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The Siberian silk moth, Dendrolimus sibiricus Tschetverikov, is a very serious pest of conifers in Russia and is an emerging threat in North America where an accidental introduction could have devastating impacts on native forest resources. Other Dendrolimus Germar species and related Eurasian lasiocampids in the genus Malacosoma (Hubner) could also present a risk to North America’s forests. Foreign vessels entering Canadian and U.S. ports are regularly inspected for Lymantria dispar (Linnaeus) and for the presence of other potentially invasive insects, including suspicious lasiocampid eggs. However, eggs are difficult to identify based on morphological features alone. Here, we report on the development of two TaqMan (Roche Molecular Systems, Inc., Rotkreuz, Switzerland) assays designed to assist regulatory agencies in their identification of these insects. Developed using the barcode region of the cytochrome c oxidase I (COI) gene and run in triplex format, the first assay can detect Dendrolimus and Malacosoma DNA, and can distinguish North American from Eurasian Malacosoma species. The second assay is based on markers identified within the internal transcribed spacer 2 (ITS2) region and was designed to specifically identify D. sibiricus, while discriminating closely related Dendrolimus taxa. In addition to providing direct species identification in the context of its use in North America, the D. sibiricus assay should prove useful for monitoring the spread of this pest in Eurasia, where its range overlaps with those of the morphologically identical D. superans (Butler) and similar D. pini (Linnaeus). The assays described here can be performed either in the lab on a benchtop instrument, or on-site using a portable machine.

Nowadays researchers provide more and more evidence that it is necessary to develop an ecologically friendly approach to pest control. This is reflected in a sharp increase in the value of the biological insecticide market in recent decades. In our study, we found a virus strain belonging to the genus Cypovirus (Reoviridae); the strain was isolated from Dendrolimus sibiricus: that possesses attractive features as a candidate for mass production of biological agents for lepidopteran-pest control. We describe morphological, molecular, and ecological features of the new Cypovirus strain. This strain was found to be highly virulent to D. sibiricus (half-lethal dose is 68 occlusion bodies per second-instar larva) and to have a relatively wide host range (infects representatives of five families of Lepidoptera: Erebidae, Sphingidae, Pieridae, Noctuidae, and Lasiocampidae). The virus strain showed a strong interaction with a nontoxic adjuvant (optical brightener), which decreased the lethal dose for both main and alternative hosts, decreased lethal time, and may expand the host range. Moreover, we demonstrated that the insecticidal features were preserved after passaging through the most economically suitable host. By providing strong arguments for possible usefulness of this strain in pest control, we call on virologists, pest control specialists and molecular biologists to give more attention to the Cypovirus genus, which may lead to new insights in the field of pest control research and may provide significant advantages to compare with baculoviruses and Bacillus thuringiensis products which are nowadays main source of bioinsecticides.Significance statementWithin this article we are describing unique set of features of newly discovered cypovirus strain which possess by significant premises for modern biological insecticides requirements: high potency, universality, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, ecologically friendly. Basing on genome alignment we suggest that increasing of host range of new strain is the sequence of evolutionary event which was occurred after coinfection of different CPV species within same host. This finding open new perspective to consider CPVs as perspective agent of biocontrol products.

Fungal infections and toxicoses caused by insecticides may alter microbial communities and immune responses in the insect gut. We investigated the effects of Metarhizium robertsii fungus and avermectins on the midgut physiology of Colorado potato beetle larvae. We analyzed changes in the bacterial community, immunity- and stress-related gene expression, reactive oxygen species (ROS) production, and detoxification enzyme activity in response to topical infection with the M. robertsii fungus, oral administration of avermectins, and a combination of the two treatments. Avermectin treatment led to a reduction in microbiota diversity and an enhancement in the abundance of enterobacteria, and these changes were followed by the downregulation of Stat and Hsp90, upregulation of transcription factors for the Toll and IMD pathways and activation of detoxification enzymes. Fungal infection also led to a decrease in microbiota diversity, although the changes in community structure were not significant, except for the enhancement of Serratia. Fungal infection decreased the production of ROS but did not affect the gene expression of the immune pathways. In the combined treatment, fungal infection inhibited the activation of detoxification enzymes and prevented the downregulation of the JAK-STAT pathway caused by avermectins. The results of this study suggest that fungal infection modulates physiological responses to avermectins and that fungal infection may increase avermectin toxicosis by blocking detoxification enzymes in the gut.

