Biological control of Lygus lineolaris by Peristenus spp. in strawberry

Peristenus digoneutis Loan (Hymenoptera: Braconidae) was introduced to the US for biological control of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), and has since spread through much of the northeast. The purpose of this study was to determine if P. digoneutis and a native congener, Peristenus pallipes (Curtis), parasitize L. lineolaris in strawberry (where it is a key pest), and what factors relate to parasitism levels. During 1997–1999 we monitored parasitism on 17 strawberry farms in 14 counties in eastern and western New York State. We found that in eastern NY (where P. digoneutis has been established since the early 1990s), overall mean parasitism was 19.7% (ranging from 0 to 70%), mostly by P. digoneutis. Mean parasitism was significantly lower (12.3%, ranging from 0 to 58%) in western NY (where P. digoneutis was first recorded in 1999), and was mostly by P. pallipes. P. pallipes parasitism was significantly lower in eastern than western NY, suggesting the potential for competitive interaction with P. digoneutis. The insecticide regime of a farm was an important factor influencing parasitism rate, which was 5- to 6.5-fold higher on organic or casually sprayed farms than on intensely treated farms, though pest density under these three regimes was not significantly different. L. lineolaris density, and parasitism rate in nearby alfalfa and abandoned fields were also significant factors for parasitism in strawberry.     

Comparative analysis of the reproductive biology of two Peristenus species (Hymenoptera: Braconidae), biological control agents of Lygus plant bugs (Hemiptera: Miridae)

Peristenus digoneutis Loan and Peristenus stygicus Loan, parasitoids of the European tarnished plant bug Lygus rugulipennis Poppius, are established in the United States for biological control of native North American Lygus species, and are being considered for deliberate release in Canada. High lifetime fecundity of parasitoids is considered a desirable attribute of biological control agents and therefore, an understanding of parasitoid reproductive biology is required. In the present study, the potential lifetime fecundity of both agents was compared under laboratory conditions to estimate the potential impact of Peristenus species on Lygus. Synovigenic P. digoneutis and P. stygicus females oviposited most actively in the first two weeks of their lifetime, with a maximum average daily oviposition rate after five days. The maximum number of eggs laid per day was 83 eggs for P. stygicus, and 36 eggs for P. digoneutis. P. digoneutis has an average potential lifetime fecundity of 385 ± 35 SE eggs produced over 22 ± 3 SE days. In contrast, P. stygicus females have a 50% higher mean potential lifetime fecundity reaching 782 ± 65 SE eggs over 28 ± 1 SE days. A positive correlation between lifetime fecundity and body size was found only for P. stygicus, and both species showed a significant relationship between lifetime fecundity and oviposition period. The present study demonstrates that the fecundity of P. digoneutis and P. stygicus is considerably higher than previously reported. Based on these findings, P. stygicus appears to be the most effective biological control agent for Lygus lineolaris (Palisot de Beauvois) when only fecundity is taken into consideration.     

Changes in abundance of native and introduced parasites (Hymenoptera: Braconidae), and of the target and non-target plant bug species (Hemiptera: Miridae), during two classical biological control programs in alfalfa.

High numbers of tarnished plant bugs [Lygus lineolaris (Palisot)], were once common in alfalfa, as was a low level of parasitism (9%) by the native Peristenus pallipes (Curtis). After the bivoltine European parasite Peristenus digoneutis Loan became well established, average parasitism of the first and second generations increased to 64%, and tarnished plant bug numbers dropped by 65%. This reduced host density eventually caused a decline in total parasitism by both parasite species to 22%. A few P. digoneutis also attacked the alfalfa plant bug, Adelphocoris lineolatus (Goeze), but did not reduce this pest or increase its parasitism rate. At another location, where P. digoneutis is not established, parasitism of first generation alfalfa plant bugs, an adventive (accidently introduced) pest, was increased to 21% by the introduced univoltine parasite, Peristenus conradi Marsh, and a slight reduction in the pest may have resulted. P. digoneutis did not parasitize the meadow plant bug, Leptopterna dolabrata (L.), an adventive pest of forage grasses, so did not affect this mirid or its parasite. Neither introduced parasite eliminated the native parasites of the tarnished or alfalfa plant bugs. The narrow host ranges of the braconid parasites of mirid nymphs are contrasted with the broad host range of the native tachinid parasite [Phasia robertsoni (Towns.)] of adult mirids. The major changes in mirid abundance and their mortality by parasites that slowly occurred during this 19-year study demonstrate the need for long-term field research, to adequately document and understand these complex interactions.     

