Rapid and Repeated Climate Adaptation Involving Chromosome Inversions following Invasion of an Insect.

Following invasion, insects can become adapted to conditions experienced in their invasive range, but there are few studies on the speed of adaptation and its genomic basis. Here, we examine a small insect pest, Thrips palmi, following its contemporary range expansion across a sharp climate gradient from the subtropics to temperate areas. We first found a geographically associated population genetic structure and inferred a stepping-stone dispersal pattern in this pest from the open fields of southern China to greenhouse environments of northern regions, with limited gene flow after colonization. In common garden experiments, both the field and greenhouse groups exhibited clinal patterns in thermal tolerance as measured by critical thermal maximum (CTmax) closely linked with latitude and temperature variables. A selection experiment reinforced the evolutionary potential of CTmax with an estimated h2 of 6.8% for the trait. We identified 3 inversions in the genome that were closely associated with CTmax, accounting for 49.9%, 19.6%, and 8.6% of the variance in CTmax among populations. Other genomic variations in CTmax outside the inversion region were specific to certain populations but functionally conserved. These findings highlight rapid adaptation to CTmax in both open field and greenhouse populations and reiterate the importance of inversions behaving as large-effect alleles in climate adaptation.

Bt maize can provide non-chemical pest control and enhance food safety in China.

China is the world's second-largest maize producer and consumer. In recent years, the invasive fall armyworm Spodoptera frugiperda (J.E. Smith) has adversely affected maize productivity and compromised food security. To mitigate pest-inflicted food shortages, China's Government issued biosafety certificates for two genetically modified (GM) Bt maize hybrids, Bt-Cry1Ab DBN9936 and Bt-Cry1Ab/Cry2Aj Ruifeng 125, in 2019. Here, we quantitatively assess the impact of both Bt maize hybrids on pest feeding damage, crop yield and food safety throughout China's maize belt. Without a need to resort to synthetic insecticides, Bt maize could mitigate lepidopteran pest pressure by 61.9-97.3%, avoid yield loss by 16.4-21.3% (range -11.9-99.2%) and lower mycotoxin contamination by 85.5-95.5% as compared to the prevailing non-Bt hybrids. Yield loss avoidance varied considerably between experimental sites and years, as mediated by on-site infestation pressure and pest identity. For either seed mixtures or block refuge arrangements, pest pressure was kept below established thresholds at 90% Bt maize coverage in Yunnan (where S. frugiperda was the dominant species) and 70% Bt maize coverage in other sites dominated by Helicoverpa armigera (Hübner) and Ostrinia furnacalis (Guenée). Drawing on experiences from other crop/pest systems, Bt maize in se can provide area-wide pest management and thus, contribute to a progressive phase-down of chemical pesticide use. Hence, when consciously paired with agroecological and biodiversity-based measures, GM insecticidal crops can ensure food and nutrition security, contribute to the sustainable intensification of China's agriculture and reduce food systems' environmental footprint.

Testing for adaptive changes linked to range expansion following a single introduction of the fall webworm.

Adaptive evolution following colonization can affect the impact of invasive species. The fall webworm (FWW) invaded China 40 years ago through a single introduction event involving a severe bottleneck and subsequently diverged into two genetic groups. The well-recorded invasion history of FWW, coupled with a clear pattern of genetic divergence, provides an opportunity to investigate whether there is any sign of adaptive evolution following the invasion. Based on genome-wide SNPs, we identified genetically separated western and eastern groups of FWW and correlated spatial variation in SNPs with geographical and climatic factors. Geographical factors explained a similar proportion of the genetic variation across all populations compared with climatic factors. However, when the two population groups were analysed separately, environmental factors explained more variation than geographical factors. SNP outliers in populations of the western group had relatively stronger response to precipitation than temperature-related variables. Functional annotation of SNP outliers identified genes associated with insect cuticle protein potentially related to desiccation adaptation in the western group and genes associated with lipase biosynthesis potentially related to temperature adaptation in the eastern group. Our study suggests that invasive species may maintain the evolutionary potential to adapt to heterogeneous environments despite a single invasion event. The molecular data suggest that quantitative trait comparisons across environments would be worthwhile.

Flight Performance of Mamestra brassicae (Lepidoptera: Noctuidae) Under Different Biotic and Abiotic Conditions.

