Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.

First report of 'Candidatus Phytoplasma rubi' associated with Rubus stunt disease of raspberry and blackberry in the Czech Republic.

Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to 'Candidatus Phytoplasma rubi' strain RS (GenBank Accession No. CP114006). To further characterize the 'Ca. P. rubi' strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with 'Ca. P. rubi' RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % 'Ca. Phytoplasma' strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference 'Ca. P. rubi' RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of 'Ca. P. rubi' from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.

Different maize varieties have greater impact on Tetranychus urticae (Acari: Tetranychidae) than GE maize expressing Cry3Bb1 insecticidal protein.

Genetically engineered (GE) maize expressing Bacillus thuringiensis Cry3Bb1 protein was developed to control Diabrotica spp. (Coleoptera: Chrysomelidae). However, Cry proteins have been reported to have effects on non-target arthropods. We therefore investigated whether the non-target pest Tetranychus urticae (Acari: Tetranychidae) was negatively affected by GE maize expressing the insecticidal Cry3Bb1 protein. Five treatments were used in the laboratory tests to determine the life history parameters of T. urticae on leaves of field-grown maize varieties: (1) GE maize MON 88017, (2) isogenic maize, (3) isogenic maize protected with the soil applied insecticide chlorpyrifos (Dursban 10G), and two unrelated varieties (4) Kipous and (5) PR38N86. Newly emerged T. urticae larvae were individually released on the upper surface of leaf discs placed on water saturated cotton wool. Immatures and adults survival, duration of developmental stages and female fecundity were recorded daily until T. urticae died. Age-stage, two-sex life table method and test for trends, revealed no significant differences in 13 of 18 studied parameters. The unrelated varieties Kipous and PR38N86 on one side and maize with the same genetic background, namely GE maize and isogenic maize with or without insecticide protection, on the other side, showed significant variations in male longevity, larval survival rate, preoviposition period, and fecundity. In addition to the differences between varieties, GE maize and insecticide-protected isogenic maize showed a substantial difference in age-specific fecundity, but not in the mean number of eggs laid by females. The obtained results do not indicate that consumption of Cry3Bb1 has negative effect on T. urticae and suggest that GE maize does not pose a risk with respect to the non-target mite pest T. urticae. The results may have implications for the approval and renewal of import and cultivation for GE crop in the European Union.

Known and Potential Invertebrate Vectors of Raspberry Viruses.

The estimated global production of raspberry from year 2016 to 2020 averaged 846,515 tons. The most common cultivated Rubus spp. is European red raspberry (Rubus idaeus L. subsp. idaeus). Often cultivated for its high nutritional value, the red raspberry (Rubus idaeus) is susceptible to multiple viruses that lead to yield loss. These viruses are transmitted through different mechanisms, of which one is invertebrate vectors. Aphids and nematodes are known to be vectors of specific raspberry viruses. However, there are still other potential raspberry virus vectors that are not well-studied. This review aimed to provide an overview of studies related to this topic. All the known invertebrates feeding on raspberry were summarized. Eight species of aphids and seven species of plant-parasitic nematodes were the only proven raspberry virus vectors. In addition, the eriophyid mite, Phyllocoptes gracilis, has been suggested as the natural vector of raspberry leaf blotch virus based on the current available evidence. Interactions between vector and non-vector herbivore may promote the spread of raspberry viruses. As a conclusion, there are still multiple aspects of this topic that require further studies to get a better understanding of the interactions among the viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better pest management strategies.

Virulence of Beauveria bassiana Strains Isolated from Cadavers of Colorado Potato Beetle, Leptinotarsa decemlineata.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a serious, widely distributed pest of potato and other crops. This pest is able to defoliate the host plant and cause severe yield loss. Moreover, the pest quickly becomes resistant to many chemical pesticides. Therefore, the development of novel biopesticides targeting this pest is urgently needed. The purpose of this study was to obtain new strains of the entomopathogenic fungus Beauveria bassiana and assess their efficacy against L. decemlineata adults under laboratory conditions. Twelve strains were isolated from cadavers of Colorado potato beetles collected in potato fields in the Czech Republic. Test beetles were treated by suspensions of conidia at the concentration of 1 × 107 spores per milliliter and their survival was recorded daily for three weeks. The results of the bioassays revealed that all new native strains were pathogenic to L. decemlineata adults and caused mortality up to 100% at the end of the trial period with an LT50 of about 7 days. These strains were more virulent than a reference strain GHA and some of them can be recommended for the development of a new mycoinsecticide against L. decemlineata. Our findings also highlight the importance of searching for perspective strains of entomopathogenic fungi among naturally infected hosts.