A large-scale rearing method for Peristenus digoneutis (Hymenoptera: Braconidae), a biological control agent of Lygus lineolaris (Hemiptera: Miridae)

Releases of Peristenus digoneutis against Lygus spp. in North America have been conducted for many years; however, no published procedures for mass production of the biological control agent were available. A laboratory rearing method was developed using Lygus lineolaris as the host to enhance establishment efforts and provide large numbers of wasps for inundative releases into high value fruit crops. Experiments were conducted to determine optimum host:parasitoid density and rearing temperature. The effects of nymph:wasp ratios and temperature on parasitism and wasp survival showed a 20:1 ratio at 20°C provided high parasitism (256 parasitized nymphs/wasp over lifetime) and excellent wasp survival of 27 days. Experiments on diapause-inducing conditions for P. digoneutis demonstrated that fluctuating temperatures of 23°C (day) and <16°C (night) and corresponding photo phases of 16 h light, for rearing parasitized nymphs, produced 100% diapausing parasitoids whereas non-diapausing parasitoids were only produced at more than 16 h light. Furthermore, parasitized Lygus nymphs need to be transferred to short day conditions no later than 10 days after parasitism to produce diapausing parasitoids. Critical life stages for exposure to conditions inducing diapause, the egg, first and second instar parasitoid larva, occurred from 0 to 10 days at 24°C constant temperature. Increased time in cold storage reduced the number of days to first emergence of parasitoids from diapausing cocoons when transferred to warm temperatures. The optimum storage time for diapausing P. digoneutis is between 25 and 44 weeks, depending upon the length of time that cocoons remain at warm conditions prior to chilling.     

A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp.

Lygus Hahn (Hemiptera: Miridae) are serious pests of agricultural and greenhouse crops throughout North America. In Europe, bivoltine Peristenus Förster (Hymenoptera: Braconidae) species have a significant impact on Lygus populations. Release and establishment of European P. digoneutis Loan in Lygus lineolaris (Palisot de Beauvois) populations in northeastern USA has renewed interest in the intended liberation of European parasitoids for Lygus control in Canada. Accurate identification of natural enemies is the cornerstone of biological control but conventional methods for identifying Peristenus species and estimating parasitism rates rely on tedious and time-consuming dissection and rearing methods. The present study describes species-specific PCR primers for three species of Peristenus, and the use of a multiplex PCR assay to detect P. digoneutis and P. stygicus Loan eggs and larvae from Lygus rugulipennis Poppius nymphs. Results indicate that the primers amplify uniquely sized, species-specific PCR products for the three species and are capable of detecting single eggs in parasitized nymphs within 3 days post-parasitism. Using a multiplex PCR assay, the primers maintain specificity and sensitivity, and allow detection of each of the three species in a single reaction. Although molecular diagnostics have previously been used in the identification of parasitoids and estimation of parasitism rates, this is the first time a single-step multiplex PCR protocol has been described.     

