Cnidium monnieri (L.) Cusson, an annual herb with many flowers, blooms from May to July. C. monnieri can support several predatory natural enemies, and the addition of C. monnieri strips increases the density of Harmonia axyridis  and improves the biological control of apple aphids in an apple orchard. H. axyridis is also the most important natural enemy in wheat aphid biocontrol and is attracted to healthy and aphid-infested C. monnieri plants. In addition, adult Propylaea japonica Thunberg survives significantly longer on C. monnieri flowers than on a water-only diet. In this study, a laboratory experiment was conducted to assess (i) the effect of nutritional supplements derived from C. monnieri flowers on the development and reproduction of H. axyridis under a wheat aphids-only diet; (ii) the effect of C. monnieri flowers on H. axyridis adult reproduction performance. We compared the larval durations, survival, weight, adult longevity, and reproduction of H. axyridis reared on wheat aphids-only and aphids plus C. monnieri flower diets. The results showed that H. axyridis larvae reared on aphids plus flowers had significantly greater weights and survival rates, shorter larval durations, and produced 1.62 times more eggs than those reared on wheat aphids-only diets. H. axyridis adults ingesting a C. monnieri flowers plus an aphid diet increased egg production 1.44 times compared to the aphids-only diet. Our study demonstrates that C. monnieri flowers as a supplementary food positively affect the survival, development, and reproduction performance of H. axyridis.Predaceous ladybeetles are highly polyphagous predators that ingest supplementary food from flowering plants. Flowering plants widely grown in agroecosystems can sustain multiple natural enemies of agricultural pests, and the pollen and nectar resources from flowering plants may have a positive role in natural enemies.  Harmonia axyridis  (Coleoptera: Coccinellidae) is a species native to central and eastern Asia and has been established in America, Europe, and Africa [H. axyridis is a highly polyphagous predator that consumes mainly aphids but can also feed on Tetranychidae, Psyllidae, Coccoidea, Chrysomelidae, Curculionidae, and Lepidoptera. It has been recognized as a biological control agent for several species of aphid and coccid pests and has provided biocontrol services in various agroecosystems  [H. axyridis and Propylaea japonica Thunberg are the most important natural enemies for cereal aphids and cotton aphid biocontrol [H. axyridis adult ingests nectar from wild flowers as well as extrafloral nectar [H. axyridis adults frequently visit and feed on floral resources such as pollen in fields [H. axyridis.The multicolored Asian ladybeetle d Africa ,2. H. axd pines) ,4. In noocontrol ,6. H. axl nectar . Gut conn fields ,8. LundgLikewise, in agroecosystems, the availability of floral resources is crucial for natural enemies . FloweriPerilla frutescens (Lamiaceae) flowers increased the longevity and fecundity of H. axyridis adults [Ephestia kuehniella (Lepidoptera: Phycitidae) eggs significantly improved the longevity and reproductive performance of H. axyridis adults [Vicia sativa (Fabaceae), Fagopyrum esculentum (Polygonaceae), and Coriandrum sativum (Apiaceae) improved the survival and reproduction of H. axyridis adults [Brassica and Sonchus flowers, as supplementary foods (pollen and nectar), can improve the reproductive performance of Hippodamia variegata, specifically under conditions of prey limitation [The effects of flowering plants on natural enemies vary greatly among plant species. Thus, selecting suitable flowering plants to target natural enemies is crucial to improving their fitness and enhancing their biological control capacity . For exas adults . In paras adults . Vicia ss adults . Brassicmitation . Brumoides foudrasii (Coleoptera: Coccinellidae) and appeared to exert an inhibitory effect on oviposition behavior [Helianthus annus L., proved fatal to H. axyridis larvae and adults [H. axyridis reared on Ageratum conyzoides or Bidens pilosa in combination with Anagasta kuehniella (Zeller)  had no oviposition [By contrast, Mauricio Silva de Lima revealed that pollen did not provide suitable nutrients for ovarian development in behavior . Michaudd adults . H. axyrposition . Floweriposition , and floposition . TherefoCnidium monnieri (L.) Cusson (Apiaceae: Cnidium) is a traditional Chinese herbal medicine that has been grown and used in China, Japan, Korea, and Vietnam. In China, it is widely grown in Hebei, Jiangsu, Zhejiang, Shandong, and Sichuan provinces [C. monnieri naturally regrows from seeds in fields and roadsides and requires no particular care. In agricultural fields, it is sown in rows for seed harvesting. Our previous study found that C. monnieri is characterized by early blossom, long inflorescence, and high pollen yields and produces prolific small white flowers with shallow corollas that make pollen more easily foraged by natural enemies. It can sustain several predatory natural enemies, such as H. axyridis and P. japonica, during its flowering period [C. monnieri significantly increased the density of predators  on apple trees and significantly decreased the density of spirea aphids [C. monnieri flower strips at the border of wheat-maize rotation fields served as a bridge habitat to conserve ladybeetles in wheat fields during harvest and helped the predator to immigrate to adjacent maize fields for pest control [rovinces . C. monng period ,26. In aa aphids . In a fi control .H. axyridis adults were attracted to healthy