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.     

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.     

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.

     

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.     

Hyperparasitism by Mesochorus spp. (Hymenoptera: Ichneumonidae) in Peristenus sp. (Hymenoptera: Braconidae) and development of PCR primers for hyperparasitoid detection

Peristenus sp. pupae collected from Lygus spp. nymphs in 2001 and 2002 were over-wintered in the laboratory. In both years, more than 30% of adults emerging from over-wintering pupae were identified as ichneumonid hyperparasitoids, Mesochorus curvulus Thomson and Meschorus sp. (Hymenoptera: Ichneumonidae). At the end of the over-wintering period, Peristenus sp. males emerged first followed by Peristenus sp. females and finally Mesochorus spp. The male:female ratio in emerging Peristenus sp. adults was skewed towards males. The Internal Transcribed Spacer (ITS) region and the cytochrome oxidase I (COI) gene from Mesochorus spp. were sequenced. ITS sequences were used to develop PCR primers to detect Mesochorus spp. hyperparasitism in the primary host, Lygus spp. PCR analysis of field-collected Lygus spp. nymphs gave similar estimates of Mesochorus spp. hyperparasitism to the rearing protocols (25–28%). Sequence analysis of COI and ITS regions and subsequent restriction endonuclease analysis of ITS PCR products from Mesochorus spp. indicate the presence of two genotypes in the population. The possibility that these two genotypes represent separate or cyrptic species is discussed.     

A survey of Lygus parasitoids in Sweden

Lygus spp. (tarnished plant bugs) are generalist herbivores and occur as pests on a wide range of crops. In the development of conservation biological control strategies for Lygus spp. in Sweden, more information is needed on the impact of different natural enemies. In this study, we determined the occurrence and the degree of parasitism on adults and nymphs of the most common Lygus species in alfalfa, barley, red clover and oilseed rape in Uppsala in Central Sweden and in Umeå in Northern Sweden. Nymphs and adults of Lygus spp. were collected by sweep netting for estimation of their parasitism level (by dissection) and identification of parasitoid species (from reared Lygus specimens). The dominant Lygus species in both locations was L. rugulipennis (75–99%). Parasitism by Phasia obesa (Tachnidae) on overwintering Lygus adults was recorded in almost every field sampled at both locations. The parasitism level was low at overwintering sites but increased in arable fields during summer. Lygus nymphs collected in Umeå were parasitised by Peristenus pallipes (Braconidae) and in Uppsala by P. relictus, P. pallipes and P. varisae. The hyperparasite Mesochorus globulator (Ichneumonidae) was recorded in Umeå.     

Impact of the introduced parasitoid Peristenus digoneutis (Hymenoptera: Braconidae) on tarnished plant bug (Hemiptera: Miridae) infesting strawberries in northwestern New Jersey,USA

Lygus lineolaris populations in the northeastern USA have been markedly reduced by the introduced parasitoid Peristenus digoneutis. A 3-year study in NJ where P. digoneutis has been established for many years demonstrated that parasitism of Lygus nymphs in strawberries averaged 30% (mostly P. digoneutis). Strawberry damage by L. lineolaris ranged from 19 to 33%.     

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.     

Does host plant influence parasitism and parasitoid species composition in Lygus rugulipennis? A molecular approach

Lygus Hahn plant bugs (Hemiptera: Miridae) are serious pests of a wide variety of economically important crops in North America. European Peristenus digoneutis Loan and P. relictus Ruthe (Hymenoptera: Braconidae) are being considered for release in Canada as part of a classical biological control program for Lygus. The attractiveness of different host plants to European Peristenus has not been addressed, but may be an important consideration prior to parasitoid release. Lygus rugulipennis Poppius nymphs were collected in the Northern Temperate Atlantic (NTA) ecoregion on red clover (Trifolium pratense L.; Fabaceae) and chamomile (Matricaria recutita L.; Asteraceae), and in the Western European Broadleaf Forest (WEBF) ecoregion on red clover and alfalfa (Medicago sativa L.; Fabaceae). Parasitism levels and parasitoid species were determined using a multiplex PCR assay for P. digoneutis, P. relictus, and P. pallipes Curtis. Mean parasitism levels in L. rugulipennis were 45-49% in the NTA ecoregion and 25-32% in the WEBF ecoregion. However, in neither ecoregion were parasitism levels and parasitoid species compositions significantly different in nymphs from different host plant species. Furthermore, multiparasitism was low despite the fact that P. digoneutis and P. relictus share the same host species.     

