Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.     

Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.     

Acidic precipitation increases egg survival in Neodiprion sertifer

The effects of simulated acidic precipitation on the egg viability of the European pine sawfly, Neodiprion sertifer Geoffroy (Hymenoptera: Diprionidae) were studied by spraying egg clusters with a mixture of sulphuric and nitric acid (1:1, pH 2, 3, 4, 5, 6, distilled water, natural precipitation) for a month in April–May. Egg viability analyses showed that the proportion of hatched first instar larvae increased with increasing acidity. The better survival of sawfly eggs in pine needles in more acidic conditions may be partly due to changes in needle physiology affecting egg mortality. However, when the eggs were reared without needles in Petri-dishes, the proportion of successfully hatched larvae likewise increased with increasing acidity indicating direct acid-induced changes in the eggs themselves. Acidic precipitation as a stress factor of the host plant apparently changes population dynamics of pine sawflies and may be an important factor in triggering outbreaks.     

Johnston's organ in Neodiprion sertifer (Insecta: Hymenoptera)

The fine structure of Johnston's organ in the pine sawfly, Neodiprion sertifer, was studied by electron microscopy to determine if there exists a dimorphism in this organ corresponding to the sexual dimorphism in antennal shape and surface area. The organ is made up of scolopidia that are ultrastructurally similar to those of other insects. The scolopidia, identical in both sexes, comprise three sensory cells bearing two types of sensory processes: Two are shorter and smaller in diameter than the third, which extends into the cuticle of the membrane connecting pedicel and flagellum and terminates at an epicuticular invagination. The dendrites and sensory processes are surrounded by two types of enveloping (glial) cells-a scolopale cell and an attachment cell. Other enveloping cells occur at different levels of the scolopidium. Sexual dimorphism is evident only in the numbers of scolopidial groups: Males have more groups with fewer scolopidia, but both sexes possess about the same total number of scolopidia.     

Quantifying Neodiprion sertifer nucleopolyhedrovirus DNA from insects,foliage and forest litter using the quantitative real‐time polymerase chain reaction

• 1 Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio‐insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV.
• 2 In the present study, we developed real‐time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real‐time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real‐time assay is 0.013 pg of viral DNA (0.013×10^?12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample.
• 3 qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy‐ or bioassay‐based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying.
• 4 This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).

     

Chemical compositions and larvicidal effectiveness of Rhododendron ponticum essential oil on Neodiprion sertifer larvae

In many countries throughout the world, the European pine sawfly, Neodiprion sertifer (Geoffroy, 1785) (Hymenoptera: Diprionidae) is known as a significant forest pest that damages pine needles. As a result of the insect damage, young pine trees, especially, might dry up totally. Mechanical, biotechnical, biological, and chemical approaches are applied in research to keep it under control. Due to the increasing danger of chemical contamination associated with the use of synthetic pesticides to control insects, alternative control strategies have been sought, and studies on essential oils have been prioritized. In this research, in addition to determining the chemical analysis of Rhododendron ponticum L. (Ericales; Ericaceae) essential oil by GC–MS, the larvicidal effects of R. ponticum on N. setifer in the six larval stages were also investigated. The research was conducted in the Artvin Çoruh University Central Laboratory under laboratory settings (25 ± 1 °C, 65 ± 5% RH, and 14L:10D h photoperiods) during 2020–21. 1-Methyl-2-pyrrolidone (38.41%), Phenylethy alcohol (10.07%), Linalool (8.01%), Myrtenol 5.68%), β-Pinene (4.26%), Citronellol (4.05%), α-Pinene (3.02%) were found to be the major components of R. ponticum oil. The administration of R. ponticum essential oil at 10, 15, and 20 μL/Petri doses, as well as controls at the 24th, 48th, 72nd, and 96th hours, produced various rates of death (19.6–100%) on six larval stages of N. sertifer. According to LD₅₀ and LD₉₀ values, the highest toxicity was determined as L₁ (0.58 μL/larva) and the lowest toxicity was as L₆ (10.44 μL/larva). In conclusion, R. ponticum essential oil showed high toxicity against L₁ and L₂ larval stages of N. sertifer.     