and aphid-infested C. monnieri plants. In addition, H. axyridis adults were attracted to 1,2-diethylbenzene and p-diethylbenzene synthetic volatile compounds detected among the volatile blends emanating from healthy and aphid-infested plants in the GC–MS and GC-EAD trials [C. monnieri as a floral food for P. japonica when prey is insufficient could significantly affect the longevity of P. japonica adults compared to a water-only diet [C. monnieri flowers as a supplementary food on larval development and reproduction of H. axyridis adults has not been studied.Furthermore, a Y-tube olfactometer laboratory experiment indicated that D trials . A labornly diet . HoweverH. axyridis longevity and fecundity instead of a single pollen diet.Previous studies have focused on the effects of pollen and sugar diets or their mixed diets with prey on ladybeetle longevity and fecundity ,21,28,29Rhopalosiphum padi and Sitobion avenae (Fabricius) the critical wheat aphids in winter wheat, and ladybeetles of H. axyridis and P. japonica are the most important natural enemies in wheat aphid biocontrol [R. padi is adequate prey and that H. axyridis larvae fed on R. padi can complete development and produce hatchable eggs. However, under the same laboratory conditions, compared with R. padi., Acyrthosiphon pisum Harris and Schizaphis graminum Rondani were the most suitable prey for H. axyridis [C. monnieri flower can prolong larval and adult survival but did not fulfill larval development and adult reproduction of H. axyridis. These experiments addressed the following two questions: (1) Are C.monnieri flowers a suitable food for Harmonia axyridis? (2) Do the C. monnieri flowers enhance the development and reproduction performance of Harmonia axyridis? In this study, we measured the effect of nutritional supplements derived from C. monnieri fresh flowers on the development and reproduction of H. axyridis under wheat aphids-only diet conditions. First, the benefit of C. monnieri flowers on the development and reproduction of H. axyridis was compared with a wheat aphids-only diet. Second, the effects of C. monnieri as supplementary food on the longevity and fecundity of H. axyridis adults were assessed.In northern China, ocontrol . Furtheraxyridis . MoreoveH. axyridis. When reared on a wheat aphids-only diet, only 77.3% of the larvae successfully reached adulthood, whereas the percentage was 90.7% for larvae offered a mixed diet of wheat aphids plus flowers  . There were no significant differences in first instars (p = 0.984) and second instars (p = 0.554) among treatments.C. monnieri flowers significantly improved the larval survival rate of  0.0015) . The lar 0.0015)    (< 0.001) a,b. H. a= 0.001) c,d. The t-test: t = −3.309, df = 59, p = 0.002)   b. The lo= 0.071) a. C. monnieri flowers as supplementary food to H. axyridis adults, the longevity of female ladybeetles and male ladybeetles was not significantly different between treatments   a,b.Reproduction was characterized using four parameters: the pre-oviposition period, the oviposition period, fecundity, and the hatching rate of eggs.t-test: t = −3.794, df = 58, p < 0.001)   (p = 0.317)  c. C. mon= 0.007) e. Furthe= 0.001) f on the = 0.317) d.H. axyridis and increased egg production by 1.41 times. Females reared on the aphids plus flowers diet laid a mean of 1027.13 ± 83.11 eggs with 731.37 ± 64.99 eggs reared on the aphids-only diet      c. Howeve= 0.471) d, the ov= 0.398) e or the = 0.215) f.H. axyridis is a polyphagous predator that has been recognized as a biological control agent for several insect pests. H. axyridis ingests nectar, and pollen from flowering plants, and the floral resources are used by coccinellids to increase survival, reduce mortality, supply energy, and enhance reproductive capacity. In this study, we found that the flowering plants C. monnieri provide supplementary food that affects the development, longevity, and fecundity of H. axyridis. H. axyridis larvae completed development in both diets, with shorter developmental times, higher larval survival rates, heavier adult weights, larger body sizes, and more egg production resulting from the provision of C. monnieri flowers. H. axyridis larvae reared on the aphid plus flower diet had a significantly shorter larval duration and greater pupal weight and adult body length than the aphids-only treatment. This may indicate that the mixed diet of aphids and C. monnieri flowers is more suitable to H. axyridis larvae. J.P. Michaud wisely suggested that diets that support completed development are suitable diet, and the faster the development and/or the larger the adult the more suitable diet [Larval performance was substantially enhanced when reared on the aphid plus flower diet compared to the aphids-only diet. Experiment I showed that ble diet . Similarble diet ,33,34. C. monnieri flowers were beneficial to H. axyridis. Hodek and Honek suggested that many polyphagous coccinellids may have a mixed feeding habit, in which they select a favorable balance of important nutrients from various foods, including plant materials [H. axyridis fed both flowers  and prey  developed better than larvae fed each diet separately [Adalia bipunctata (L.) (Coleoptera: Coccinellidae) fed on a mixed diet of frozen bee pollen and Ephestia kuehniella (Lepidoptera: Phycitidae) eggs had a faster development than on a diet of only E. kuehniella eggs [Coleomegilla maculata (DeGeer) developed better on a mixture of aphids and corn pollen than on aphids or pollen alone [E. kuehniella eggs with bee pollen only shortened the pre-oviposition period