First report of a hymenopteran parasitoid complex on jackfruit shoot and fruit borer Diaphania caesalis (Lepidoptera: Crambidae) from India

ABSTRACT

A survey of the species composition and relative abundance of Diaphania caesalis Walker (Lepidoptera: Crambidae) in plantations of jackfruit (Artocarpus heterophyllus Lam.) in three southern states along India’s western coast recorded thirteen species of primary parasitoids and one species of a hyperparasitoid, namely Tetrastichus pantnagerensis Khan (Hymenoptera: Eulophidae). Apanteles stantoni Ashmead (Hymenoptera: Braconidae) was the most dominant parasitoid and found in 35% of the parasitised host larvae in Karnataka, 38.5% in Kerala, and 33.1% in Tamil Nadu whereas the overall level of parasitisation, involving all the different parasitoids recorded, was approximately 41%–45% and did not vary significantly with location.     

Endoparasitoids of larval Anomis privata (Lepidoptera: Noctuidae), major pest of Hibiscus syriacus (Columniferae: Malvaceae)

Endoparasitoids of Anomis privata larvae include five species in three families of two orders. In this work, two species of Hymenoptera Braconidae (Cotesia sp., Microplitis sp.), one species of Ichneumonidae (Mesochorus vittator) and two species of Diptera Tachinidae (Exorista (Podotachina) sorbillans, Timavia amoena) were investigated. Of the 261 larvae of A. privata examined, 32 had a parasite, so the rate of parasitism was 12.26%. Parasitism by taxon was the highest, at 10.35% (27 individuals), in Cotesia sp. in Hymenoptera Braconidae. Parasitoids of Braconidae and Ichneumonidae were larval parasitoids. A parasitic insect of Tachinidae was a larva–pupal parasitoid. Solitary parasitoids included Microplitis sp. in Braconidae and E. sorbillans in Tachinidae. Gregarious parasitoids included Cotesia sp. in Braconidae, M. vittator in Ichneumonidae and T. amoena in Tachinidae. There was also a multiparasitoid (T. amoena) and two superparasitoids (Cotesia sp., M. vittator). A larva of A. privata sought feed even after it was parasitized every parasitoid investigated in this study, so five species of parasitoids were all koinobiont.     

Interspecific competition between the braconid endoparasitoids Glyptapanteles porthetriae and Glyptapanteles liparidis in Lymantria dispar larvae

The effect of interspecific competition between the solitary endoparasitoid Glyptapanteles porthetriae Muesebeck (Hymenoptera: Braconidae) and the gregarious Glyptapanteles liparidis Bouché (Hymenoptera: Braconidae), was investigated in larvae of Lymantria dispar L. (Lepidoptera: Lymantriidae). Host larvae were parasitized by both wasp species simultaneously in premolt to the 2^nd or the 3^rd host instar or in an additional approach with a 4‐day delay in parasitization by the second wasp species. Host acceptance experiments revealed that both wasp species do not discriminate between unparasitized host larvae and larvae parasitized previously by the same or the other species. In more than 90% female wasps parasitized the larva they encountered first. During the period of endoparasitic development, larvae of the competing parasitoid species never attacked the egg stage of the other species. When host larvae were parasitized simultaneously by both wasp species, the rate of successful development of both species depended on the age of the host larva at the time of its parasitization; G. liparidis emerged successfully from 44% of host larvae parasitized during the premolt to 2^nd instar, G. porthetriae from 28%, and in 20% of the hosts both parasitoid species were able to develop in one gypsy moth larva. However, when host larvae were parasitized simultaneously during premolt to the 3^rd instar, G. liparidis was successful in 90% of the hosts, compared to 8% from which only G. porthetriae emerged. In the experiments with delayed oviposition, generally the species that oviposited first succeeded in completing its larval development. Larvae of the species ovipositing with four days delay were frequently attacked and killed by larvae of the first parasitizing species or suffered reduced growth. As the secondary parasitoid species, G. porthetriae‐larvae were never able to complete their development, whereas G. liparidis developed successfully in at least 12,5% of the multiparasitized host larvae. Thus, multiparasitism of gypsy moth larvae by both Glyptapanteles species corresponds to the contest type; however, G. porthetriae is only able to develop successfully as the primary parasitoid of young host larvae.     

