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.     

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 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.     

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.     

Parasitism activity of Peristenus spp. (Hymenoptera: Braconidae) on Lygus lineolaris (Hemiptera: Miridae) nymphs prior to the establishment of P. digoneutis in southwestern Quebec

Parasitism of tarnished plant bug nymphs, Lygus lineolaris (Palisot de Beauvois) was investigated on different weeds and on lettuce, celery and alfalfa-clover, in a muck soil area of southwestern Quebec. The phenology of parasitism, defined as the time during which female parasitoids are active in the field, was evaluated. Two nymphal parasitoid species were found: Peristenus pallipes (Curtis) and P. pseudopallipes (Loan). Lygus lineolaris was most abundant on stinging-nettle with levels of parasitism of 17% (P. pallipes) and 27% (P. pseudopallipes) and on alfalfa-clover with levels of parasitism of 25% (P. pallipes) and 8% (P. pseudopallipes). On lettuce, parasitism by P. pseudopallipes was below 4% while no parasitism was observed on celery. The dissection and rearing methods were used to estimate parasitism, and dissection usually yielded parasitism levels higher than rearing. Degree-days requirements of both host and parasitoids were used to calculate the period of activity of parasitoid adults. The estimates of adult activity were 47 days from late May to early July for P. pallipes, and 42 days from mid-July to late August for P. pseudopallipes.     

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.     

Functional response and mutual interference of Peristenus spretus (Hymenoptera: Braconidae), a parasitoid of Apolygus lucorum (Heteroptera: Miridae)

Peristenus spretus Chen & van Achterberg (Hymenoptera: Braconidae) is a solitary endoparasitoid, which is considered for augmentative biological control of Apolygus lucorum Meyer-Dür (Heteroptera: Miridae) in Chinese cotton fields. Since the association of P. spretus with A. lucorum was only recently discovered, the biology of the parasitoid remains unknown. In order to understand its reproductive biology, the mutual interference and functional response of P. spretus were investigated by altering either the parasitoid or the host density while keeping the other constant. In both experiments, the effects of parasitoid and host densities on parasitism, superparasitism, progeny production and sex ratio were assessed. P. spretus exhibited a Holling type II functional response to changing host densities, indicating that parasitism increases with increasing host density until the parasitoid reaches its maximum reproductive capacity. The model suggested that a single P. spretus female could parasitise a maximum of 88 nymphs per day or four nymphs per hour. Increasing the wasp-nymph ratio from 1:10 to 1:80 significantly increased the offspring production more than fivefold from ±5.8 to ±35.6; further increasing the host densities (above 80 nymphs) did not significantly increase offspring production. Strong mutual interference of foraging P. spretus females occurred only at high parasitoid densities. Parasitoids foraging alone produced an average progeny of 33.4, whereas parasitoids foraging in groups of 16 produced only 2.6. The optimal wasp-nymph ratio for mass-rearing P. spretus is 4:100, given that resources of parasitoids and nymphs are unlimited.     

Seasonality and species composition of Peristenus (Hymenoptera: Braconidae) species,and Lygus (Hemiptera: Miridae) species parasitism in southern Alberta,Canada

We report a long-term survey summarising the seasonal variation in species composition of Peristenus species, and their levels of parasitism in an assemblage of Lygus spp., over more than 10 years in southern Alberta in western Canada. Plants sampled include alfalfa (Medicago sativa L.), canola (Brassica napus L.), and stands of different weed and grass species commonly found on crop field edges. Three native Peristenus species (Peristenus dayi, Peristenus mellipes, and Peristenus howardi) were recorded in 2003–2011 and 2013–2015. In laboratory trials, P. mellipes emerged 13 days earlier than P. howardi. Degree-days for field-collected individuals suggested a sequential occurrence during the growing season: P. dayi appears early in the season, followed by P. mellipes, and later on by P. howardi. Studies of species composition and phenology of native parasitoids of insect pests provide valuable baseline information when considering introduction of exotic agents, and contribute to the management and improvement of conservation biological control by endemic natural enemies in agricultural systems.     

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.     

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.     

