The Impact of a Cypovirus on Parental and Filial Generations of Lymantria dispar L.

Recently, we found that the spongy moth Lymantria dispar L. is susceptible to infection by a Dendrolimus sibiricus cytoplasmic polyhedrosis virus (DsCPV-1). In the present study, we evaluated the pathogenicity of DsCPV-1 against L. dispar larvae and its impact on surviving insects after the infection. Offspring of virally challenged insects were tested for susceptibility to a stress factor (starvation). In addition, we used light microscopy and quantitative polymerase chain reaction (qPCR) to test the ability of DsCPV-1 to be transmitted vertically. We found insect mortality of the L. dispar parents following the infection was positively associated with DsCPV-1 dose. DsCPV-1 was lethal to second-instar L. dispar larvae with a 50% lethal dose (LD50) of 1687 occlusion bodies per larva. No vertical transmission of DsCPV-1 to offspring larvae was detected, while the majority of insect deaths among offspring larvae were caused by microsporidia (Vairimorpha lymantriae), which was harbored by the parents. The offspring of virally challenged parents exhibited a higher number of detected microsporidia compared to the control. Our findings suggest that the application of DsCPV-1 is effective in controlling pests in terms of transgenerational impact following virus exposure.

Phenological Features of the Spongy Moth, Lymantria dispar (L.) (Lepidoptera: Erebidae), in the Northernmost Portions of Its Eurasian Range.

The spongy moth, Lymatria dispar, is a classic example of an invasive pest accidentally introduced from Europe to North America, where it has become one of the most serious forest defoliators, as in its native range. The present study was aimed at (i) identifying the current northern limit of L. dispar's Eurasian range and exploring its northward expansion in Canada using pheromone trap data, and (ii) comparing northern Eurasian populations with those from central and southern regions with respect to male flight phenology, the sums of effective temperatures (SETs) above the 7 °C threshold necessary for development to the adult stage, and heat availability. We show that the range of L. dispar in Eurasia now reaches the 61st parallel, and comparisons with historical data identify the average speed of spread as 50 km/year. We also document the northern progression of L. dispar in southern Canada, where the actual northern boundary of its range remains to be identified. We show that the median date of male flight does not vary greatly between northern and southern regions of the spongy moth range in Eurasia despite climate differences. Synchronization of flight at different latitudes of the range is associated with an acceleration of larval development in northern Eurasian populations. Similar changes in developmental rate along a latitudinal gradient have not been documented for North American populations. Thus, we argue that this feature of spongy moths from northern Eurasia poses a significant invasive threat to North America in terms of enhanced risks for rapid northward range expansion.

Appearances are deceptive: Three RNA viruses co-infected with the nucleopolyhedrovirus in host Lymantria dispar.

The virus infection, which visually looks like typical monoinfection, in fact may hide a great complex of different species. Without detailed analysis, we may miss the important interaction between pathogens, including new species. In the current study, we found the new species inside the mix of cubic and polyhedral occlusion bodies (OBs) isolated from the gypsy moth, Lymantria dispar L. (Ld). Transmission electron microscopy (TEM) revealed that into the one cadaver were OBs which belonged to baculovirus and cypoviruses. The baculovirus produced polyhedral OBs, while cypoviruses produced polyhedral and cubic OBs. Genomic analysis detected the multiple Ld nucleopolyhedroviruses, and cypoviruses were Hubei lepidoptera virus 3 and Dendrolimus punctatus cypovirus 1. This represents the first isolation of the Hubei lepidoptera virus 3 from the gypsy moth, proposed as "Lymantria dispar cypovirus 3". The RNAseq analysis also revealed the presence of Lymantria dispar iflavirus 1. The insecticidal activity of the mixed infection was comparable to that of typical baculovirus monoinfection. Thus, we demonstrate that i) the shape of OBs identified by light microscopy cannot be a robust indicator of viral species infecting the host; ii) only specific analysis may reveal the true composition of viral infection.

A Comparison of the Vertical Transmission of High- and Low-Virulence Nucleopolyhedrovirus Strains in Lymantria Dispar L.