Interspecific competition between the braconid endoparasitoids Glyptapanteles porthetriae and Glyptapanteles liparidis in Lymantria dispar larvae

The effect of interspecific competition between the solitary endoparasitoid Glyptapanteles porthetriae Muesebeck (Hymenoptera: Braconidae) and the gregarious Glyptapanteles liparidis Bouché (Hymenoptera: Braconidae), was investigated in larvae of Lymantria dispar L. (Lepidoptera: Lymantriidae). Host larvae were parasitized by both wasp species simultaneously in premolt to the 2^nd or the 3^rd host instar or in an additional approach with a 4-day delay in parasitization by the second wasp species. Host acceptance experiments revealed that both wasp species do not discriminate between unparasitized host larvae and larvae parasitized previously by the same or the other species. In more than 90% female wasps parasitized the larva they encountered first. During the period of endoparasitic development, larvae of the competing parasitoid species never attacked the egg stage of the other species. When host larvae were parasitized simultaneously by both wasp species, the rate of successful development of both species depended on the age of the host larva at the time of its parasitization; G. liparidis emerged successfully from 44% of host larvae parasitized during the premolt to 2^nd instar, G. porthetriae from 28%, and in 20% of the hosts both parasitoid species were able to develop in one gypsy moth larva. However, when host larvae were parasitized simultaneously during premolt to the 3^rd instar, G. liparidis was successful in 90% of the hosts, compared to 8% from which only G. porthetriae emerged. In the experiments with delayed oviposition, generally the species that oviposited first succeeded in completing its larval development. Larvae of the species ovipositing with four days delay were frequently attacked and killed by larvae of the first parasitizing species or suffered reduced growth. As the secondary parasitoid species, G. porthetriae-larvae were never able to complete their development, whereas G. liparidis developed successfully in at least 12,5% of the multiparasitized host larvae. Thus, multiparasitism of gypsy moth larvae by both Glyptapanteles species corresponds to the contest type; however, G. porthetriae is only able to develop successfully as the primary parasitoid of young host larvae.     

Superparasitism strategies by a native and an exotic parasitoid species attacking the Mexican fruit fly,Anastrepha ludens (Diptera: Tephritidae)

Superparasitism refers to the action of parasitoids ovipositing eggs in hosts that are already parasitized; this inevitably results in the elimination of supernumerary larvae in solitary parasitoids. Here, we investigated superparasitism performed by two species of solitary parasitoids on the larvae of Anastrepha ludens (Loew; Diptera: Tephritidae): a native species, Doryctobracon crawfordi (Viereck; Hymenoptera: Braconidae), and an exotic species, Diachasmimorpha tryoni (Cameron; Hymenoptera: Braconidae). Tests were conducted under laboratory conditions evaluating the behaviour of females acting alone (self-superparasitism) or in groups (conspecific superparasitism). Parasitism strategies were different between these two species. In D. crawfordi, the number of first instar larvae found in each dissected host pupa was never greater than two, regardless of the number of oviposition scars observed per pupa. In contrast, there was a positive correlation between the number of oviposition scars and the number of first instar larvae in D. tryoni. The survival and fecundity of D. crawfordi females emerging from pupae with one scar was higher than in females emerging from pupae with more scars. In D. tryoni, the number of oviposition scars did not show deleterious effects on life history traits and was positively correlated with the proportion of emerging females. An understanding of the superparasitism strategy adopted by parasitoid species could be of great interest to augmentative biological control programmes because the mass rearing of natural enemies could be negatively or positively affected by this condition.     

Potential for classical biological control of the potato bug Closterotomus norwegicus (Hemiptera: Miridae): description, parasitism and host specificity of Peristenus closterotomae sp. n. (Hymenoptera: Braconidae)

The potato bug, Closterotomus norwegicus (Gmelin) (Hemiptera: Miridae) is an introduced pest of lucerne, white clover and lotus seed crops in New Zealand and a key pest of pistachios in California, USA. Efforts were made to identify potential biological control agents of C. norwegicus in Europe. A total of eight parasitoids, including six primary parasitoids from the genus Peristenus (Hymenoptera: Braconidae) and two hyperparasitoids from the genus Mesochorus (Hymenoptera: Ichneumonidae), were reared from C. norwegicus nymphs collected in various habitats in northern Germany. With a proportion of more than 85% of all C. norwegicus parasitoids, Peristenus closterotomae (Hymenoptera: Braconidae), a new species, was the most dominant parasitoid, whereas other parasitoid species only occurred sporadically. Peristenus closterotomae did not fit in the keys to any described species and is described as new to science. Parasitism caused by P. closterotomae was on average 24% (maximum 77%). To assess the host specificity of parasitoids associated with C. norwegicus, the parasitoid complexes of various Miridae occurring simultaneously with C. norwegicus were studied. Peristenus closterotomae was frequently reared from Calocoris affinis (Herrich-Schaeffer), and a few specimens were reared from Calocoris roseomaculatus (De Geer) and the meadow plant bug, Leptopterna dolobrata (Linnaeus) (all Hemiptera: Miridae). The remaining primary parasitoids associated with C. norwegicus were found to be dominant in hosts other than C. norwegicus. Whether nymphal parasitoids may potentially be used in a classical biological control initiative against the potato bug in countries where it is introduced and considered to be a pest is discussed.     

