Physiological and Nutritional Effects of a Fluorescent Brightener on Nuclear Polyhedrosis Virus-Infected Lymantria dispar (L.) Larvae (Lepidoptera: Lymantriidae)

Recent studies with gypsy moths (Lymantria dispar L.) show that the fluorescent brightener, Tinopal LPW, acts synergistically with the L. dispar nuclear polyhedrosis virus (LdMNPV), lowering the 50% lethality concentration of the virus (LC₅₀) by 1000-fold. As a continuation of this work, we investigated the effects of ingestion of LdMNPV/Tinopal on larval midgut pH, hemolymph pH, and nutritional indices. Larval feeding for 48 h on diet treated with either LdMNPV or Tinopal did not affect midgut luminal pH, which averaged 10.3. In contrast, after 48 h of larval feeding on LdMNPV/ Tinopal diet, midgut pH averaged 8.5, and frass production, weight gain, and larval nutritional indices were significantly reduced. These effects appeared to be irreversible after the first 24 h of feeding ad libitum on LdMNPV/Tinopal diet, and the reduction in midgut pH was not due to feeding cessation per se. No significant effects on hemolymph pH were detected with any treatment. No treatment effects were observed when "SITS," a related fluorescent brightener, was substituted for Tinopal. The severe biological perturbations resulting from LdMNPV/Tinopal consumption are interpreted in light of current understanding of larval lepidopteran midgut physiology.     

Interactions between a fungal pathogen, foliar properties, and generalist herbivores

American [Castanea dentata (Marsh) Borkh.] × Chinese [Castanea mollissima Blume] chestnut (Fagac, ae) hybrids are a novel system in which to study influences of phytopathogenic fungi and woody plant hybridization on herbivore susceptibility, as the hybrids are well characterized with regard to resistance to the chestnut blight fungus [Cryphonectria parasita (Murr) Barr (Endothia) Diaporthales: Valsaceae] and variability is present. We chose two groups of resistance‐rated backcross chestnut that shared an F1 parent and had different American parents. Foliage from both backcross groups and the parent trees was sampled on three dates for use in feeding assays with gypsy moth larvae [Lymantria dispar (L.) [Lepidoptera: Lymantriidae], adult Japanese beetles [Popillia japonica Newman (Coleoptera: Scarabaeidae)], and fall webworm larvae [Hyphantria cunea Drury (Lepidoptera: Arctiidae)], respectively. Foliar analyses were performed concurrently and included carbohydrate, tannin, and nitrogen content, toughness, and density. Blight resistance had almost no effect on herbivore performance or foliar chemistry. When the parent trees and backcross groups were compared, however, significant differences in gypsy moth performance and Japanese beetle consumption were evident. There were no differences in fall webworm consumption. Most foliar characteristics measured differed among chestnut genotypes at some point in the season, and all varied seasonally. No clear pattern emerges with respect to the relationship among blight resistance, herbivore susceptibility, foliar properties, and plant genotype, and more research is needed to separate these effects.     

Genotype‐by‐genotype interactions between an insect and its pathogen

Genotype‐by‐genotype (G×G) interactions are an essential requirement for the coevolution of hosts and parasites, but have only been documented in a small number of animal model systems. G×G effects arise from interactions between host and pathogen genotypes, such that some pathogen strains are more infectious in certain hosts and some hosts are more susceptible to certain pathogen strains. We tested for G×G interactions in the gypsy moth (Lymantria dispar) and its baculovirus. We infected 21 full‐sib families of gypsy moths with each of 16 isolates of baculovirus and measured the between‐isolate correlations of infection rate across host families for all pairwise combinations of isolates. Mean infectiousness varied among isolates and disease susceptibility varied among host families. Between‐isolate correlations of infection rate were generally less than one, indicating nonadditive effects of host and pathogen type consistent with G×G interactions. Our results support the presence of G×G effects in the gypsy moth–baculovirus interaction and provide empirical evidence that correlations in infection rates between field‐collected isolates are consistent with values that mathematical models have previously shown to increase the likelihood of pathogen polymorphism.     