Low Efficacy of Isaria fumosorosea against Box Tree Moth Cydalima perspectalis: Are Host Plant Phytochemicals Involved in Herbivore Defence against Fungal Pathogens?

Buxus sp. is an important native and ornamental tree in Europe threatened by a serious invasive pest Cydalima perspectalis. The larvae of this moth are able to defoliate box trees and cause their death. The development of novel biopesticides targeting this pest might help protect Buxus trees grown wildly or in city parks. Laboratory experiments were conducted to assess the efficacy of entomopathogenic fungus Isaria fumosorosea strain CCM 8367 against C. perspectalis. The last-instar larvae of the box tree moth were treated by the suspension of fungus conidia at concentrations ranging from 1 × 104 to 1 × 108 spores per 1 mL. Fungus infection was observed mostly in pupae, but the maximum mortality did not exceed 60%, indicating a very low susceptibility of C. perspectalis to I. fumosorosea. Furthermore, a number of ungerminated fungal conidia were found on larval cuticles using a low-temperature scanning electron microscopy. Our data also reveal that the hydroalcoholic extract from B. sempervirens leaves significantly inhibits both the germination of I. fumosorosea conidia and fungus growth. It can be speculated that the strain CCM 8367 of I. fumosorosea is not a potent biocontrol agent against C. perspectalis and low virulence of the fungus might be due to the accumulation of host plant phytochemicals having antimicrobial activity in larval cuticle of the pest.

Dissemination of Isaria fumosorosea Spores by Steinernema feltiae and Heterorhabditis bacteriophora.

Entomopathogenic nematodes and fungi are globally distributed soil organisms that are frequently used as bioagents in biological control and integrated pest management. Many studies have demonstrated that the combination of biocontrol agents can increase their efficacy against target hosts. In our study, we focused on another potential benefit of the synergy of two species of nematodes, Steinernema feltiae and Heterorhabditis bacteriophora, and the fungus Isaria fumosorosea. According to our hypothesis, these nematodes may be able to disseminate this fungus into the environment. To test this hypothesis, we studied fungal dispersal by the nematodes in different arenas, including potato dextrose agar (PDA) plates, sand heaps, sand barriers, and glass tubes filled with soil. The results of our study showed, for the first time, that the spreading of both conidia and blastospores of I. fumosorosea is significantly enhanced by the presence of entomopathogenic nematodes, but the efficacy of dissemination is negatively influenced by the heterogeneity of the testing arena. We also found that H. bacteriophora spread fungi more effectively than S. feltiae. This phenomenon could be explained by the differences in the presence and persistence of second-stage cuticles or by different foraging behavior. Finally, we observed that blastospores are disseminated more effectively than conidia, which might be due to the different adherence of these spores (conidia are hydrophobic, while blastospores are hydrophilic). The obtained results showed that entomopathogenic nematodes (EPNs) can enhance the efficiency of fungal dispersal.

Adipokinetic hormone promotes infection with entomopathogenic fungus Isaria fumosorosea in the cockroach Periplaneta americana.

The adipokinetic hormones (AKHs) are known to be involved in insect immunity, thus their role in the cockroach Periplaneta americana infected with the entomopathogenic fungus Isaria fumosorosea was examined in this study. The application of I. fumosorosea resulted in a significant increase in both Akh gene expression and AKH peptide levels. Further, co-application of I. fumosorosea with Peram-CAH-II significantly enhanced cockroach mortality compared with the application of I. fumosorosea alone. The mechanism of AKH action could involve metabolic stimulation, which was indicated by a significant increase in carbon dioxide production; this effect can increase the turnover and thus efficacy of toxins produced by I. fumosorosea in the cockroach's body. I. fumosorosea treatment resulted in a significant decrease in haemolymph nutrients (carbohydrates and lipids), but co-application with Peram-CAH-II restored control level of lipids or even further increased the level of carbohydrates. Such nutritional abundance could enhance the growth and development of I. fumosorosea. Further, both I. fumosorosea and Peram-CAH-II probably affected oxidative stress: I. fumosorosea alone curbed the activity of catalase in the cockroach's gut, but co-application with Peram-CAH-II stimulated it. Interestingly, the hormone alone had no effect on catalase activity. Taken together, the results of the present study demonstrate the interactions between the fungus and AKH activity; understanding this relationship could provide insight into AKH action and may have practical implications for insect pest control in the future.