Host preferences of introduced and native parasites (Hymenoptera: Braconidae) of phytophagous plant bugs (Hemiptera: Miridae) in alfalfa-grass fields in the northeastern USA

During an eight-year field study at two widely-separated locations (NJ & DE, USA), seven species of phytophagous mirid plant bugs were found in alfalfa and alfalfa-grass fields grown for animal forage. Five species of parasites were reared from these mirids. Two parasite species introduced from Europe killed significantly higher proportions of nymphs of the two important mirid pests of alfalfa than did native parasites. The introduced Peristenus digoneutis Loan parasitized an average of 31% of first and second generation tarnished plant bugs, Lygus lineolaris, a native insect; and the introduced Peristenus conradi parasitized 22% of first generation alfalfa plant bugs, Adelphocoris lineolatus, an introduced species. In addition, Peristenus pallipes significantly parasitized nymphs of Trigonotylus caelestialium (43%) and Leptopterna dolabrata (37%); both mirids are immigrant species. Because the parasite P. pallipes significantly attacked only these two non-native mirids, and is present in Europe, it also may be an accidental introduction to North America. A native wasp, Leiophron uniformis, heavily parasitized (49%) the native garden fleahopper, Halticus bractatus. A third native species, P. pseudopallipes, occasionally parasitized a few Lygus lineolaris in alfalfa at one location. Two other mirids, Stenotus binotatus and Megaloceroea recticornis, both accidently-introduced grass-feeding species, were not parasitized by native or introduced species. It is noteworthy that the effective host ranges of all the parasites in alfalfa-grass fields were limited: four of the five parasite species significantly parasitized only one of the seven mirid species, and the other parasite significantly parasitized only two mirids. Activity of the four common parasites was correlated with the mirids' host plant: three species principally parasitized alfalfa-feeding mirids, and one species principally parasitized grass-feeding mirids.     

Native braconid parasitism of the Tarnished plant bug (Hemiptera: Miridae) in Southern Ontario

Three agricultural regions in southern Ontario (London, Niagara and Guelph) were sampled weekly from May to September for Lygus spp. and their parasitoids in 1998, 1999 and only one region in 2000. Alfalfa was the primary crop sampled, including both “clean” and weedy alfalfa fields. Lygus lineolaris (Palisot de Beauvois) accounted for 99% of all Lygus spp. collected by sweepnet. Lygus lineolaris had two generations (mid-June and late July) on alfalfa with a partial third generation in early September which contributes to the over-wintering adults. In 1998 and 2000, an early first generation peak of Lygus nymphs was observed in mid-May on the weeds, chickweed and shepherd's purse. Overall rates of parasitism (from dissections) for native parasitoids were consistent from year to year and the means of the three regions in each growing season were below 11%. Both nymphs and adult Lygus were parasitized, with the highly mobile adults being a potential means of dispersing the parasitoids. In general weedy fields were more highly parasitized than fields of weed-free alfalfa. Six species of native braconid parasitoids were collected from L. lineolaris in southern Ontario (in decreasing order of occurrence): Peristenus pallipes (Curtis), Peristenus pseudopallipes (Loan), Leiophron lygivorus (Loan), L. solidaginis Loan, L. uniformis (Gahan), and Leiophron sp. near brevipetiolatus Loan. The large populations of Lygus and the low percent baseline parasitism in southern Ontario, particularly of second generation Lygus, support the need for introduction of a multivoltine parasitoid species in this region.     

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%.     

Delayed development of the whitefly (Bemisia tabaci) and increased parasitism by Encarsia bimaculata in response to sublethal doses of piperonyl butoxide