Behaviour of male pine sawflies, Neodiprion sertifer, released downwind from pheromone sources

This study investigated the behaviour of male European pine sawflies, Neodiprion sertifer Geoffroy (Hym., Diprionidae), that were released and observed downwind from pheromone traps baited with 100 g of the sex pheromone, (2S,3S,7S)-3,7-dimethyl-2-pentadecyl acetate. Releases were done at three distances; either at 5 m from one trap, or at 50 or 200 m from five traps, placed in a line perpendicular to the current wind direction. As control, males were released identically but without any pheromone source present. The behaviour of the males prior to take-off was studied. A total of 1729 males were released, and 80% of them took flight. Males took off significantly faster in the presence of pheromone. Grooming was significantly more frequent in presence of pheromone compared with control. In all pheromone experiments significantly more males displayed grooming, wing fanning and take-off towards the wind compared with the control. Weather data was simultaneously collected at the study site. Wing fanning was negatively correlated with wind speed. Grooming was not influenced by wind speed. Reduced levels of incoming short-wave radiation lowered the take-off frequency significantly. Pheromone-induced behaviour in diprionids seems to be less distinct than in other insects, e.g., Lepidoptera.     

Virulence of a nucleopolyhedrovirus to Neodiprion abietis (Hymenoptera: Diprionidae)

Different larval stages of balsam fir sawfly, Neodiprion abietis (Harris) (Hymenoptera: Diprionidae), were challenged by different concentrations of a nucleopolyhedrovirus in the laboratory to determine larval susceptibility to the virus and to test treatment effects of the virus on the sawfly survivors. The results indicated that younger larvae were more susceptible to the virus than older instars. The speed of larval death depended on the larval age and the virus concentration. Generally, the virus killed second or third instars in approximately 5 d and fourth or fifth instars in 10-12 d at concentrations of 10(7) polyhedral inclusion bodies (PIB) /ml. The virus had profound treatment effects on sawfly survivors. Feeding activity of the survivors was reduced by > 40% compared with that of the control group, pupal weight by approximately 25%, and adult emergence by > 30%. There was also a higher percentage of male adults in the virus-treated groups than in the control.     

Dispersal of the nuclear polyhedrosis virus of Neodiprion sertifer from soil to pine foliage with dust

Inclusion bodies (PIB) of nuclear polyhedrosis viruses (NPV) remain infectious for long periods in forest soils, but their transfer from soil to foliage is not well understood. Two instances of dispersal of the NPV of Neodiprion sertifer (Fourcroy) (Hymenoptera: Diprionidae) by dust are reported. The distributioo of diseased larval colonies in a young Scots pine stand indicated that dust caused by logging during winter could have started an epizootic. At another site there was a higher frequency of infected colonies near a forest road than further into the stand. The dust transmission hypothesis was tested by bioassays of road surface material and of samples of airborne dust collected on membrane filters. Both soil and air samples contained viable PIBs. Thus, virus dispersal with dust provided a likely explanation for the observed distributions of diseased larval colonies.

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Zusammenfassung Mortalität von N.sertifer Larven durch das wirtsspezifische Kernpolyedervirus ist zwischen den Massenvermehrungen der Sägewespe selten. Eine ständige Infektionskette bleibt nicht erhalten. Das einzige bekannte Reservoir von Virusinoculum zwischen den Massenvermehrungen ist der Waldboden. Die Übertragung von Virus vom Boden auf Kiefernnadeln wird als seltenes Ereignis betrachtet. Sie dürfte häufiger sein, wenn die bodenbedeckende Vegetation gestört ist. Es wird von zwei Studien berichtet, in denen offenbar NPV in windverfrachtetem Staub die Krankheit bei Larven verursachte.In einem jungen Bestand schottischer Kiefer, in dem keine neueren Meldungen von Virusauftreten vorlagen, wurden Proben genommen. Die Proben stammten aus verschiedenen Distanzen von einem ausgewachsenen, im vorherigen Winter gefällten Bestand. Die Befallsintensität sank hier scharf ab mit zunehmender Distanz vom Rand.Ferner wurde der Krankheitsbefall in Larvenkolonien an kleinen Bäumen nahe einer schmalen Forst-strasse und an Bäumen im Bestandesinnern untersucht: In Strassennähe war der Befall höher. Luftmuster wurden nahe der Strasse durch Membranfiltern gesaugt und in einem Biotest auf Virus untersucht. Wenn die Strassenoberfläche ungestört war, wurde kein Virus festgestellt. Wurde Staub aufgewirbelt, konnten infektiöse PIBs gefunden werden. Material von der Strassenoberfläche hatte eine hohe Virusaktivität.Die Übertragung des Virus vom Boden auf Kiefernnadeln dürfte demnach wichtig sein, um Krankheitsausbrüche auszulösen. Die beschriebenen Störungen waren vom Menschen verursacht. Ähnliches könnte sich unter andern Umständen ereignen, z.B. bei Windfall oder bei der Aktivität grösserer Tiere.