ANALYSIS OF HELICOVERPA ARMIGERA SINGLE NUCLEOPOLYHEDROVIRUS IMMEDIATE EARLY 1 (IE‐1) GENE*

Abstract A 6.12 kb Xbal‐H fragment of the Helicoverpa armigem single nucleopolyhedrovirus (HaSNPV) gemone was cloned and the complete sequence of this fragment was sequenced by random sequencing method. Sequence comparison and analysis revealed an ORF13 which was homologous to ie‐1 of Auiographa California nucleopolyhedrovirus (AcMNPV). The homologous encoding gene is ie‐1. The total length of the encoding region of HaSNPV gene was 1986 bp and was predicted to encode 661 amino acid protein(IE‐1) with molecular weight of 76.5 kD. The alingment of putative HaSNPV IE‐1 amino acid sequence with those of other 9 reported baculoviruses IE‐Is showed that the HaSNPV IE‐1 was most closely related to Helicoverpa zea nucleopolyhedrovirus (HzNPV) IE‐1, with 97% amino acid identidy. But it showed a low degree of sequence similarity to those of AcMNPV, Bombyx mori nucleopolyhedrovirus (BmNPV), Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV), Lymantria dispar nucleopolyhedrovirus (LdMNPV), Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV), Spodoptera exigua nucleopolyhedrovirus (SeMNPV), Plutella xylostella granulovirus(PxGV) and Xestia c‐nigrum granulovirus (XcGV), with 23%, 23%, 23%, 25%, 23%, 14%, 27% and 7% amino acid identity, respectively. A phylogenetic tree of ten baculoviruses IE‐1 was also given.     

Molecular identification and phylogenetic analyses of multiple nucleopolyhedrovirus isolated from <Emphasis Type="Italic">Lymantria obfuscata</Emphasis> (Lepidoptera: Lymantriidae) in India

The Lymantria obfuscata Walker (Lyob) multiple (M) nucleopolyhedrovirus (NPV) (LyobMNPV) has been isolated and successfully applied for the management of the Indian gypsy moth, L. obfuscata in Jammu and Kashmir (J&K), India. The present work aimed to investigate the variability of LyobMNPV isolates from six localities of J&K through molecular [amplification of the polyhedrin (polh), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes] and biological (bioassays) characterization. To identify the position of LyobMNPV in the phylogenetic tree of baculoviruses, partial sequences of the polh, lef-8 and lef-9 genes were determined by using the DNA sequences within their coding regions by optimizing the polymerase chain reaction with degenerate primers. The sequence alignment revealed that LyobMNPV isolates exhibited seven, five and eleven single nucleotide polymorphic sites in the case of polh, lef-8 and lef-9, respectively. The phylogenetic analyses supported placing LyobMNPV with the Lymantria dispar L. MNPV (LdMNPV) isolates from different countries, and showed that it was more closely related to LdMNPV than to Lymantria xylina Swinhoe NPV and Lymantria monacha L. NPV. The contaminated diet plug bioassays using 2nd instar larvae indicated that the median lethal dose (LD₅₀) and median survival time (ST₅₀) of different isolates of LyobMNPV against L. obfuscata were lower than those of LdMNPV against L. dispar. LyobMNPV was more closely related to LdMNPV but its LD₅₀ and ST₅₀ were lower than those of LdMNPV. The study provides novel information on the position of LyobMNPV in the phylogenetic tree of baculoviruses and about biological and genetic variation of Lymantria species’ NPV isolates from different parts of the world.     

Non-target effects of transgenic blight-resistant American chestnut (Fagales: Fagaceae) on insect herbivores

American chestnut [Castanea dentata (Marshall) Borkhausen], a canopy dominant species across wide swaths of eastern North America, was reduced to an understory shrub after introduction of the blight fungus [Cryphonectria parasitica (Murrill) Barr] in the early 1900s. Restoration of American chestnut by using biotechnology is promising, but the imprecise nature of transgenesis may inadvertently alter tree phenotype, thus potentially impacting ecologically dependent organisms. We quantified effects of genetic engineering and fungal inoculation of trees on insect herbivores by using transgenic American chestnuts expressing an oxalate oxidase gene and wild-type American and Chinese (C. mollissima Blume) chestnuts. Of three generalist folivores bioassayed, only gypsy moth [Lymantria dispar (L.)] was affected by genetic modification, exhibiting faster growth on transgenic than on wild-type chestnuts, whereas growth of polyphemus moth [Antheraea polyphemus (Cramer)] differed between wild-type species, and fall webworm [Hyphantria cunea (Drury)] performed equally on all trees. Inoculation of chestnuts with blight fungus had no effect on the growth of two herbivores assayed (polyphemus moth and fall webworm). Enhanced fitness of gypsy moth on genetically modified trees may hinder restoration efforts if this invasive herbivore's growth is improved because of transgene expression.     