Bioclimatic analyses of distributions of a parasitoid Peristenus digoneutis and its host species Lygus spp. in Europe and North America

• 1 Peristenus digoneutis Loan is a parasitoid of Lygus plant bugs, which was successfully introduced from Europe into North America in the 1980s for controlling native Lygus populations. Surveys confirmed that P. digoneutis populations have become established throughout eastern North America and that the spread of the parasitoid continues. For unknown reasons, previous releases of P. digoneutis in Western Canada were not successful.
• 2 A bioclimate (climex ®; Hearne Scientific Software Pty Ltd, Australia) model for P. digoneutis in North America was developed, based on climate and ecological parameters, and then validated with actual distribution records. The current distribution of P. digoneutis in eastern North America was consistent with the predicted distribution. The model suggests that P. digoneutis will probably continue its spread westwards throughout the U.S.A. along the Great Lakes.
• 3 The southern distribution of P. digoneutis is expected to be limited by hot summer temperatures, whereas its northern range is limited by the number of Lygus host generations rather than cold stress.
• 4 Peristenus digoneutis has the potential to occur in the southern parts of the prairie ecozone of western Canada; however, Ecoclimatic Index values in the prairies indicate mainly marginal or unfavourable conditions, which may explain why earlier releases of P. digoneutis in Western Canada failed.

     

The natural occurrence of Pandora heteropterae (Zygomycetes: Entomophthorales) infecting Lygus lineolaris (Hemiptera: Miridae)

An unknown fungal pathogen was recovered from Lygus lineolaris (Palisot de Beauvois) during a survey of parasitic and pathogenic natural enemies conducted in Franklin County, Arkansas. The pathogen was identified as Pandora heteropterae (Ba?azy) Keller based on characteristics of the morphology, as well as growth and sporulation on hosts. The fungus infected 11 of the 3405 (0.32%) wild L. lineolaris collected. In a laboratory host-range bioassay, five of seven hemipteran species from the families Miridae, Coreidae, Lygaeidae, and Pentatomidae were successfully infected. P. heteropterae was previously reported only once, from an unidentified host species in Poland. Here we describe the morphology and growth of P. heteropterae and discuss its potential impact on L. lineolaris in the field.     

Detection of European Peristenus digoneutis loan in mirid populations in southern Ontario

Lygus spp. Hahn (Hemiptera: Miridae) are serious pests of a wide variety of economically important crops in North America. A European parasitoid, Peristenus digoneutis Loan (Hymenoptera: Braconidae), released in the northeastern USA for the biological control of Lygus, has successfully established in nine states and in eastern Canada, including southeastern Ontario, southern Quebec and Nova Scotia. To determine the extent to which P. digoneutis has dispersed and established in mirid populations in Ontario, a single-step multiplex PCR assay, designed to differentiate European species P. digoneutis and P. relictus from native North American Peristenus species, was used to identify parasitoid larvae dissected from field-collected Miridae. A total of 222 parasitoid larvae were analysed with the multiplex assay. Most (172) were identified as members of the P. pallipes Curtis complex; however, three from the Niagara region were identified as P. digoneutis. Specimens that did not amplify using the Peristenus multiplex PCR assay were screened with a PCR primer set designed to detect Leiophron (Hymenoptera: Braconidae) species, and most were confirmed to be Leiophron larvae. This study confirms the presence of P. digoneutis species in southern Ontario, and is another example of the utility of molecular methods for the detection of newly introduced or dispersed parasitoids. The presence of exotic P. digoneutis in southern Ontario may expedite future releases to augment already-established populations. Following such releases, the Peristenus multiplex PCR assay will be a useful component of post-release studies to evaluate the success of the biological control programme for Lygus plant bugs in Canada.     