Strains of entomopathogenic fungi may have substantial differences in their final stages of mycosis. Insect cadavers are usually overgrown with mycelium after colonization of the insect body, but in many cases, bacterial decomposition of the colonized hosts occurs. We used two Metarhizium robertsii strains in the work: Mak-1 (cadavers become overgrown with mycelium and conidia) and P-72 (cadavers decay after fungal colonization). We conducted a comparative analysis of gut and cadaver microbiota in Colorado potato beetle larvae using 16S rRNA gene sequencing after infection with these strains. In addition, we estimated the content of different forms of nitrogen in cadavers and the influence of cadavers on the growth of Solanum lycopersicum on sand substrates under laboratory conditions. It was shown that infections did not lead to a significant shift in the midgut bacterial communities of infected insects compared to those of untreated insects. Importantly, bacterial communities were similar in both types of cadaver, with predominantly enterobacteria. Decomposing cadavers (P-72) were characterized by increased nitrate and ammonium, and they had a stronger growth-promoting effect on plants compared to cadavers overgrown with mycelium and conidia (Mak-1). We also estimated the colonization and growth of plants after treatment with conidia of both strains cultivated on artificial medium. Both cultures successfully colonized plants, but strain P-72 showed stronger growth promotion than Mak-1. We propose that the use of deviant strains that are unable to sporulate on cadavers leads to a faster (though only passive) flow of nitrogen from killed insects to plants.

The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae.Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes.Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB. © 2017 Society of Chemical Industry.

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.

Susceptibility to the fungus Metarhizium robertsii and changes in host defences were evaluated in different stages of the intermoult period (4–6 h, 34–36 h and 84–86 h post moult in IV larval instars) of the Colorado potato beetle. A significant thickening of the cuticle during larval growth was accompanied by decreases in cuticle melanization, phenoloxidase activity and epicuticular hydrocarbon contents (C28-C32). At the same time, a decrease in the conidial adhesion rate and an increase in resistance to the fungus were observed. In addition, we recorded significant elevation of the encapsulation rate and total haemocyte counts in the haemolymph during the specified period. The activity of detoxification enzymes decreased in the haemolymph but increased in the fat body during larval growth. No significant differences in the fatty acid content in the epicuticle were observed. The role of developmental disorders in susceptibility to entomopathogenic fungi is also discussed.

Thirty-four isolates of Metarhizium spp. from Russian collections were genotyped using 5′ EF-1α gene sequence analysis. Four species were identified, of which M. robertsii and M. brunneum were the most frequent, whereas M. anisopliae and M. pemphigum were sporadic. Radial growth studies in the temperature range of 10–40 °C revealed that growth at high temperatures (35–37.5 °C) was inherent for M. robertsii isolates but not for M. brunneum isolates. In contrast, M. brunneum isolates were more active at cold temperatures (10 °C) compared to M. robertsii. Virulence was evaluated against larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, under two regimes: humid (21 °C, 80% relative humidity (RH)) and arid (31 °C, 55% RH). M. brunneum isolates were less virulent compared to M. robertsii under both regimes. M. robertsii activity did not differ under the two regimes, but M. brunneum was less virulent under the arid regime compared to the humid one. A field experiment under natural conditions (steppe zone of Western Siberia) with daily ranges of 10–43 °C and 13–98% RH showed that M. robertsii was significantly more active than M. brunneum against CPB larvae.

Baculoviruses are a family of insect-specific pathogenic viruses can persist outside for long periods through the formation of occlusion bodies. In spite of this ability, the UV of sunlight is an essential factor that limits the survival of baculoviruses outside the host. In the current study, we compared the UV tolerance of two strains of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), which were isolated in spatially different regions (LdMNPV-27/0 in Western Siberia (Russia) and LdMNPV-45/0 in North America (USA)) and dramatically differ in their potency. We exposed the studied strains to sunlight in an open area for 0.25, 0.5, 1, and 2 hours and later perorally inoculated host larvae with the same doses of virus (5x105) and with doses leading to same effect (LD90). We observed that strain LdMNPV-45/0, which previously showed high virulence against L. dispar larvae, was more sensitive to UV irradiation (estimated as the relative rate of inactivation (r, h -1) and as the half-life of the virus (τ1/2, h)) compared to LdMNPV-27/0. Exposure to sunlight induced a significant delay of LdMNPV-45/0-induced pathogenesis already after 0.25 h of sunlight exposure, while for LdMNPV-27/0 this delay was occurred only after 2 h exposure in spite of used concentrations. We also compared the sequences of the main structural proteins of the studied strains as UV light contributes not only to genome damage in viruses but also to structural protein damage. The most prominent genetic difference between the structural proteins of the strains was related to the loss of the virus enhancin factor-1 (vef-1) gene in the LdMNPV-27/0 strain. Thus initially highly potent viral strain (such as LdMNPV-45/0) is not recommend to use in the regions (or forest stand density) with high UV load. The role of virus enhancin factor-1 in baculovirus tolerance to UV needs for following studies.