Dynamics of twolined chestnut borer Agrilus bilineatus as influenced by defoliation and selection thinning

1 The twolined chestnut borer, Agrilus bilineatus (Coleoptera: Buprestidae), is a major mortality agent of stressed oak trees. However, patterns of abundance and population change are not well understood. 2 We studied the spatial and temporal variation in abundance of twolined chestnut borer adults during a gypsy moth, Lymantria dispar (Lepidoptera: Lymnatriidae), outbreak and examined the influence of both defoliation and thinning on twolined chestnut borer abundance. 3 In stands that were defoliated by gypsy moth, extensive defoliation occurred in one year, and major overstory tree mortality followed in the next. Most mortality occurred in the year preceding the peak year of twolined chestnut borer abundance and abundance of twolined chestnut borer was positively associated with defoliation and mortality in the previous year. 4 Twolined chestnut borers were more frequently associated with poor or fair crown condition trees than trees with good crown condition and were more abundant on members of the red oak group than the white oak group.     

Restoration of threatened species: a noble cause for transgenic trees

Some of the first applications of transgenic trees in North America may be for the conservation or restoration of threatened forest trees that have been devastated by fungal pathogens or insect pests. In some cases, where resistance has yet to be found in the natural population of a tree species, incorporating genes from other organisms may offer the only hope for restoration. In others, transgenics may play a role as part of an integrated approach, along with conventional breeding or biocontrol agents. American chestnut (Castanea dentata) was wiped out as a canopy species by a fungal disease accidentally introduced into the United States around 1900. Similarly, American elm (Ulmus americana) virtually disappeared as a favored street tree from Northeastern U.S. cities after the introduction of the Dutch elm disease fungus in the 1940s. In both cases, progress has been made toward restoration via conventional techniques such as selection and propagation of tolerant cultivars (American elm) or breeding with a related resistant species (American chestnut). Recently, progress has also been made with development of systems for engineering antifungal candidate genes into these “heritage trees.” An Agrobacterium-leaf disk system has been used to produce transgenic American elm trees engineered with an antimicrobial peptide gene that may enhance resistance to Dutch elm disease. Two gene transfer systems have been developed for American chestnut using Agrobacterium-mediated transformation of embryogenic cultures, setting the stage for the first tests of potential antifungal genes for their ability to confer resistance to the chestnut blight fungus. Despite the promise of transgenic approaches for restoration of these heritage trees, a number of technical, environmental, economic, and ethical questions remain to be addressed before such trees can be deployed, and the debate around these questions may be quite different from that associated with transgenic trees developed for other purposes.     

Comparisons of Ectomycorrhizal Colonization of Transgenic American Chestnut with Those of the Wild Type,a Conventionally Bred Hybrid,and Related Fagaceae Species

American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.     

Fitness‐related offspring sex allocation of Anastatus disparis,a gypsy moth egg parasitoid,on different‐sized host species

Anastatus disparis (Ruschka) (Hymenoptera: Eupelmidae) is an egg parasitoid and considered a potential biological control agent of the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). Only male offspring of A. disparis emerge from single eggs of L. dispar in the laboratory, and A. disparis exhibits low parasitism on L. dispar in the field. We therefore selected several lepidopteran species with various body sizes to evaluate the optimal egg size for hosting A. disparis. In addition, we explored whether the nutritional content of a single L. dispar egg influences the sex of A. disparis offspring and why female offspring can be reared from L. dispar eggs in the field. The results indicated that host egg size decisively influenced the body size and sex ratio of the parasitoid offspring. Therefore, larger hosts, especially the largest eggs of Antheraea pernyi Guérin‐Méneville (Saturniidae), might increase the fitness of A. disparis females. Lymantria dispar eggs concealed in the larger egg shell of A. pernyi produced female A. disparis, suggesting that adult A. disparis should prefer hosts with larger bodies and that the nutritional content of L. dispar eggs did not play a decisive role in the sex allocation of A. disparis. The results also indicated that the egg mass and the fur cover of L. dispar egg masses might be the key factors inducing female A. disparis to lay female offspring in L. dispar eggs.     