PCR-based,rapid diagnosis of parasitism of Lygus spp. (Hemiptera: Miridae) by Peristenus relictus (Hymenoptera: Braconidae)

Several species of Lygus (Heteroptera: Miridae: Mirini) are serious crop pests in North America where their parasitism rate by native nymphal parasitoids is generally lower than in Europe. Peristenus relictus (Ruthe) (formerly P. stygicus Loan) (Hymenoptera: Braconidae: Euphorinae) is the predominant nymphal parasitoid of several Lygus spp. in the warm Mediterranean region and has been a candidate for introduction against Lygus hesperus Knight and L. lineolaris (Palisot de Beauvois) in the southern US. We report a rapid, sensitive, and specific PCR-based assay for diagnosis of P. relictus immature stages within Lygus nymphs that entails three steps: DNA extraction, PCR of the partial mitochondrial COI gene, and agarose gel electrophoresis. The PCR-based methodology is species-specific because the target DNA of other sympatric, congeneric species was not amplified with use of the primers developed for P. relictus diagnosis. The sensitivity of the PCR method, assessed through spike tests, was established by the detection of a ratio of 1:10,000 P. relictus DNA to Lygus DNA. Molecular diagnosis of parasitism of field collected nymphs is achievable in one day, eliminating the need to rear nymphs to obtain adult parasitoids for morphological identification.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the tarnished plant bug, Lygus lineolaris L., in strawberries

Beauveria bassiana has a high insecticidal potential to control the tarnished plant bug, Lygus lineolaris, a significant pest of strawberries. Screening experiments showed that L. lineolaris adults were susceptible to several B. bassiana isolates. Another screening test with Coleomegilla maculata, a natural enemy found in strawberries, was also performed in order to select the isolate having lower entomopathogenic impact on this insect. Based on data obtained from both insect species and on the ecozone origin of the B. bassiana isolates, INRS‐IP and INRS‐CFL isolates were selected for further experiments. The LC₅₀ values of these two isolates against L. lineolaris adults were 7.8 × 10⁵ and 5.3 × 10⁵ conidia/ml, and average survival time (AST) values were 4.46 and 4.37 days at a concentration of 1 × 10⁸ conidia/ml respectively. Results also indicated that L. lineolaris nymphs are susceptible to the selected isolates. During field experiments, using a randomized block design with four replicates, INRS‐IP and INRS‐CFL isolates were applied at two rates (1 × 10¹¹ and 1 × 10¹³ conidia/ha) weekly during a period of 4 weeks. These multiple applications triggered a significant reduction of L. lineolaris nymphal populations in strawberries. Twenty‐four days after the first application, a significant difference was observed between the mean population densities of surviving nymphs in all B. bassiana‐treated plots (less than one insect per five plants) compared with those in control plots (four insects per five plants). During the field experiment, persistence of insecticidal activity and viability of B. bassiana conidia were also monitored. The results showed the presence of viable and infective conidia up to 6 days after each application on strawberry foliage. Moreover, the multiple applications of B. bassiana at the rate of 1 × 10¹³ conidia/ha triggered a significant reduction in strawberry fruit injuries induced by L. lineolaris feeding behaviour compared with the control plots.     

Parasitism ofLygus lineolaris eggs onCoronilla varia,solanum tuberosum,and three host weeds in southeastern Québec

Parasitism ofLygus lineolaris (Palisot de Beauvois) eggs by three species ofMymaridae, Anaphes iole Girault,Erythmelus miridiphagus Dozier andPolynema pratensiphagum Walley and one species ofScelionidae, Telenomus sp., was investigated at Ste-Anne-de-Bellevue, Québec. The maximum level of field parasitism ofL. lineolaris eggs by individual species was 15.4, 53.8, 70.0 and 16,7%, respectively. Parasitoids were retrieved from eggs ofL. lineolaris inserted in stems and branches ofAmaranthus retroflexus L.,Chenopodium album L.,Coronilla varia L.,Rumex obtusifolius L. andSolanum tuberosum L. Amaranthus retroflexus andS. tuberosum sustained a large population ofL. lineolaris and egg parasitism was high enough to consider it as a potential control factor.      

Effects of different Beauveria bassiana isolates on field populations of Lygus lineolaris in pigweed (Amaranthus spp.)