Mamestra brassicae L. is an important, regionally migratory pest of vegetable crops in Europe and Asia. Its migratory activity contributes significantly to population outbreaks, causing severe crop yield losses. Because an in-depth understanding of flight performance is key to revealing migratory patterns, here we used a computer-linked flight mill and stroboscope to study the flight ability and wingbeat frequency (WBF) of M. brassicae in relation to sex, age, temperature, and relative humidity (RH). The results showed that age significantly affected the flight ability and WBF of M. brassicae, and 3-d-old individuals performed the strongest performance (total flight distance: 45.6 ± 2.5 km; total flight duration: 9.3 ± 0.3 h; WBF: 44.0 ± 0.5 Hz at 24°C and 75% RH). The age for optimal flight was considered to be 2-3 d old. Temperature and RH also significantly affected flight ability and WBF; flight was optimal from 23°C to 25°C and 64-75% RH. Because M. brassicae thus has great potential to undertake long-distance migration, better knowledge of its flight behavior and migration will help establish a pest forecasting and early-warning system.

Genome-wide identification and characterization of putative lncRNAs in the diamondback moth, Plutella xylostella (L.).

Long non-coding RNAs (lncRNAs) are of particular interest because of their contributions to many biological processes. Here, we present the genome-wide identification and characterization of putative lncRNAs in a global insect pest, Plutella xylostella. A total of 8096 lncRNAs were identified and classified into three groups. The average length of exons in lncRNAs was longer than that in coding genes and the GC content was lower than that in mRNAs. Most lncRNAs were flanked by canonical splice sites, similar to mRNAs. Expression profiling identified 114 differentially expressed lncRNAs during the DBM development and found that majority were temporally specific. While the biological functions of lncRNAs remain uncharacterized, many are microRNA precursors or competing endogenous RNAs involved in micro-RNA regulatory pathways. This work provides a valuable resource for further studies on molecular bases for development of DBM and lay the foundation for discovery of lncRNA functions in P. xylostella.

Chromosome-level genome assembly of the Japanese sawyer beetle Monochamus alternatus.

The Japanese sawyer beetle Monochamus alternatus (Coleoptera: Cerambycidae) is a pest in pine forests and acts as a vector for the pine wood nematode Bursaphelenchus xylophilus, which causes the pine wilt disease. We assembled a high-quality genome of M. alternatus at the chromosomal level using Illumina, Nanopore, and Hi-C sequencing technologies. The assembled genome is 767.12 Mb, with a scaffold N50 of 82.0 Mb. All contigs were assembled into ten pseudo-chromosomes. The genome contains 63.95% repeat sequences. We identify 16, 284 protein-coding genes in the genome, of which 11,244 were functionally annotated. The high-quality genome of M. alternatus provides an invaluable resource for the biological, ecological, and genetic study of this beetle and opens new avenues for understanding the transmission of pine wood nematode by insect vectors.

Stable Isotopes Indicate Seasonal Changes in Natal Geographic Origins and Host Plants of Ostrinia furnacalis (Guenée) Migrants Across the Bohai Strait in China.

The Asian corn borer, Ostrinia furnacalis (Guenée), is a notorious pest of maize that migrates seasonally in Asia. Two migration peaks were found on Beihuang island in the Bohai Strait of China by observing the number of migrants. However, the origins and host plants of the migrants in the two migration periods remain unclear. Here, stable hydrogen (δ2H) and carbon (δ13C) isotope levels were measured to infer the origin and host plants of the O. furnacalis captured on Beihuang island in 2017-2019. δ2H in wings of spring-summer O. furnacalis captured from May to June ranged from -99 to -56‰, while that of autumn migrants from August to September ranged from -127 to -81‰. Based on the linear relationship between δ2H in the wing of migrants (δ2Hw) and δ2H in precipitation (δ2Hp), the spring-summer O. furnacalis likely originated from the summer maize area in the Huang-Huai-Hai Plain in China. In contrast, the autumn migrants came from the northern spring maize area in Liaoning, Jilin and Inner Mongolia. Based on δ13C, the spring-summer migrants fed on both C3 plants such as wheat (47.76%) and C4 weeds or belonged to the over winter individuals in maize field (52.24%), while the autumn migrants mainly fed on maize (C4, 91.21%). The results point to a northward migration in spring-summer and southward migration in autumn of O. furnacalis. Our study gives an important knowledge for improving the forecasting and management level of this pest.

A comprehensive assessment of insecticide resistance mutations in source and immigrant populations of the diamondback moth Plutella xylostella (L.).