Laboratory Evaluation of Isaria fumosorosea CCM 8367 and Steinernema feltiae Ustinov against Immature Stages of the Colorado Potato Beetle.

The Colorado potato beetle, Leptinotarsa decemlineata, has developed resistance to most registered pesticides and has become one of the most difficult insect pests to control. Development of new biopesticides targeting this pest might solve the resistance problem and contribute to sustainable crop production. Laboratory experiments were conducted to assess the efficacy of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) strain CCM 8367 against L. decemlineata when applied alone or combined with the entomopathogenic nematode Steinernema feltiae. The last-instar larvae of the Colorado potato beetle showed the highest susceptibility to I. fumosorosea followed by pre-pupae and pupae. The median lethal concentration (LC50) was estimated to be 1.03×106 blastospores/ml. The strain CCM 8367 was more virulent, causing 92.6% mortality of larvae (LT50 = 5.0 days) compared to the reference strain Apopka 97, which caused 54.5% mortality (LT50 = 7.0 days). The combined application of the fungus with the nematodes increased the mortality up to 98.0%. The best results were obtained when S. feltiae was applied simultaneously with I. fumosorosea (LT50 = 2.0 days); later application negatively affected both the penetration rate and the development of the nematodes. We can conclude that the strain CCM 8367 of I. fumosorosea is a prospective biocontrol agent against immature stages of L. decemlineata. For higher efficacy, application together with an entomopathogenic nematode is recommended.

A novel function of red pigment-concentrating hormone in crustaceans: Porcellio scaber (Isopoda) as a model species.

The RP HPLC and LC/MS QTOF analyses of the methanolic CNS extract from isopod crustacean the woodlouse, Porcellio scaber revealed a presence of the red pigment-concentrating hormone (Panbo-RPCH) in this species. It has been shown that this neuropeptide plays a role in mobilization of energy stores: topical treatments of P. scaber individuals by Panbo-RPCH in a concentration 20 pmol/microl increased the level of glucose in haemolymph about 4 times, while the level of trehalose was only doubled. The results demonstrated that glucose was the main carbohydrate mobilized by the Panbo-RPCH treatment: glucose was responsible for about 97% of total carbohydrate increasing. Despite the demonstration of hyperglycaemic activity of Panbo-RPCH, no stimulatory effect of this hormone on the locomotory activity of P. scaber was observed. The present study is the first discovery of an occurrence of Panbo-RPCH and its hyperglycaemic activity in the representative of the isopod crustaceans. The relationship of the function of Panbo-RPCH in P. scaber to the role of this neuropeptide and adipokinetic hormones in insects is discussed.

Stimulatory effects of bioamines norepinephrine and dopamine on locomotion of Pyrrhocoris apterus (L.): is the adipokinetic hormone involved?

In the present paper we studied the effects of five biogenic amines - norepinephrine, dopamine, octopamine, serotonin and histamine - on the locomotory activity and mobilization of lipids in the adult females of the firebug, Pyrrhocoris apterus (L.). We tested the hypothesis (1) whether the stimulation of walking activity in the bugs injected with the bioamines is associated also with their hyperlipaemic effects, like in the case of adipokinetic hormones (AKHs), and (2) whether these effects are direct or mediated through a release of the AKHs into the hemolymph. The results demonstrated that all five tested biogenic amines mobilized the fat body lipids, but only norepinephrine and dopamine were capable to enhance the walking activity simultaneously with an elevation of the lipid level in the hemolymph. Those two amines had no effect on the level of AKHs in CNS, but modulated the AKHs level in hemolymph: norepinephrine increased it, while dopamine decreased it. The results indicate an apparent feedback between AKH characteristics and dopamine and norepinephrine actions occurring in this insect species. While the stimulatory effects of norepinephrine on lipid mobilization and walking activity could involve the release of bug's own AKHs, dopamine probably employs an independent stimulatory pathway.

Effects of metabolic inhibitors on activity of Cry1Ab toxin to inhibit growth of Ephestia kuehniella larvae.