The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is a pest of various fruit, vegetable, fiber, and seed crops; including cotton. Lygus spp. populations often build on alternate host plants before moving to cotton, and in the midsouthern U.S. wild host plants, such as pigweed (Amaranthus spp.), play a major role in L. lineolaris population development. Three isolates of the entomopathogenic fungus Beauveria bassiana (Balsamo) were evaluated for L. lineolaris control in redroot pigweed (Amaranthus retroflexus L.): one from L. lineolaris in Mississippi (TPB3); one from Lygus hesperus (Knight) in California (WTPB2); and one commercial isolate from Mycotrol^® (GHA). Fungal applications resulted in moderate to high mycosis in adults (33 to 80%) and moderate mycosis in nymphs (36 to 53%) that were collected from field plots at 2 days post-treatment and incubated under laboratory conditions. Although TPB3 was previously found to be more pathogenic in laboratory bioassays, there was not a consistent separation of this isolate from the other two isolates in field trials. Where differences in adult mycosis or mortality were observed, TPB3 was the most pathogenic. However, in one field trial 7 day mortality for nymphs treated with GHA was higher than those treated with TPB3 or WTPB2. Infection rates at 2, 7, and 14 days post-treatment from caged and non-caged adults suggested that movement of adults among plots occurred, which could have masked some treatment effects. Fungal treatments did not significantly reduce populations relative to controls. This may have been caused by delayed mortality rates under field conditions and/or difficulties with estimating population change under field conditions characteristic of wild host plant populations (e.g., heterogeneous populations, adult movement, and small plot size). Further work evaluating time–dose–mortality over dynamic temperatures, spring and fall field trials on this and other wild hosts, and improved methods for estimating populations on wild hosts are needed.     

Plant-mediated decisions by an herbivore affect oviposition pattern and subsequent egg parasitism

Natural enemies are important mortality factors for herbivores and thus may influence herbivore population dynamics. In response to natural enemy pressure, herbivores can alter life history decisions, such as oviposition behavior, so that offspring are protected from natural enemies. One such strategy is to deposit eggs into structures where vulnerability to natural enemies is reduced or eliminated, i.e., use enemy-free space. The plant bug, Lygus lineolaris (Palisot de Beauvois), is native to North America and has a broad host range (>350 plant species), including crops. This bug’s eggs are attacked by a native parasitoid, Anaphes iole Girault, and parasitism levels vary greatly among host plant species. Weed hosts are critical to contemporary L. lineolaris life history because they serve as an ecological bridge from one crop growing season to the next. We investigated the egg distribution pattern of L. lineolaris on 11 host plant species (nine weeds and two crops), and parasitism by A. iole, to determine whether oviposition choices by L. lineolaris females protect their eggs from parasitism and to demonstrate the mechanism of this protection. Our results indicate that the reproductive structures of Erigeron annuus, as well as those of several other host plant species, provide a refuge from parasitism for most L. lineolaris eggs. This refuge is due to the deposition of host eggs deeper in plant tissue than the length of the ovipositor of A. iole. Also, overall parasitism levels were greater on non-Asteraceae host plant species compared with host plant species belonging to Asteraceae. Oviposition site choice by female bugs appears to be a selective strategy to take advantage of enemy-free space.     

Characterization of eight polymorphic microsatellite markers in the tarnished plant bug,Lygus lineolaris (Palisot de Beauvois)

A partial genomic library of the tarnished plant bug, Lygus lineolaris, enriched for microsatellite sequences was screened to identify marker loci. Eight polymorphic loci suitable for population genetic studies were identified by screening 192 field‐collected insects. The observed number of alleles ranged from four to 21 with an average of 12.25 (SE ± 1.94) while the effective number of alleles ranged from 1.23 to 11.05 with an average of 4.49 (SE ± 1.15). No linkage disequilibria or significant deviations from Hardy–Weinberg expectations were detected at any of the loci. Seven of the eight L. lineolaris microsatellite loci were transferable to Lygus hesperus.     