Belowground infections of the invasive Phytophthora plurivora pathogen enhance the suitability of red oak leaves to the generalist herbivore Lymantria dispar

1. Globally, vast areas of forest are currently threatened by Lymantria dispar L. and Phytophthora species, which cause widespread declines and cascading ecological impacts. One important aim of evolutionary and ecological studies is to understand their interactions. 2. The present study tests whether Quercus rubra L. trees naturally infected with P. plurivora T. Jung & T.I. Burgess or free of infection are more suitable for L. dispar herbivory, and if relationships between L. dispar performance and herbivory may vary depending on whether trees are infected or free of infection. 3. In choice tests, the consumed area of leaves from trees infected by P. plurivora was four times larger than that from non‐infected trees, probably because the increased values of N, soluble protein, and water content observed in the leaves of infected trees enhanced acceptability. Although larval performance was better in Phytophthora‐infected trees, relationships between larval performance and defoliation did not significantly interact with the health status of trees. 4. The present results suggest that the impact of P. plurivora on natural and managed ecosystems may generate a positive feedback loop for oak decline. The link between the behavioural and physiological responses of L. dispar to infected trees and the population growth in nature deserves further investigation.     

Genetic structure and demographic history of Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae) in its area of origin and adjacent areas

We analyzed the population genetic structure and demographic history of 20 Lymantria dispar populations from Far East Asia using microsatellite loci and mitochondrial genes. In the microsatellite analysis, the genetic distances based on pairwise F_ST values ranged from 0.0087 to 0.1171. A NeighborNet network based on pairwise F_ST genetic distances showed that the 20 regional populations were divided into five groups. Bayesian clustering analysis (K = 3) demonstrated the same groupings. The populations in the Korean Peninsula and adjacent regions, in particular, showed a mixed genetic pattern. In the mitochondrial genetic analysis based on 98 haplotypes, the median‐joining network exhibited a star shape that was focused on three high‐frequency haplotypes (Haplotype 1: central Korea and adjacent regions, Group 1; Haplotype 37: southern Korea, Group 2; and Haplotype 90: Hokkaido area, Group 3) connected by low‐frequency haplotypes. The mismatch distribution dividing the three groups was unimodal. In the neutral test, Tajima's D and Fu's FS tests were negative. We can thus infer that the Far East Asian populations of L. dispar underwent a sudden population expansion. Based on the age expansion parameter, the expansion time was inferred to be approximately 53,652 years before present (ybp) for Group 1, approximately 65,043 ybp for Group 2, and approximately 76,086 ybp for Group 3. We propose that the mixed genetic pattern of the inland populations of Far East Asia is due to these expansions and that the inland populations of the region should be treated as valid subspecies that are distinguishable from other subspecies by genetic traits.     

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.     

Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.     

The natural enemy complex of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in different phases of its population dynamics in eastern Austria and Slovakia – a comparative study