BACKGROUND: The diamondback moth (DBM) Plutella xylostella has developed resistance to almost all insecticides used to control it. Populations of DBM in temperate regions mainly migrate from annual breeding areas. However, the distribution pattern of insecticide resistance of DBM within the context of long-distance migration remains unclear. RESULTS: In this study, we examined the frequency of 14 resistance mutations for 52 populations of DBM collected in 2010, 2011, 2017 and 2018 across China using a high-throughput KASP genotyping method. Mutations L1041F and T929I conferring pyrethroid resistance, and mutations G4946E and E1338D conferring chlorantraniliprole resistance were near fixation in most populations, whereas resistant alleles of F1020S, M918I, A309V and F1845Y were uncommon or absent in most populations. Resistance allele frequencies were relatively stable among different years, although the frequency of two mutations decreased. Principal component analysis based on resistant allele frequencies separated a southern population as an outlier, whereas the immigrants clustered with other populations, congruent with the migration pattern of northern immigrants coming from the Sichuan area of southwestern China. Most resistant mutations deviated from Hardy-Weinberg equilibrium due to a lower than expected frequency of heterozygotes. The deviation index of heterozygosity for resistant alleles was significantly higher than the index obtained from single nucleotide polymorphisms across the genome. These findings suggest heterogeneous selection pressures on resistant mutations. CONCLUSION: Our results provide a picture of resistant mutation patterns in DBM shaped by insecticide usage and migration of this pest, and highlight the widespread distribution of resistance alleles in DBM. © 2022 Society of Chemical Industry.

A high-throughput KASP assay provides insights into the evolution of multiple resistant mutations in populations of the two-spotted spider mite Tetranychus urticae across China.

BACKGROUND: The two-spotted spider mite (TSSM), Tetranychus urticae (Acari: Tetranychidae), is a cosmopolitan phytophagous pest in agriculture and horticulture. It has developed resistance to many acaricides by target-site mutations. Understanding the status and evolution of resistant mutations in the field is essential for resistance management. Here, we applied a high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) method for detecting six mutations conferring resistance to four acaricides of the TSSM. We genotyped 3274 female adults of TSSM from 43 populations collected across China in 2017, 2020, and 2021. RESULTS: The KASP genotyping of 24 testing individuals showed 99% agreement with Sanger sequencing results. KASP assays showed that most populations had a high frequency of mutations conferring avermectin (G314D and G326E) and pyridaben (H92R) resistance. The frequency of mutation conferring bifenazate (A269V and G126S) and etoxazole (I1017F) resistance was relatively low. Multiple mutations were common in the TSSM, with 70.2% and 24.6% of individuals having 2-6 and 7-10 of 10 possible resistant alleles, respectively. No loci were linked in most populations among the six mutations, indicating the development of multiple resistance is mainly by independent selection. However, G314D and I1017F on the nuclear genome deviated from Hardy-Weinberg equilibrium in most populations, indicating significant selective pressure on TSSM populations by acaricides or fitness cost of the mutations in the absence of acaricide selection. CONCLUSION: Our study revealed that the high frequency of TSSMs evolved multiple resistant mutations in population and individual levels by independent selection across China, alarming for managing multiple-acaricides resistance. © 2023 Society of Chemical Industry.

KASP genotyping and semi-quantitation of G275E mutation in the α6 subunit of Thrips palmi nAChR gene conferring spinetoram resistance.

BACKGROUND: Pesticide resistance is a long-standing and growing problem in the chemical control of invertebrate pests. Molecular diagnostic methods can facilitate pesticide resistance management by accurately and efficiently detecting resistant mutations and their frequency. In this study, the kompetitive allele specific PCR (KASP) approach, a technology for high-throughput single nucleotide polymorphism (SNP) genotyping, is validated as a useful method for characterizing genotypes at a pesticide-resistance locus for the first time. We focus on the spinetoram resistance mutation of G275E in the nicotinic acetylcholine receptor alpha 6 (nAChR α6) subunit gene of Thrips palmi. RESULTS: Of the 341 individuals of Thrips palmi tested, 98.24% were successfully genotyped, with 100% concordance with Sanger sequencing results. We then quantitatively mixed genomic DNA of known genotypes to establish 21 DNA mixtures with a resistant allele frequency ranging from 0 to 100% at steps of 5%. The linear discriminant analysis (LDA) showed that 75.8% of original grouped cases were correctly classified; six groups had no overlap in membership (resistant allele frequency: 0%, 5%, 10-75%, 80-85%, 90-95%, and 100%). When we chose 11 pooled samples with 10% steps for LDA, 84.4% of original grouped cases were correctly classified; seven groups had no overlap in membership (0%, 10%, 20-30%, 40-70%, 80%, 90%, 100%). The results indicated that KASP applied to pooled samples may provide a semi-quantitative estimate of resistance. CONCLUSIONS: Our study points to the suitability of KASP for high-throughput genotyping of genotypes affecting pesticide resistance and semi-quantitative assessments of resistance allele frequencies in populations. © 2023 Society of Chemical Industry.