Strategy for orchard use of Bacillus thuringiensis while minimizing impact on Choristoneura rosaceana parasitoids

Trials were conducted to study how spring Bacillus thuringiensis Berliner subsp. kurstaki treatments on apple may be timed to maximize the survival of parasitoids of the obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae), found in the southern interior of British Columbia, Canada. Orchard collections verified that second through fourth instar obliquebanded leafrollers were found in varying proportions from pink through the petal fall stage of apple development when spring B. thuringiensis treatments are applied vs. lepidopteran pests. Laboratory‐reared second through fourth instar obliquebanded leafrollers, unparasitized and parasitized by one of three native parasitoid species, were fed untreated apple leaves or leaves treated with B. thuringiensis. The highest mortality of unparasitized obliquebanded leafrollers occurred when fourth instars were exposed to B. thuringiensis‐treated leaves; B. thuringiensis‐induced mortality in the unparasitized second and third instars was less than 50%. The consumption of B. thuringiensis‐treated leaves by host larvae significantly increased the percentage of dead host larvae in all parasitized and unparasitized treatments. However, because of the low susceptibility of this leafroller species to B. thuringiensis, relatively high numbers (38–43%) of three obliquebanded leafroller parasitoid species were able to survive the consumption of B. thuringiensis by second and third instar host larvae. Fourth instar obliquebanded leafrollers were found at the full bloom and petal fall stage of apple development in the orchard, at which time B. thuringiensis treatments are recommended for optimal leafroller control. The highest parasitoid mortality due to host mortality was recorded in Apophua simplicipes Cresson (Hymenoptera: Ichneumonidae) and Macrocentrus linearis (Nees) (Hymenoptera: Braconidae), when the hosts were treated as fourth instars. Both of these parasitoids emerge from fifth and sixth instar obliquebanded leafrollers. Bacillus thuringiensis did not have as negative an impact on Apanteles polychrosidis Viereck (Hymenoptera: Braconidae), which emerges when the host is in the fourth instar. When leafroller mortality and parasitism were combined, the B. thuringiensis treatment did not significantly increase host elimination above that of parasitism alone, except for larvae parasitized by A. simplicipes that were in the fourth instar. The consumption of B. thuringiensis by unparasitized larvae was shown to slow larval development.     

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.     

Post-release dispersal of the introduced lygus bug parasitoid Peristenus relictus in California

Lygus spp. (Heteroptera: Miridae) are serious pests of numerous field and fruit crops in North America. In an effort to reduce crop damage, parasitoids known to attack these species in Europe were introduced into the USA beginning in the 1970s. Permanent populations of the nymphal endoparasitoid Peristenus relictus (Hymenoptera: Braconidae) were established at two locations in California during the 2000s. Both populations are associated with significant reductions in lygus bugs attacking alfalfa, commercially produced strawberries grown organically and in wild vegetation. Beginning in 2009, in an effort to determine the extent to which P. relictus has spread from the Sacramento Valley and Monterey Bay region, populations of lygus bug were sampled at increasing distances from their original release sites. P. relictus has dispersed at least 213 km in the central region of California and 150 km along the coastal mountains. These respective populations have averaged 16.6 km/year and 17.7 km/year since they were released. While not directly examined, the spread of P. relictus south into the central and major growing region of California, the San Joaquin Valley, where previous releases have failed, suggests this population may be evolving greater heat tolerance, relative to the populations originally introduced into California.     

Molecular Markers for Identification of the Hyperparasitoids Dendrocerus carpenteri and Alloxysta xanthopsis in Lysiphlebus testaceipes Parasitizing Cereal Aphids

Polymerase chain reaction (PCR)-based molecular markers have been developed to detect the presence of primary parasitoids in cereal aphids and used to estimate primary parasitism rates. However, the presence of secondary parasitoids (hyperparasitoids) may lead to underestimates of primary parasitism rates based on PCR markers. This is because even though they kill the primary parasitoid, it’s DNA can still be amplified, leading to an erroneous interpretation of a positive result. Another issue with secondary parasitoids is that adults are extremely difficult to identify using morphological characters. Therefore, we developed species-specific molecular markers to detect hyperparasitoids. A 16S ribosomal RNA mitochondrial gene fragment was amplified by PCR and sequenced from two secondary parasitoid species, Dendrocerus carpenteri (Curtis) (Hymenoptera: Megaspilidae) and Alloxysta xanthopsis (Ashmead) (Hymenoptera: Charipidae), four geographic isolates of the primary parasitoid, Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae), and six aphid species common to cereal crops. Species-specific PCR primers were designed for each insect on the basis of these 16S rRNA gene sequences. Amplification of template DNA, followed by agarose gel electrophoresis, successfully distinguished D. carpenteri and A. xanthopsis from all four isolates of L. testaceipes and all six cereal aphid species in this laboratory test.