Parasitism and pathogen mortality of Lymantria dispar were compared between host populations of different densities in Slovakia and Austria. Over a period of 4 years, L. dispar eggs, larvae, and pupae were collected in a stage‐specific manner at three mixed oak stands in each country and reared in the laboratory to assess parasitoid and pathogen mortality. At sites with low host densities, L. dispar abundance was artificially augmented by exposing egg masses and young larvae. We ascertained marked differences between the natural enemy complexes of L. dispar populations in Slovakia and Austria. Overall, pathogens caused highest mortality in the former and parasitoids in the latter. Moreover, the species composition differed significantly between both countries. High variation was also observed between years and host populations at different densities. The investigations revealed that egg parasitization was low in Slovak L. dispar populations, and no egg parasitoids were found in Austria. Larval and pupal parasitism was low at sites with outbreak populations, but higher at the Austrian than at the Slovak site. The tachinid Parasetigena silvestris was the dominant species at elevated host densities. Nuclear polyhedrosis virus also caused significant mortality in these cases. An increase in parasitism by several species was noticed after the breakdown of L. dispar populations. The ichneumonid Phobocampe spp. caused high levels of parasitism during the post‐culmination years. At sites where host abundance increased in the first year of the study, significant differences between the Austrian and the Slovak locality could be ascertained. Mortality was low in the population at the latter site, and host abundance increased to outbreak levels the year after. The population at the Austrian site suffered high parasitism by the tachinids P. silvestris and Blepharipa pratensis and this might have contributed to the prevention of a further increase in L. dispar abundance in this locality. Braconids showed a strong reaction to the local, artificial increase in host density at study plots with innocuous L. dispar populations. Particularly high parasitism by Glyptapanteles liparidis was observed at the Austrian site, but G. porthetriae and Cotesia melanoscela also accounted for significant mortality in both low‐density populations. The artificial L. dispar populations were usually eradicated by natural enemies before pupation of the hosts.     

Pheromone‐trapping the nun moth,Lymantria monacha (Lepidoptera: Lymantriidae) in Inner Mongolia,China

The nun moth, Lymantria monacha L., is one of the most important defoliators of Eurasian coniferous forests. Outbreaks during 2011–2015 in the natural/planted larch, and larch‐birch mixed forests of the Greater Khingan Range in Inner Mongolia, China, caused tremendous timber losses from severe defoliation and tree mortality. A series of trapping experiments were conducted in these outbreak areas to evaluate the efficacy of a synthetic species‐specific pheromone lure based on the female pheromone blend of European nun moth populations. Our results clearly show that the nun moth in Inner Mongolia is highly and specifically attracted to this synthetic pheromone, with few gypsy moths (Lymantria dispar) captured. Flight activity monitoring of L. monacha male moths using pheromone‐baited Unitraps at 2 locations during the summer of 2015 indicated that the flight period started in mid‐July, peaking in early August at both locations. Based on male moth captures, there was a strong diurnal rhythm of flight activity throughout the entire scotophase, peaking between 22:00 and 24:00. Unitraps and wing traps had significantly and surprisingly higher catches than the gypsy moth traps. Unitraps fastened to tree trunks 2 m above ground caught significantly more male moths than those at the ground level or at 5 m height. Male L. monacha moths can be attracted to pheromone‐baited traps in open areas 150–200 m distant from the infested forest edge. Our data should allow improvement on the performance of pheromone‐baited traps for monitoring or mass‐trapping to combat outbreaks of this pest in northeastern China.     

Effects of elicitation treatment and genotypic variation on induced resistance in Populus: impacts on gypsy moth (Lepidoptera: Lymantriidae) development and feeding behavior

We examined the effects of various wounding treatments and genotypic variation on induced resistance in Populus (Salicales: Salicaceae) against herbivory by the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae). Second-instar larvae grew and consumed less on leaves from induced than non-induced trees. Likewise, larvae preferred leaf disks from non-induced trees. Among induction treatments, gypsy moth feeding had the strongest and most consistent effect in behavioral choice tests. Mechanical wounding of leaves and mechanical wounding plus application of gypsy moth regurgitant had intermediate effects, while application of jasmonic acid had the weakest overall effect. Under no-choice conditions, there were no consistent trends across clones in the ability of various treatments to elicit plant responses affecting the herbivore. Levels of constitutive and inducible resistance to herbivory varied significantly among 12 Populus clones. Larvae grew up to 30-fold more, and consumed up to 250-fold more on the most suitable than the least suitable clone. Prior feeding by gypsy moths reduced larval feeding up to 71.4% on the most highly inducible clone, but it had little or no effect for the least inducible clones. There was no evidence for a relationship between levels of inducible and constitutive resistance, or between inducible resistance and phylogenetic relatedness among clones. We discuss implications for the ecology and evolution of plant-insect interactions and the management of insect pests. Received: 12 October 1998 / Accepted: 22 March 1999     

Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.