     

Neodiprion sertifer defoliation causes long-term systemic changes of oxidative enzyme activities in Scots pine needles

The aim of the experiments reported here was to study possible long-term effects of Neodiprion sertifer Geoff. (Hymenoptera: Diprionidae) herbivory, or artificial defoliation, on oxidative enzyme activities in Scots pine (Pinus sylvestris L.) needles as a consequence of induced defense responses. During year 1 (the first season), defoliation by N. sertifer, which feeds on previous season’s needles, did not result in statistically significant changes in polyphenol oxidase activity in the current year’s needles. In contrast, defoliation did lead to increased peroxidase activity in those needles. In the second season (year 2) N. sertifer defoliation of pine seedlings, also defoliated in the previous season either by larvae or artificially, resulted in a decrease of peroxidase activity in the current year’s needles. No significant differences between treatments carried out in year 1 were found in year 2 for peroxidase activity in the previous year’s needles. However, defoliation in year 1 by N. sertifer resulted in decreased needle consumption, and higher mortality of larvae, in year two. These results indicate the existence of long-term changes in needle oxidative enzyme activities as a consequence of N. sertifer feeding.     

Sex pheromone perception in male pine sawflies,Neodiprion sertifer (Hymenoptera; Diprionidae)

Summary Electroantennographic and single sensillum recordings were performed on male pine sawfly, Neodiprion sertifer, antennae. Responses to the sex pheromone component (2S, 3S, 7S)- 3,7-dimethyl-2-pentadecenyl (diprionyl) acetate (SSS:OAc), to the behavioral inhibitor (2S, 3R, 7R)-diprionyl acetate (SRR:OAc), to the six other enantiomers of diprionyl acetate, and to the biosynthetic precursor diprionol were recorded. Responses to trans-perillenal, a monoterpene identified in female gland extracts and to (2S, 3S, 7S)-diprionyl propionate (SSS:OPr), a field attractant for N. sertifer and some related sawfly species were also recorded.EAG recordings demonstrated a high antennal sensitivity to SSS:OAc and to SSS:OPr. A somewhat lower response was elicited by SRR:OAc.Single sensillum recordings revealed 8–12 different cells firing in each sensillum, corresponding to the number of cells observed in earlier morphological investigations. Out of these cells all, except one, responded to SSS:OAc and to SSS:OPr. No differences in the response to the two components could be observed. The largest amplitude cell in each sensillum was specifically tuned to the behavioral antagonist, SRR:OAc. The pheromone perception system encountered in male pine sawflies thus differs clearly from that observed in moths.Abbreviation EAG electroantennogram - OAc acetate - OPr propionate     

Geographic variation in winter freezing susceptibility in the eggs of the European pine sawfly (Neodiprion sertifer)

Abstract 1 The European pine sawfly, Neodiprion sertifer (Geoffroy) (Hymenoptera, Diprionidae), frequently defoliates Scots pine (Pinus sylvestris L.) forests in northern Europe. It overwinters as an egg. It has been proposed that the high egg mortality caused by low winter temperatures limits the occurrence of outbreaks to the southern part of Fennoscandia. 2 In this study, variation in freezing avoidance by egg supercooling between four Finnish populations (originating between latitudes 60°N and 69°N) of N. sertifer was tested by differential thermal analysis. Offspring of 20 females within each population were selected for the study. The freezing avoidance of parasitized eggs was also examined. 3 The northernmost Inari population was found to be the cold hardiest, and the southernmost (Hanko) was the least hardy population. The within‐population variation between females was greatest in the population from Inari, and the next greatest in the one from Hanko. The inland populations in Eastern Finland had the smallest within‐population variation in freezing avoidance. 4 The high variation in freezing avoidance of eggs will enable N. sertifer to adapt to the predicted climate change and to spread its distribution northwards. This may also change the risk for outbreaks in this area. Parasitized eggs froze at higher temperature than healthy eggs. This observation indicates that N. sertifer may experience reduced egg parasitism in certain winter climate conditions.     

Synthesis and Field Test of a Pheromone Mimic of the Pine Sawfly (Neodiprion sertifer)

A stereochemically pure mimic of the sex pheromone components of the pine sawfly was synthesized. Acetates of (2S,3S)- and (2S,3R)-3-methylpentadecan-2-ol were prepared, and the pheromone activity of their mixture was compared with that of a true pheromone mixture of the acetates of (2S,3S,7S)- and (2S,3R,7R)-3,7-dimethylpentadecan-2-ol. Although the potency of the true pheromone system was 50 times as high as that of the pheromone mimic, the mimic could attract a sufficient number of male sawflies in the field test.     