RNA interference‐mediated knockdown of IAP in Lygus lineolaris induces mortality in adult and pre‐adult life stages

In recent years, RNA interference (RNAi) has been validated as a viable approach for functional genetic studies in non‐model organisms. In this report we demonstrate the efficacy of RNAi in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). A L. lineolaris inhibitor of apoptosis gene (LlIAP) has been identified and cloned. The translated sequence encodes a 381 amino acid protein similar to other insect IAPs and contains two conserved baculovirus inhibitor of apoptosis protein repeat (BIR) domains. Microinjection of double stranded RNA (dsRNA) corresponding to two disparate portions of the gene resulted in decreased LlIAP mRNA quantities relative to controls. Both nymphs and adult specimens injected with IAP dsRNA exhibited significantly reduced lifespan compared with those injected with non‐insect dsRNA (eGFP). Thus, RNAi‐mediated knockdown of LlIAP expression has been correlated with a lethal phenotype in adults and nymphs.     

Local and landscape scale drivers of Euschistus servus and Lygus lineolaris in North Carolina small grain agroecosystems

1. Crop production sequences influence arthropod populations in temporally unstable row crop systems. Winter wheat (Triticum aestivum L.) represents one of the earliest abundant crops in south-eastern United States. This study aims to understand primary source habitats driving brown stink bug, Euschistus servus (Say), and tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), population abundance in wheat.
2. To better understand these relationships, adult and nymphal densities were in wheat fields weekly from flowering through harvest in 2019 and 2020. Geospatial data were used to measure landscape composition surrounding sampled fields. We investigated the influence of landscape predictors on E. servus and L. lineolaris abundance using generalized linear mixed modelling.
3. Field size, proportion of agriculture, proportion of wheat area, and proportion of soybean Glycine max L.) area from the previous year in the surrounding landscape were associated with E. servus abundance in wheat. Similarly, L. lineolaris abundance was associated with proportion of wheat area and soybean area from the previous year.
4. These results reveal the influence of soybean area planted the previous year on insect pest densities the following spring in wheat. Further, results suggest agricultural landscapes dominated by wheat are associated with decreased pest abundance across the sampled region.

     

Polygalacturonase causes lygus-like damage on plants: cloning and identification of western tarnished plant bug (Lygus hesperus) polygalacturonases secreted during feeding

Polygalacturonase (PG), an enzyme that degrades pectin within the plant tissue cell wall, has been postulated as the chemical cause of damage to plants by the mirid Lygus hesperus. Micro-injection of two pure recombinant Aspergillus niger PG II protein forms, the wild type enzymically active and the mutant inactive one, into alfalfa (Medicago sativa L.) florets, demonstrates that the enzymatic activity rather than the PG protein structure per se elicits damage symptoms. A PG gene family has been described for the tarnished plant bug, L. lineolaris. Here we report cloning members of the L. hesperus PG gene family, Lhpg2, obtained with L. lineolaris PG-specific primers and a novel Lhpg4, amplified with degenerate primers that were designed based, in part on the N-terminal sequence from an active, partially purified L. hesperus salivary gland PG protein. Proteomic analyses revealed that the salivary gland PGs encoded by Lhpg2 and Lhpg4 are detected in a diet into which L. hesperus has extruded its saliva when feeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: Henryk Czosnek     

Relative preference of Lygus hesperus (Hemiptera: Miridae) to selected host plants in the field

Abstract Information on host plant preference of agriculturally important insect pests, such as Lygus hesperus (Knight), can be helpful in predicting its occurrence and future movement among crop and non-crop host plants. A field study was conducted during 2005 and 2006 to evaluate the host preference of Lygus to cotton and four other host plants in the Texas High Plains, including alfalfa (Medicago sativa L.), wild sunflower (Helianthus annuus L.), Russian thistle (Salsola iberica L.) and pigweed (Amaranthus palmeri L.). Sampling for both nymphs and adults during 2005 (July to November) and 2006 (June to November) showed that alfalfa and Russian thistle were the two most preferred hosts out of the five hosts evaluated. Abundance of nymphs (numbers per 50 sweeps per host plant) during the sampling period also indicated the superior reproductive suitability of alfalfa and Russian thistle. Cotton appeared to be the least attractive host plant for Lygus when Russian thistle and alfalfa were available in the host mosaic. Seasonal abundance of Lygus was found to be lower during 2006 compared to 2005, which may be explained by the difference in rainfall patterns during these two years. In terms of species dominance, L. hesperus was the most dominant species in the sampled population followed by an inconsiderable fraction of L. elisus and L. lineolaris.     