BACKGROUND: Although the toxins of Bacillus thuringiensis Berliner (Bt) are frequently used to control lepidopteran pests, the tolerance or resistance of some lepidopteran moth strains may limit Bt applications. In this study, insecticidal cocktails consisting of sublethal doses of Cry1Ab toxin and additive compounds were tested for their suppressive effect on larval relative growth rate (RGR) in Ephestia kuehniella Zeller under laboratory conditions. RESULTS: In the first step, the suppressive effect of diflubenzuron, soybean trypsin inhibitor (STI) and chitinase on RGR was confirmed. In the second step, these compounds were incorporated into a mixture of crushed kernels of Bt maize hybrid MON 810-YieldGard and its isoline with concentrations of Cry1Ab toxin ranging from 0.011 to 0.091 microg g(-1) diet. An additive effect on the suppression of larval RGR in E. kuehniella was found in a combination of diflubenzuron, STI and STI + chitinase as secondary compounds in insecticidal cocktails. Chitinase showed no additive effect on RGR. CONCLUSION: The highest suppression level was found in cocktails with STI + chitinase as a secondary compound. It is hypothesized that the protease inhibitor (STI) protects both chitinase and Cry1Ab proteins from endogenous proteases in the larval midgut and prolongs their insecticidal activities. The possible application of insecticidal cocktails in the control of E. kuehniella is discussed.

The influence of conspecific chemical cues on walking behavior of Ixodes ricinus males.

The influence of female and male cues on the male searching behavior of the sheep tick, Ixodes ricinus (Acari: Ixodidae), was studied under constant laboratory conditions by means of a computerized video tracking system. Males were released into an open glass arena in which one half had been exposed for 1 h to 5 freely walking unfed females or five males. The obtained track data were analyzed to reveal if male walking pattern was random or affected by cues of conspecifics left on the arena surface. The results showed that males spent significantly more time and walked a significantly longer distance in the area with female cues compared to the control area. Moreover, the walking path of males was more tortuous on the cues half than the control half. In contrast, walking behavior of males was not affected by male cues in any observed parameter. These results provide the first evidence that unfed females leave sex-specific semiochemicals on a substrate during their walk and that these cues have an effect on male searching behavior. Since this effect was not observed when female cues were 24-h old, the semiochemicals seem to either evaporate or are subject to degradation. The biological significance of these findings for mate-searching in I. ricinus males is discussed.

Perspectives for the biological control of Cameraria ohridella.

The horse chestnut leaf-miner, Cameraria ohridella Deschka et Dimic (Lepidoptera, Gracillariidae) is a serious invasive pest of Aesculus hippocastanum in Europe. The larvae of this species feed on leaf parenchyma and can reduce the tree growth. We studied the impact of parasitoids on C. ohridella in the Czech Republic and also searched for entomopathogenic fungi associated with this pest. The results showed that the rate of parasitism varied between 5% and 15% in most cases. The most parasitized stages of C. ohridella were spinning stages and especially pupae. The most abundant parasitoid species were Minotetrastichus frontalis, Pnigalio sp. and Pediobius saulius (Hymenoptera, Eulophidae). All species are polyphagous. Using the Galleria-bait method we isolated many strains of entomopathogenic fungi. Dominant species were Paecilomyces fumosoroseus, Paecilomyces farinosus and Beauveria bassiana. The perspectives of fungal bioagents in control of C. ohridella is discussed.

Interactions in a tritrophic acarine predator-prey metapopulation system V: within-plant dynamics of Phytoseiulus persimilis and Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae).

To investigate the relative contributions of bottom-up (plant condition) and top-down (predatory mites) factors on the dynamics of the two-spotted spider mite (Tetranychus urticae), a series of experiments were conducted in which spider mites and predatory mites were released on bean plants. Plants inoculated with 2, 4, 8, 16, and 32 adult female T. urticae were either left untreated or were inoculated with 3 or 5 adult female predators (Phytoseiulus persimilis) one week after the introduction of spider mites. Plant area, densities of T. urticae and P. persimilis, and plant injury were assessed by weekly sampling. Data were analysed by a combination of statistical methods and a tri-trophic mechanistic simulation model partly parameterised from the current experiments and partly from previous data. The results showed a clear effect of predators on the density of spider mites and on the plant injury they cause. Plant injury increased with the initial number of spider mites and decreased with the initial number of predators. Extinction of T. urticae, followed by extinction of P. persimilis, was the most likely outcome for most initial combinations of prey and predators. Eggs constituted a relatively smaller part of the prey population as plant injury increased and of the predator population as prey density decreased. We did not find statistical evidence of P. persimilis having preference for feeding on T. urticae eggs. The simulation model demonstrated that bottom-up and top-down factors interact synergistically to reduce the density of spider mites. This may have important implications for biological control of spider mites by means of predatory mites.

Interactions in a tritrophic acarine predator-prey metapopulation system III: effects of Tetranychus urticae (Acari: Tetranychidae) on host plant condition.