Interspecific and intraspecific variation in susceptibility of two co-occurring pest thrips, Frankliniella occidentalis and Thrips palmi, to nine insecticides.

BACKGROUND: Field control of pest thrips mainly relies on insecticides, but the toxicity of insecticides can vary among thrips species and populations. In this study, we examined the susceptibility of multiple field populations of two thrips pests, Frankliniella occidentalis, and Thrips palmi, that often co-occur on vegetables, to nine insecticides belonging to seven subgroups. RESULTS: The highest level of variation in susceptibility among F. occidentalis populations was for spinetoram (73.92 fold difference between most resistant and most susceptible population), followed by three neonicotinoids (8.06-15.99 fold), while among T. palmi populations, it was also for spinetoram (257.19 fold), followed by emamectin benzoate, sulfoxaflor, and acetamiprid (23.64-45.50 fold). These findings suggest evolved resistance to these insecticides in some populations of the two thrips. One population of F. occidentalis had a particularly high level of resistance overall, being the most resistant for five of the nine insecticides tested. Likewise, a population of T. palmi had high resistance to all nine insecticides, again suggesting the evolution of resistance to multiple chemicals. For F. occidentalis, the LC95 values of most populations were higher than the field-recommended dosage for all insecticides except chlorfenapyr and emamectin benzoate. For several T. palmi populations, the LC95 values also tended to be higher than recommended dosages, except in the case of emamectin benzoate and spinetoram. CONCLUSIONS: Our study found interspecific and intraspecific variations in the susceptibility of two thrips to nine insecticides and multiple resistance in some populations, highlighting the need for ongoing monitoring and resistance management. © 2023 Society of Chemical Industry.

Influence of seasonal migration on the development of the insecticide resistance of oriental armyworm (Mythimna separata) to λ-cyhalothrin.

BACKGROUND: The seasonal long-distance migration of pests amongst different seasonal habitats may encounter different degrees of insecticide selection pressure, but knowledge of the evolution of resistance and the underlying mechanisms remains sparse. Here, we show that the development of resistance of the oriental armyworm Mythimna separata (Walker), a notorious agricultural pest that migrates between northern and northeast China seasonally across the Bohai Gulf, is influenced by seasonal migration. RESULTS: There are two conspicuous migrations of M. separata, the northeastwards population in the second half of May and the returning population in the second half of August, between northern and northeast China per year, and the abundance values of migrants from April to October are significantly different. The resistance levels of seasonal migratory populations to λ-cyhalothrin vary in different months, and the resistance levels and estimated frequency of the resistance allele of the first northeastward population (second half of May, May.-2) in spring are significantly higher than those of the returning populations in autumn. Moreover, resistance decline was observed in migrating population Jul.-2, with the resistance levels in the F1 progeny higher than those in their F2 progeny. Synergism tests indicate that cytochrome P450 monooxygenases (P450s) play a major role in resistance. Enzymatic assays show that P450 activity is significantly correlated with resistance levels in migratory populations, indicating that increased P450 activity is the main mechanism of resistance. The expression levels and correlation analysis of the relative expression of P450s with resistance levels show that three (CYP9A144, CYP9G40, and CYP6B79) out of 23 genes from CYP6 and CYP9 subfamilies are potentially involved in resistance to λ-cyhalothrin. CONCLUSION: Our results show that the resistance of M. separata to λ-cyhalothrin is unstable and likely to be metabolically driven by enhanced P450 activity mediated by the overexpression of multiple P450 genes. Notably, the development of resistance is probably affected by seasonal migration.

Increasing Frequency of G275E Mutation in the Nicotinic Acetylcholine Receptor α6 Subunit Conferring Spinetoram Resistance in Invading Populations of Western Flower Thrips in China.