Abstract Effects of sublethal piperonyl butoxide (PB) on parasitization of Bemisia tabaci (Gennadius ) (Hemiptera: Aleyrodidae) by Encarsia bimaculata Heraty et Polaszek (Hymenoptera: Aphelinidae) were evaluated both in cage and greenhouse experiments. When first, second and third instar B. tabaci nymphs were treated with PB, all but the first instar were significantly prolonged. Data indicated that sublethal PB could improve E. bimaculata parasitism rates without influencing parasitoid eclosion rates. Prolonged development increased rates of parasitism by E. bimaculata, from 17.6% to 24.7% in cages, presumably by increasing the duration of host exposure. Sublethal PB combined with E. bimaculata as an integrated approach to control B. tabaci was evaluated using life table parameters under greenhouse conditions. Indices of population trend (I) calculated from life tables were estimated at 4.6 for B. tabaci exposed to PB and parasitoids compared to 14.1 with parasitoids alone and 23.5 in untreated controls. The results showed that after PB was sprayed and parasitoids introduced, development of B. tabaci was delayed and the peak of each stage was postponed. The older nymphal stage had highest mortality, primarily due to mortality caused by parasitism by E. bimaculata.     

斑衣蜡蝉若虫寄生蜂地理种群遗传差异及早期快速检测

【目的】明确不同地理种群斑衣蜡蝉Lycorma delicatula若虫寄生蜂种群遗传差异,实现若虫被寄生早期的快速准确检测,以评估其对斑衣蜡蝉种群的控制作用。【方法】利用DNA条形码技术获得不同地理种群斑衣蜡蝉若虫寄生蜂COI和28S rDNA序列,利用K2P模型计算不同地理种群间的遗传距离,以邻接(neighbor-joining,NJ)法构建系统发育树;基于COI序列设计种特异性PCR(SS-PCR)引物,利用SS-PCR对斑衣蜡蝉若虫DNA进行扩增,测定其体内是否有中华螯蜂Dryinus sinicus寄生;利用目测法观察和PCR扩增测定寄生蜂对不同采样点斑衣蜡蝉若虫的寄生率。【结果】经鉴定,不同地理种群斑衣蜡蝉若虫寄生蜂为中华螯蜂,该寄生蜂COI序列共检测到16个单倍型,28S rDNA序列共检测到4个单倍型。不同地理种群间中华螯蜂遗传距离在0.00691~0.01310之间。邻接法构建的系统发育树显示不同地理种群中华螯蜂均聚于一枝。基于COI序列设计的SS-PCR引物对中华螯蜂成虫、幼虫均具有良好的扩增效果,最低检测阈值为0.000005 ng/μL DNA。利用SS-PCR测定中华螯蜂对各采样点斑衣蜡蝉若虫的寄生率为22.54%~60.00%,显著高于目测统计的结果(5.63%~36.98%)。【结论】不同中华螯蜂地理种群间遗传差异较小;SS-PCR引物可用于不同地区中华螯蜂对斑衣蜡蝉早期寄生率的快速检测。     

Density-dependent parasitism of cowpea aphid,Aphis craccivora by Lysiphlebus fabarum,Binodoxys acalephae,and Aphidius matricariae (Hymenoptera: Braconidae: Aphidiinae)

Biological control, as a major component of pest management strategies, uses natural biological agents to reduce pest populations. Studying the interaction among Aphis craccivora and its parasitoids including, Lysiphlebus fabarum, Binodoxys acalephae, and Aphidius matricariae in 2016 and 2017 in Tehran Parke-Shahr, showed positive, significant correlations in all cases between the densities of three parasitoid species and that of aphid nymphs and adults. The density of the parasitoids increased by increasing the density of the aphids. The parasitoids showed aggregative behavior in response to different densities of the host. There was a positive density-dependent correlation between the density of A. craccivora and rate of parasitism. Parasitism rates of nymphs and adult aphids by L. fabarum, B. acalephae, and A. matricariae increased or decreased along with decline or increase in the population of the aphid host. In 2016 spring, the highest rates of parasitism on aphid nymphs by L. fabarum, B. acalephae, and A. matricariae were 46.82, 23.09, and 17.16%, respectively. In 2017 spring, the highest rates of parasitism on aphid nymphs by L. fabarum, B. acalephae, and A. matricariae were 48.97, 21.77, and 15.06%, respectively. So, given the accordance between changes in aphid population and that of parasitoids, and parasitoids’ efficacy in Tehran’s polluted air, they can be used as biological agents in the management of A. craccivora population.     