Metabolism and excretion of nitrogen during metamorphosis and egg production in the sawfly, Neodiprion sertifer

Measurements of whole-body dry matter, total nitrogen, water-soluble protein, amino acids, and uric acid were determined at successive stages during metamorphosis in Neodiprion sertifer. The major change was in the uric acid fraction: in females, it increased up to the non-pharate pupa and then decreased during the subsequent stages of adult development and egg production; in males, it continued to increase during adult development. The decline of uric acid could not be explained by the accumulation of allantoin, allantoic acid, urea, or uric acid riboside. Examination of amino acid levels in the gut revealed an accumulation in the pupa followed by a depletion at the onset of adult development. This was followed by an excretory phase marked by the progressive accumulation of large quantities of uric acid and small quantities of urea, ammonia, and amino acids during the formation of the meconium. Amino acid analysis of the meconium revealed the presence of large proportions of proline, hydroxyproline, and histidine in comparison with the other amino acids.     

Mating disruption of the pine sawfly Neodiprion sertifer (Hymenoptera: Diprionidae) in isolated pine stands

• 1 Previous applications of the mating disruption technique to Neodiprion sertifer resulted in decreased numbers of males caught in the treated area but no effect on sex ratio or overall population density.
• 2 The present study assessed the efficacy of mating disruption against N. sertifer outbreaks in pine stands surrounded by agricultural areas or pasture, and therefore isolated from other infested areas.
• 3 Pine stands were treated by placing dispensers with an erythro‐mixture of the acetate ester of 3,7‐dimethyl‐2‐pentadecanol every 10 m in a grid.
• 4 The efficacy of this technique was evaluated by comparing the number of males caught in sticky traps baited with synthetic pheromone, and by comparing the sex ratio and the population density of the sawfly in the subsequent generation between treated and control pine stands.
• 5 The number of males caught within treated areas was significantly lower than in the control area.
• 6 In the treated pine stands 46% of the egg clusters resulted in male cocoons only, compared to 3% of the clusters in the control stand.
• 7 A significant reduction of the sawfly population was observed in the treated pine stands. The results contrast to the earlier mating disruption attempts with N. sertifer and can probably be ascribed to the isolation of the experimental stands.

     

A technique for continuous rearing of Neodiprion sertifer (Geoffroy) (Hym., Diprionidae) in the laboratory

The laboratory rearing of Neodiprion sertifer (Geoffroy) on Pinus sylvestris L. plants grown in vases was successfully achieved on a continuous basis. Larvae were seen to emerge on average after 106 days from egg clusters being kept on plants held at a temperature of 15 ± 1°C and with a 10‐h photophase until emergence. These larvae were then transferred to plastic containers and kept at a temperature of 20 ± 1°C with a 12‐h photophase where, under optimum hygiene conditions and being fed fresh pine branches, they completed their development on average in 30.6 days. Adults began emerging from the cocoons after about 30 days, continuing to do so for at least 7 months. Under these conditions the insect completes its life cycle in 179 days, thus permitting to obtain two generations a year in the laboratory. This staggered adult emergence pattern makes specimens available at all stages of development for experimental purposes throughout most of the year.     

Simulated acid rain reduces the susceptibility of the European pine sawfly (Neodiprion sertifer) to its nuclear polyhedrosis virus

Summary The study dealt with the effect of simulated acid rain (both H₂SO₄ and HNO₃; acidities of pH 4 and pH 3) on the susceptibility of the larvae of Neodiprion sertifer to its nuclear polyhedrosis virus. Scots pines growing in a subarctic area with low ambient pollution levels were irrigated with simulated acid rain during two summers. Neodiprion larvae fed with foliage from the experimental trees were infected with a dilute virus suspension. The acid treatment of host trees had a significant effect on the proportion of virus-treated larvae alive 16 days after the virus application: there were almost no differences between the controls and the pH 4 irrigation group, but on the needles of pH 3-treated trees larval survival was twice as high as with other treatments. The direct spraying of acid water on the needles before they were fed to the larvae did not significantly affect the survival of virus infected larvae. Our results suggest that acid rain may reduce the susceptibility of Neodiprion larvae to virus disease via changes in the quality of pine foliage.