Baculoviruses can persist in insect host organisms after infection and may be vertically transmitted to the next generation, in which they may be reactivated. The goal of the present study was to compare the efficiency of the vertical transmission of high- and low-virulence strains and the subsequent reactivation of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) in the offspring of Lymantria dispar L. adults who survived after viral infection. As a result of parental infection, the fecundity of survived females, pupae weight, and fertility were significantly different compared to the untreated insects. However, differences in these parameters between high- and low-virulence strains were not observed. The prevalence of virus strains in the offspring measured by quantitative polymerase chain reaction also did not differ. When the larvae reached the fourth instar, they were starved to activate the vertically transmitted virus. The frequency of virus activation in the experiment was not dependent on the virulence of the virus strains. These results are helpful for understanding the strategy of virus survival in nature and for the selection of the most effective strains with transgenerational effects in the years following pest treatment.

The activity of phenoloxidase in haemolymph plasma is not a predictor of Lymantria dispar resistance to its baculovirus.

Host innate immunity is one of the factors that determines the resistance of insects to their entomopathogens. In the research reported here we studied whether or not phenoloxidase (PO), a key enzyme in the melanogenesis component of humoral immunity of insects, plays a role in the protection of Lymantria dispar larvae from infection by L. dispar multiple nucleopolyhedrovirus. We studied two types of viral infection: overt and covert. The following lines of investigation were tested: i) the intravital individual estimation of baseline PO activity in haemolymph plasma followed by virus challenging; ii) the specific inhibition of PO activity in vivo by peroral treatment of infected larvae with phenylthiourea (PTU), a competitive inhibitor of PO; iii) the evaluation of PO activity in the haemolymph plasma after larval starvation. Starvation is a stress that activates the covert infection to an overt form. All of these experiments did not show a relationship between PO activity in haemolymph plasma of L. dispar larvae and larval susceptibility to baculovirus. Moreover, starvation-induced activation of covert viral infection to an overt form occurred in 70 percent of virus-carrying larvae against the background of a dramatic increase of PO activity in haemolymph plasma in the insects studied. Our conclusion is that in L. dispar larvae PO activity is not a predictor of host resistance to baculovirus.

Phenological asynchrony between host plant and gypsy moth reduces insect gut microbiota and susceptibility to Bacillus thuringiensis.

The phenological synchrony between the emergence of overwintering herbivorous insects and the budding of host plants is considered a crucial factor in the population dynamics of herbivores. However, the mechanisms driving the interactions between the host plant, herbivores, and their pathogens are often obscure. In the current study, an artificially induced phenological asynchrony was used to investigate how the asynchrony between silver birch Betula pendula and gypsy moth Lymantria dispar affects the immunity of the insect to bacteria, its susceptibility to the entomopathogenic bacteria Bacillus thuringiensis, and the diversity in its midgut microbiota. The lysozyme-like activity in both the midgut and hemolymph plasma and the nonspecific esterase activity and antimicrobial peptide gene expression in the midgut were studied in both noninfected and B. thuringiensis-infected larvae. Our results provide the first evidence that phenologically asynchronous larvae are less susceptible to B. thuringiensis infection than phenologically synchronous larvae, and our results show that these effects are related to the high basic levels and B. thuringiensis-induced levels of lysozyme-like activities. Moreover, a 16S rRNA analysis revealed that dramatic decreases in the diversity of the larval gut bacterial consortia occurred under the effect of asynchrony. Larvae infected with B. thuringiensis presented decreased microbiota diversity if the larvae were reared synchronously with the host plant but not if they were reared asynchronously. Our study demonstrates the significant effect of phenological asynchrony on innate immunity-mediated interactions between herbivores and entomopathogenic bacteria and highlights the role of nonpathogenic gut bacteria in these interactions.

Asynchrony between Host Plant and Insects-Defoliator within a Tritrophic System: The Role of Herbivore Innate Immunity.

