insect interactions and biological control. *Curr. Opin. Insect Sci.* 44, 64–71

77. Haba, Y. and Mcbride, L. (2022) Origin and status of *Culex pipiens* mosquito ecotypes. *Curr. Biol.* 32, R237–R246

78. Safyanova, V. (1964) Laboratory cultivation of sandflies (Diptera: Phlebotominae). *Bull. World Health Organ.* 31, 573

79. Ahmad, A. et al. (2021) Insecta: Phthiraptera. In *Faunal Diversity of Biogeographic Zones of India: North-East* (Chandra, K. and Raghunathan, C. and Kosygin, L. and Gupta, D., eds), pp. 271–280, Zoological Survey of India

80. Zhang, Y. et al. (2022) Mitochondrial phylogenomics provides insights into the taxonomy and phylogeny of fleas. *Parasit. Vectors* 15, 223

81. Appelgren, A.S. et al. (2018) Gene flow and adaptive potential in a generalist ectoparasite. *BMC Evol. Biol.* 18, 1–13

82. Harnos, A. et al. (2017) Size matters for lice on birds: coevolutionary allometry of host and parasite body size. *Evolution* 71, 421–431

83. Gelderblom, H.R. (1996) Structure and classification of viruses. In *Medical Microbiology* (Baron, S., ed.), University of Texas Medical Branch at Galveston, Galveston (TX). Chapter 41, Available from: https://www.ncbi.nlm.nih.gov/books/NBK8174/.

84. Sandhu, S.K. et al. (2021) Revisiting the role of hyperparasitism in the evolution of virulence. *Am. Nat.* 197, 216–235

85. Toguebaye, B.S. et al. (2014) Ultrastructure and development of *Nosema podocotyloides* n. sp. (Microsporidia), a hyperparasite of *Podocotyloides magnatestis* (Trematoda), a parasite of *Parapristipoma octolineatum* (Teleostei). *Parasite* 21, 44

86. Wood, J. and Ashby, B. (2023) Hyperparasitism and the evolution of parasite virulence. *Evolution* 77, 2631–2641

87. Poulin, R. and Randhawa, H.S. (2015) Evolution of parasitism along convergent lines: from ecology to genomics. *Parasitology* 142, S6–S15

88. Burke, G.R. et al. (2014) Widespread genome reorganization of an obligate virus mutualist. *PLoS Genet.* 10, e1004660

89. Perry, H.M. et al. (2022) Status and management of the blue crab fishery in the Gulf of Mexico. *N. Am. J. Fish Manag.* 42, 164–179

90. Walters, E.A. et al. (2023) Salinity and temperature affect the symbiont profile and host condition of Florida USA blue crabs *Callinectes sapidus*. *J. Invertebr. Pathol.* 198, 107930

91. Holt, C.C. et al. (2019) The first clawed lobster virus *Homarus gammarus* nudivirus (HgNV n. sp.) expands the diversity of the Nudiviridae. *Sci. Rep.* 9, 10086

92. Arulmoorthy, M. et al. (2020) Major viral diseases in culturable penaeid shrimps: a review. *Aquac. Int.* 28, 1939–1967

93. Yang, Y.-T. et al. (2014) The genome and occlusion bodies of marine *Penaeus monodon* nudivirus (PmNV, also known as MBV and PemoNPV) suggest that it should be assigned to a new nudivirus genus that is distinct from the terrestrial nudiviruses. *BMC Genomics* 15, 628

94. Bojko, J. et al. (2022) Pathology and genetic connectedness of the mangrove crab (*Aratus pisonii*) – a foundation for understanding mangrove disease ecology. *Anim. Dis.* 2, 8

95. Ritter, C.J. and Bourne, D.G. (2024) Marine amphipods as integral members of global ocean ecosystems. *J. Exp. Mar. Biol. Ecol.* 572, 151985

96. Sandoval, L.A. et al. (2022) Leaf consumption and experimental discrimination of stable isotopes between mangrove leaves and the tree-climbing crab (Brachyura: Sesarmidae: *Aratus pisonii*). *Estuar. Coast. Shelf Sci.* 274, 107906

97. Bojko, J. et al. (2021) Invasive non-native crustacean symbionts: diversity and impact. *J. Invertebr. Pathol.* 186, 107482

98. Bojko, J. et al. (2018) Green crab *Carcinus maenas* symbiont profiles along a North Atlantic invasion route. *Dis. Aquat. Org.* 128, 147–168

99. Wood, L. et al. (2021) Options for the control of Dikerogammarus villosus (killer shrimp) and other invasive amphipods: invasive amphipod control. *Manag. Biol. Invasion.* 12, 662–684

100. Mancinelli, G. et al. (2021) A global occurrence database of the Atlantic blue crab *Callinectes sapidus*. *Sci. Data* 8, 111

101. Bojko, J. et al. (2019) Pathogens of Dikerogammarus haemobaphes regulate host activity and survival, but also threaten native amphipod populations in the UK. *Dis. Aquat. Org.* 136, 63–78

102. Shuttleworth, C.M. et al. (2022) An opportunistic assessment of the impact of squirrelpox disease outbreaks upon a red squirrel population sympatric with grey squirrels in Wales. *Animals* 12, 99

103. Wang, Y. et al. (2007) The genome of *Gryllus bimaculatus* nudivirus indicates an ancient diversification of baculovirus-related nonoccluded nudiviruses of insects. *J. Virol.* 81, 5395–5406

104. Rust, M.K. (2020) Recent advancements in the control of cat fleas. *Insects* 11, 668

105. Chiu, E. et al. (2012) Insect virus polyhedra, infectious protein crystals that contain virus particles. *Curr. Opin. Struct. Biol.* 22, 234–240

106. Francki, R. (1991) Fifth report of the International Committee on Taxonomy of Viruses. *Arch. Virol. Suppl.* 2, 1–450
107. Keown, J.R. et al. (2023) Atomic structure of a nudivirus occlusion body protein determined from a 70-year-old crystal sample. *Nat. Commun.* 14, 4160

108. Cheng, R.-L. et al. (2020) Nudivirus remnants in the genomes of arthropods. *Genome Biol. Evol.* 12, 578–588

109. Lin, C.-L. et al. (1999) Persistent Hz-1 virus infection in insect cells: evidence for insertion of viral DNA into host chromosomes and viral infection in a latent status. *J. Virol.* 73, 128–139

110. Weitzman, M.D. and Fradet-Turcotte, A. (2018) Virus DNA replication and the host DNA damage response. *Annu. Rev. Virol.* 5, 141–164

111. Minh, B.Q. et al. (2020) IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. *Mol. Biol. Evol.* 37, 1530–1534

112. Letunic, I. and Bork, P. (2021) Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. *Nucleic Acids Res.* 49, W293–W296