The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is an important invasive pest worldwide. Field-evolved resistance to the pesticide spinetoram is an increasing problem in the chemical control of this pest. Here, we examined changes in the frequency of a genetic mutation associated with spinetoram resistance, the G275E mutation in the acetylcholine receptor Foα6, in 62 field populations collected from 2009 to 2021 across areas of China invaded by this pest. We found a low frequency of the G275E mutation in populations collected at the early invasion stage, in contrast to a high frequency in native USA populations. However, the frequency of the G275E mutation has increased to a high level in recently collected populations, with the mutation becoming fixed in some populations. There was a correlation between the frequency of the G275E mutation and resistance to spinetoram as characterized by median lethal concentration, although two populations were outliers. These results showed that G275E mutation is one of the mechanisms conferring spinetoram resistance in many invading populations in China. Ongoing dispersal of the WFT may have facilitated a rapid increase in the G275E mutation across China. Our study highlights the rapid evolution of pesticide resistance in an invasive species and points to a useful marker for molecular diagnostics of spinetoram resistance.

Variation in the toxicity of a novel meta-diamide insecticide, broflanilide, among thrips pest species and developmental stages.

BACKGROUND: Thrips pests cause increasing damage to crops around the world. Widespread usage of some insecticides against thrips has now led to the evolution of resistance to several active ingredients, and new insecticides are required. This study examined the toxicity of the novel insecticide broflanilide to multiple populations of several thrips pests. RESULTS: Bioassays showed that thrips populations had LC50 values ranging from 0.5 to almost 300 mg·L-1 . A population of Frankliniella occidentalis had the highest LC50 value at 290.63 mg·L-1 , while a population of Echinothrips americanus had the lowest LC50 value at 0.51 mg L-1 . LC50 values among seven populations of Thrips palmi ranged from 2.5689 to 23.6754 mg·L-1 , indicating intraspecific variation in toxicity. In this species, the toxicity of broflanilide was relatively higher in adults than in larvae. More than 90% of eggs of T. palmi could not develop into larvae when treated with 5-50 mg L-1 broflanilide. Compared to five commonly used insecticides, broflanilide showed relatively high toxicity to T. palmi. Field control tests with T. palmi showed that control efficacy (from 90.44% to 93.14%) was maintained from day three to day 14 after treatment with 22.5 and 45 ga.i hm-1 broflanilide. CONCLUSION: Broflanilide is potentially a useful insecticide for controlling Thrips hawaiiensis, Frankliniella intonsa, Megalurothrips usitatus. E. americanus, and some populations of T. palmi. However, the variation in toxicity of this insecticide to different species, populations, and developmental stages indicates that target species and life stages may need to be carefully considered. © 2022 Society of Chemical Industry.

Correction: Wu, Q.-L.; et al. Estimation of the Potential Infestation Area of Newly-Invaded Fall Armyworm Spodoptera Frugiperda in the Yangtze River Valley of China. Insects 2019, 10, 298.

The authors wish to make the following correction to this paper [...].

Interspecific and Seasonal Variation in Wingbeat Frequency Among Migratory Lepidoptera in Northern China.

Many lepidopteran species rely upon active flight to migrate over long distances, thus pursuing ephemeral resources, colonizing new habitats, or escaping adverse meteorological conditions. Though their biology and ecology are often well studied, there is only scant information on their wingbeat frequency (WBF), a key aerodynamic determinant of insect flight. In this study, we assessed interspecific and seasonal variability in WBF for 85 different migratory species of Lepidoptera (11 families) under the laboratory conditions of 25 ± 1°C and 75 ± 5% RH. WBF of migrant individuals ranged between 6.7 and 84.5 Hz and substantial interspecific differences were recorded, with members of the Bombycidae exhibiting the highest mean WBFs (i.e., 55.1 ± 1.0 Hz) and Saturniidae the lowest (8.5 ± 0.2 Hz). At a species level, seasonal variation was observed in WBF for Mythimna separata (Walker) (Lepidoptera: Noctuidae), Scotogramma trifolii Rottemberg (Lepidoptera: Noctuidae), and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Our findings add to the scientific knowledge on flight biology of migratory insects, facilitate (automatic) monitoring and population forecasting, and can have broader implications for insect pest management or biodiversity conservation.

Seasonal Migration Patterns of Ostrinia furnacalis (Lepidoptera: Crambidae) Across the Bohai Strait in Northern China.