Spider mites are serious pests on many economically important plant species, because they may reduce plant productivity and, at high mite densities, overexploit and even kill the host plants. We have conducted a series of greenhouse experiments to quantify the effects of two-spotted spider mites (Tetranychus urticae) on host plants (Phaseolus vulgaris). The average amount of chlorophyll per cm2 leaf area was used as a measure of plant condition. It was shown that chlorophyll concentration decreases with plant age and intensity of spider mite feeding. Damage caused by spider mites was assessed visually, using the Leaf Damage Index (LDI) defined by Hussey and Parr (1963b), and a mathematical relationship between the visual measurements and the amount of chlorophyIV cm2 was fitted to data. The relationship may serve as a short-cut to estimate overall plant injury, expressed as the relative loss of chlorophyll/cm2 leaf area caused by spider mites (D). D takes values between 0 (no injury) and I (all leaves dead). A highly significant positive relationship between the instantaneous spider mite density and D was found, even though D is expected to reflect the cumulated density of mites (mite-days). A model of plant growth incorporating information about plant age and D predicts that plant area has a maximum when plant age is about 60 days, and that plant area decreases exponentially with an increase in D.

Interactions in a tritrophic acarine predator-prey metapopulation system IV: effects of host plant condition on Tetranychus urticae (Acari: Tetranychidae).

Feeding by spider mites can cause severe injury to a host plant and lead to a decreasing per capita growth rate and an increasing per capita emigration rate. Such density-dependent responses to local conditions are important in a metapopulation context because they allow the herbivores to colonize new host plants and thereby prolong the time until regional (metapopulation) extinction. In order to include density-dependent responses of the two-spotted spider mite (Tetranychus urticae) in a realistic metapopulation model, a series of greenhouse experiments was conducted with the purpose to quantify how the condition of bean plants (Phaseolus vulgaris) influences the demographic parameters of T. urticae. Plant age per se reduced the growth rate of the spider mites only slightly, whereas the growth rate declined significantly as the plants were injured by the mites. The relationships between plant condition (expressed by the plant injury index D) and the birth and loss (death + emigration) rates of the mites were quantified so as to predict population growth as a function of D. Maximum per capita growth rate (r) was estimated to be c. 0.21 day(-1). The growth rate is expected to be negative when D exceeds 0.8. When mites were allowed to emigrate to neighbouring plants via bridges, the per capita emigration rate increased almost exponentially with D. The proportion of eggs in the population decreased with D while the numerical ratio between immatures to adults and the sex ratio did not change with D. Overall, immatures and adults constituted 74% and 26%, respectively, of the active mites and c. 46% of the adults were males. The bridges that connected a donor plant with the surrounding recipient plants were responsible for the majority of the emigrations from donor plants. Most mites stopped after having crossed a single bridge, but a few crossed two bridges while none crossed three bridges within 24 h. The significance of the results for biological control is discussed.

Mode of action of an insect neuropeptide leucopyrokinin (LPK) on pupariation in fleshfly (Sarcophaga bullata) larvae (Diptera: Sarcophagidae).

An insect neuropeptide leucopyrokinin (LPK) (pQTSFTPRLamide) accelerates pupariation in wandering larvae of the fleshfly Sarcophaga bullata. The period of sensitivity to the action of LPK begins approximately 4 h before pupariation. Within this period the degree of acceleration of contraction into the shape of a puparium is practically independent of the age at which the larvae are injected, while acceleration of tanning is distinctly more age dependent. From ligation experiments we conclude that intact central innervation is essential for the action of LPK on puparial contraction, whereas central neurones take no part in mediation of LPK action on tanning of the cuticle. An analysis of tensiometric recordings of muscular activity revealed that the actual time of LPK accelerated puparial contraction coincides with the beginning of the immobilisation/retraction phase. LPK accelerates the switch from wandering behaviour to immobilisation/retraction behaviour but has no effect on the onset and duration of motor patterns that normally underlie puparial contraction in controls. The morphology of an accelerated puparium is normal but its formation is temporally dissociated from normal 'contraction patterns' that are performed a long time after the puparium has contracted. It means that neuromuscular activity of larvae accelerated by LPK does not cease upon formation of the white puparium, but continues until the whole motor programme of pupariation behaviour is completed. Apparently the peptide acts on the integument by stimulating it to contract and shrink, and no specific patterns of muscular contractions are needed to properly shape the puparium. This finding sheds a new light on our understanding of the mechanism of puparium formation.