Incidence of Anagrus obscurus (Hymenoptera: Mymaridae) egg parasitism on Calopteryx haemorrhoidalis and Platycnemis pennipes (Odonata: Calopterygidae: Platycnemididae) in Italy

Very little is known about the incidence of egg parasitoids in odonates, perhaps because Odonata eggs are well protected by stems or leaves, sometimes below water. In Central Italy (Pontecorvo, Frosinone Province) two damselflies, Calopteryx haemorrhoidalis and Platycnemis pennipes, occur in high densities. In August 2007 we collected 30 stems of the aquatic plant Potamogeton sp. used as substrate for oviposition and incubated eggs in the laboratory. Most stems (24 for C. haemorrhoidalis and 23 for P. pennipes) contained Odonata eggs. Parasitoids emerged from 12 stems, with a mean parasitism of 2% for C. haemorrhoidalis and 6% for P. pennipes, and a maximum of 14% and 50%, respectively. Furthermore, we observed egg‐laying of 19 females of C. haemorrhoidalis and 11 of P. pennipes, and marked the stems where oviposition was observed. Clutches remained in the river for five days and were then collected and incubated. Parasitoids emerged from 11 of 30 stems, with an average parasitism of 8% for C. haemorrhoidalis and 3% for P. pennipes (maximums of 50% and 29%, respectively). All parasitoids belonged to the family Mymaridae, and were identified as Anagrus (Anagrus) obscurus Förster, 1861, sensu Soyka, 1955 . This is the first time that this species is described as an egg parasitoid of odonates, and that the egg parasitoid of C. haemorrhoidalis and P. pennipes is identified. Our data suggest that egg parasitism might be a significant selective factor for both odonates in the studied locality, affecting female oviposition behavior.     

Insect-associated fungi from naturally mycosed vine mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae)

Vine mealybug, Planococcus ficus, is a major pest of grapevine, which is present in at least 39 countries. According to American Vineyard Foundation, P. ficus is in the top ranks among major insect-pests of grapevine. It is the ‘top priority concerns’ by grape growers and a ‘threat to the sustainability of wine industry’ demanding a ‘high priority research’. In Douro vineyards, it is considered as an occasional insect-pest; however, its importance is increasing in some localities. The present study investigates the occurrences of P. ficus-associated fungi. Vine mealybugs were observed in two of the four surveyed farms. Out of the 183 collected mealybugs, 58 were dead of which 25 had symptoms of mycosis and 13 were parasitised. Subculturing cadavers and subsequent pathogenicity test yielded 22 entomopathogenic fungi (EPF) including yeasts. The yeast Meyerozyma (=Pichia) guilliermondii, and the EPF Sarocladium kiliense and Purpureocillium lilacinum were the most abundant, i.e. representing 18.18% (N?=?4), 13.64% (N?=?3) and 13.64% (N?=?3) of the isolates, respectively. Considering biological affinities, fungal families Nectriaceae and Microascaceae had the most similar count-data profiles. To our knowledge, this work reports the first isolations of EPF from vine mealybug worldwide; and Pseudocosmospora rogersonii in Europe and as EPF worldwide. The mortality rate originated by mycoses on P. ficus was significantly higher than by its parasitoids, suggesting that fungi as P. ficus biocontrol agents are relatively more important than considered before. Overall, this report provides new insights into the development of mycoinsecticides and conservation biocontrol strategies for P. ficus pest management.