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is a notorious polyphagous insect pest in China and other regions of East Asia. The long-distance flight behavior of the ACB moths, however, is not well understood, especially in the field conditions. In this study, nightly monitoring data for multiple years (2003-2017) on the long-distance flight of adults crossing the Bohai Strait in northern China, showed that a large number of the adults frequently flew across the Bohai Strait from May to September with the peak migrations usually occurred, followed in order by September, June, July, and May, and the number of southward migrants was larger than that of northward migrants. From May to September in 2010, 2011, and 2017, a subsample of trapped ACB females was dissected (879 individuals in 2010, 197 individuals in 2011, and 247 individuals in 2017), and the results showed that the sex ratio of the trapped ACB moths was unbiased each month from May to September. While the proportion of mated females in northward populations (May to July = 92.85 ± 4.86%) was significantly higher than that in southward population (August and September = 74.53 ± 8.55%) . The seasonal pattern in the proportion of sexually mature females was similar to the above proportion of mated females. We conclude that the ACBs have a strong propensity and ability for long-distance migration, although local dispersal is generally considered to be the primary movement of the ACB. These findings may be helpful to improve the forecasting systems and the pest management schemes for the ACBs.

Estimation of the Potential Infestation Area of Newly-invaded Fall Armyworm Spodoptera Frugiperda in the Yangtze River Valley of China.

The fall armyworm (FAW), native to the Americas, has rapidly invaded the whole of Southern China since January 2019. In addition, it can survive and breed in the key maize- and rice- growing area of the Yangtze River Valley. Furthermore, this pest is also likely to continue infiltrating other cropping regions in China, where food security is facing a severe threat. To understand the potential infestation area of newly-invaded FAW from the Yangtze River Valley, we simulated and predicted the possible flight pathways and range of the populations using a numerical trajectory modelling method combining meteorological data and self-powered flight behavior parameters of FAW. Our results indicate that the emigration of the first and second generations of newly-invaded FAW initiating from the Yangtze River Valley started on 20 May 2019 and ended on 30 July 2019. The spread of migratory FAW benefitted from transport on the southerly summer monsoon so that FAW emigrants from the Yangtze River Valley can reach northern China. The maize-cropping areas of Northeastern China, the Korean Peninsula and Japan are at a high risk. This study provides a basis for early warning and a broad picture of FAW migration from the Yangtze River Valley.

Cardiac allograft tolerance induced by isogeneic CD4+CD25+ regulatory T cells.

OBJECTIVES: To evaluate the effect of isogeneic CD4+CD25+ regulatory T cells on cardiac allograft tolerance in heterotopic heart transplant from Balb/c to C57BL/6 mice. MATERIALS AND METHODS: Isogeneic and allogeneic CD4+CD25+ regulatory T cells were obtained from pregnant C57BL/6 mice crossed with male Balb/c mice and from regular Balb/c mice. Recipient C57BL/6 mice were treated with sublethal radiation (2 Gy) and an infusion of isogeneic CD4+CD25+ regulatory T cells, allogeneic CD4+CD25+ regulatory T cells, or phosphate-buffered saline alone 1 day before a Balb/c-to-C57BL/6 heterotopic heart transplant. At 10 days after the transplant, cardiac allografts and the sera of recipients were evaluated with histology and cytokine analysis. Splenocytes of recipients were collected to determine chimerism on the day of the cessation of allograft heartbeat. RESULTS: Mice that received an infusion of isogeneic CD4+CD25+ regulatory T cells had significantly greater mean median survival time, greater degree of chimerism, decreased levels of cytokines (monokine induced by interferon-γ, interleukin 6, interleukin 10, and regulated upon activation, normal T cell expressed and secreted protein), and decreased lymphocytic infiltration than did mice that received phosphate-buffered saline alone. The effects on allograft tolerance were stronger in mice that received the isogeneic than the allogeneic CD4+CD25+ regulatory T cells. CONCLUSIONS: Isogeneic CD4+CD25+ regulatory T cells may establish cardiac allograft tolerance by inducing mixed chimerism and suppressing immune responses. Infusion with isogeneic CD4+CD25+ regulatory T cells combined with radiation (sublethal dose, 2 Gy) may establish allograft tolerance in mice. Therefore, further study is warranted because isogeneic CD4+CD25+ regulatory T cells may have therapeutic benefits.