The effects of asynchrony in the phenology of spring-feeding insect-defoliators and their host plants on insects' fitness, as well as the importance of this effect for the population dynamics of outbreaking species of insects, is a widespread and well-documented phenomenon. However, the spreading of this phenomenon through the food chain, and especially those mechanisms operating this spreading, are still unclear. In this paper, we study the effect of seasonally declined leafquality (estimated in terms of phenolics and nitrogen content) on herbivore fitness, immune parameters and resistance against pathogen by using the silver birch Betula pendula--gypsy moth Lymantria dispar--nucleopolyhedrovirus as the tritrophic system. We show that a phenological mismatch induced by the delay in the emergence of gypsy moth larvae and following feeding on mature leaves has negative effects on the female pupal weight, on the rate of larval development and on the activity of phenoloxidase in the plasma of haemolymph. In addition, the larval susceptibility to exogenous nucleopolyhydrovirus infection as well as covert virus activation were both enhanced due to the phenological mismatch. The observed effects of phenological mismatch on insect-baculovirus interaction may partially explain the strong and fast fluctuations in the population dynamics of the gypsy moth that is often observed in the studied part of the defoliator area. This study also reveals some indirect mechanisms of effect related to host plant quality, which operate through the insect innate immune status and affect resistance to both exogenous and endogenous virus.

Leaf surface lipophilic compounds as one of the factors of silver birch chemical defense against larvae of gypsy moth.

Plant chemical defense against herbivores is a complex process which involves a number of secondary compounds. It is known that the concentration of leaf surface lipophilic compounds (SLCs), particularly those of flavonoid aglycones are increased with the defoliation treatment of silver birch Betula pendula. In this study we investigated how the alteration of SLCs concentration in the food affects the fitness and innate immunity of the gypsy moth Lymantria dispar. We found that a low SLCs concentrations in consumed leaves led to a rapid larval development and increased females' pupae weight (= fecundity) compared to larvae fed with leaves with high SLCs content. Inversely, increasing the compounds concentration in an artificial diet produced the reverse effects: decreases in both larval weight and larval survival. Low SLCs concentrations in tree leaves differently affected larval innate immunity parameters. For both sexes, total hemocytes count in the hemolymph increased, while the activity of plasma phenoloxidase decreased when larvae consume leaves with reduced content of SLCs. Our results clearly demonstrate that the concentration of SLCs in silver birch leaves affects not only gypsy moth fitness but also their innate immune status which might alter the potential resistance of insects against infections and/or parasitoids.

The effects of defoliation-induced delayed changes in silver birch foliar chemistry on gypsy moth fitness, immune response, and resistance to baculovirus infection.

We tested the effects of defoliation-induced changes in silver birch, Betula pendula, foliar chemistry (delayed induced resistance, DIR) on the fitness and immune defense of the gypsy moth, Lymantria dispar. We measured larval developmental time, pupal weight, rate of survival to the adult stage, and five characteristics of larval immune defense: (1) encapsulation response; (2) phenoloxidase activity; (3) hemocyte concentration and (4) lysozyme-like activity in the hemolymph; and (5) resistance to infection by L. dispar nucleopolyhedrovirus (LdMNPV). The latter is an entomopathogenic baculovirus that often causes epizootics during outbreaks of L. dispar. We also measured the involvement of foliage non-tannin phenolic compounds in resistance of B. pendula to herbivory as well as the relationship between the compounds we identified and L. dispar development, growth, and survival. Leaves of B. pendula with previous defoliation history contained increased levels of myricetin glycoside, two flavonoid aglycones (acacetin and tetrahydroxy-flavone dimethyl ether), as well as one unidentified simple phenolic. The concentrations of two glycosides of quercetin, as well as the content of one unidentified flavonoid glycoside were significantly decreased under defoliation treatment. DIR of B. pendula retarded larval growth rate and increased lysozyme-like activity in the hemolymph, but did not affect encapsulation response, phenoloxidase activity, or hemocyte count. We did not find any DIR-mediated tritrophic interactions among birch, gypsy moth, and LdMNPV. After viral inoculation, the mean hemocyte counts in larvae reared on an individual tree correlated significantly with the survival of larvae reared on that same tree, indicating that hemocyte density in hemolymph might be associated with resistance to viral infection. We found a strong positive correlation between the concentration of 1-(4″-hydroxyphenyl)-3'-oxopropyl-ß-D-glucopyranose and L. dispar survival rate, which may indicate an unlikely role of this dominant non-tannin phenolic in B. pendula defense against L. dispar. Our study also shows that several immune characteristics of insects that function as barriers against different groups of parasites are differently affected by plant induced defenses. This underscores the importance of considering multiple factors when characterizing barriers to insect immunity.