Host suitability of different instars of the whitefly Bemisia tabaci `biotype Q' for Eretmocerus mundus

Eretmocerus mundus Mercet is a parasitoidof Bemisia tabaci (Genn.) indigenous tothe Mediterranean and is used commercially foraugmentative biological control in Spain andelsewhere. A better understanding of thesuitability of different host instars wouldhelp optimize production and field application.Incidence of parasitism, development time,survivorship and sex ratio were evaluated whendifferent nymphal instars of the sweetpotatowhitefly Bemisia tabaci biotype `Q' wereoffered for parasitization. Experiments wereconducted on sweet pepper at 25 °C, 75%RH and 16:8 (L:D) photoperiod. E. mundusoviposited in all nymphal instars of B.tabaci except the mature 4th instar orpharate adult (previously designated, `pupa').Incidence of parasitism was greatest (33.8± 5.1 parasitized nymphs) and developmenttime shortest (14.1 ± 0.1 d) whenoviposition occurred under 2nd and3rd instar nymphs compared to 1st or4th instars. Survivorship (85%) andoffspring sex ratio (39.8% female) did notdiffer statistically for parasitoids developingin whiteflies that were parasitized asdifferent instars. Although 2nd and3rd instars were clearly the mostfavorable host stage for E. mundus, itscapacity to parasitize and develop on a widerange of host stages is a favorablecharacteristic for both rearing and fieldapplication.     

Effect of mating combinations on the host parasitisation and sex allocation in solitary endoparasitoid,Aenasius arizonensis (Hymenoptera: Encyrtidae)

Aenasius arizonensis (Girault) is an important solitary endoparasitoid of Phenacoccus solenopsis Tinsley. To optimise the mass production of high-quality females, it is important to assess the influence of mating regimes on the progeny fitness and sex allocation. We, therefore, hypothesise that mating combinations in A. arizonensis adults emerged from different host instars may influence parasitism and sex allocation in the subsequent generation. Therefore, we compared three nymphal instars (1st, 2nd and 3rd) and adults host stages of P. solenopsis for parasitism and sex allocation by A. arizonensis. Further, F₁ female progeny of the parasitoid emerged from different host instars was henceforth evaluated for its fitness in six mating combinations. A. arizonensis females parasitised all the host stages except the 1st instar nymphs. The parasitised 2nd instar nymphs yielded only males, while the sex ratio in the later host instars was strongly female-biased. The parasitoid females preferred 3rd instar nymphs with respect to higher parasitism (74.0–84.0%) and produced more females in the F₁ progeny as compared to other host stages. F₁ females that emerged from 3rd instar nymphs produced significantly higher parasitism (74.0–79.0%). These mating combinations also yielded more female progeny in the F₂ generation. However, parasitism by F₁ females was significantly lower (9.0–12.0%) when mated with males that emerged from 2nd instar P. solenopsis nymphs. Moreover, latter combinations yielded only male progeny in F₂ generation. These findings can be used in laboratory mass rearing of this parasitoid vis-à-vis biological control of P. solenopsis.     

The effects of aggregation on survival and growth rate in the wood‐feeding cockroach Panesthia angustipennis spadica (Blaberidae)

The wood‐feeding cockroach Panesthia angustipennis spadica Shiraki (Blaberidae) is a gregarious species, and its groups containing both nymphs and adults have often been observed in the field. To clarify the effects of aggregation with a parent and siblings on nymphal development in P. angustipennis spadica, we raised nymphs of this species in the laboratory under the following three experimental categories: (a) aggregate with a female adult; (b) aggregate without a female adult; and (c) solitary. Survival rates did not significantly differ among the three categories. Unexpectedly, our results clearly showed that nymphs raised in aggregates with a female adult were smaller, lighter, and reached a lower instar than those raised without a female adult. These results indicate that the presence of a female adult does not positively affect nymphal development; i.e. there may be no direct parental care in this species as indicated by previous studies. Moreover, solitary nymphs grew faster and larger than nymphs in aggregates, showing that the aggregation with siblings also has no positive effect on nymphal development. Thus, it seems likely that external factors, e.g. predation and environmental conditions, may drive aggregating behavior in P. angustipennis spadica.