of H. axyridis but did not affect other developmental and reproductive parameters [C. monnieri flowers may provide floral resources for H. axyridis larvae, which contain nutrients that are not abundant in wheat aphids. The nutrition of C. monnieri flowers helps to improve some essential foods to promote the development of H. axyridis larvae. In this study, the essential nutrients provided by the pollen or nectar of aterials . Flora raterials ,36. Protaterials . Moreoveaterials . There wparately . Adalia lla eggs . Likewisen alone . Howeverrameters . In concH. axyridis. This may indicate that the mixed diet showed high fitness for larval development and contributed to a heavier adult weight. This may allow more nutrients to be reserved in the adult body, and the nutrients reserved in the larvae may promote adult longevity. Similarly, body weight and adult longevity studies showed that heavier adults also had the longest lifespan [Lasioderma serricorne (F.) (Coleoptera: Anobiidae Fleming) is positively correlated with the fresh weight of newly emerged adults [C. monnieri flowers provide floral resources that cannot prolong the longevity of H. axyridis adults directly, but the longevity performance of H. axyridis could be improved through the nutrition reserved at the larval development stage.Floral resources available only at the adult stage showed no significant differences in the longevity of either male or female ladybeetles among treatments. However, flower resources are constantly provided since the first instar significantly prolongs the longevity of male lifespan . The qualifespan ,43. The d adults . Furtherd adults ,44. In cC. monnieri flowers significantly enhanced the adult reproductive performance of H. axyridis. The fecundity of H. axyridis was significantly enhanced, regardless of whether C. monnieri flowers were provided at the adult stage or during the entire lifespan. In experiment II, females reared on an R. padi-only diet laid a mean of 731.37 eggs. Similar to our study, under laboratory conditions, H. axyridis fed on R. padi produced a mean of 791 eggs [C. monnieri flowers provided at the entire lifespan of H. axyridis increased egg production 1.62 times, and those only provided at the adult stage increased egg production 1.44 times. Moreover, the number of oviposition days and egg hatchability were significantly higher in the aphid plus flower diet than in the aphids-only diet when flower resources were provided from the first instars of H. axyridis. This may indicate that C. monnieri flowers provide sufficient pollen for H. axyridis, and the protein of the pollen promoted the reproduction of H. axyridis adults. The availability of 791 eggs . C. monnColeomegilla maculate fuscilabris (Mulsant), even though a diet including animal protein usually results in higher fecundity [A. bipunctata (L.) fed a mixed diet of frozen bee pollen and E. kuehniella eggs had higher oviposition and egg hatching rates than E. kuehniella eggs only [Orius majusculus (Anthocoridae: Orius) continued to lay eggs in the presence of alyssum species, but the maximum reproduction of O. majusculus was only possible under conditions of simultaneous availability of prey food [E. kuehniella eggs is a suitable diet for rearing H. axyridis, significantly increasing egg hatchability [C. monnieri flowers significantly enhance the adult reproduction performance of H. axyridis. H. axyridis can compensate for a suboptimal diet of prey food by supplementary feeding on C. monnieri flowers. Reproduction requires food resources that are high in protein, and a mixture diet of prey and non-prey food usually supports greater reproduction output ,16,45,46ecundity . Likewisggs only . Orius mrey food . A mixedhability . When pohability . In concC. monnieri flowers are a suitable supplementary food for promoting the development and reproduction of ladybeetles. Therefore, C. monnieri may be utilized as a candidate flowering plant to enhance the population of H. axyridis in crop systems where H. axyridis is the dominant natural enemy. Moreover, C. monnieri can attract predatory natural enemies, such as Propylaea japonica, Hippodamia variegata, Chrysoperla sinica, and Episyrphus balteata, especially during the flowering period [C. monnieri flowers for other predators and evaluate the effects of C. monnieri plantings on pest suppression in a diversity of adjacent crops.Understanding the mechanisms of floral resources on ladybeetle longevity and fecundity is essential to designing agroecosystems that support predator populations for pest control . The preg period . Floral g period ,50,51. FRhopalosiphum padi Linnaeus used in the experiments were collected from a wheat field in Jiyang, Shandong Province, China. Wheat aphids were reared on wheat seedlings as prey provided to H. axyridis.H. axyridis were reared under laboratory conditions in nylon mesh net cages (55 × 80 × 32 cm). They were fed ad libitum with R. padi, reared on wheat seedlings, and allowed to reproduce freely. The colony was maintained in an environmentally controlled chamber at 25 ± 2 °C, 65 ± 5% relative humidity (RH), and an L16:D8 h photoperiod. All pupae used in Experiment II were offspring of the third generation (the fourth generation). All newly hatched first instars of H. axyridis used in Experiment I were the offspring of the fourth (fifth) generation.C. monnieri was planted in Jiyang, Shandong Province. Every five days, fresh flowers were cut from the fields and preserved in a plastic bucket filled with water. For the flower plus aphid diet treatment, flowers were provided in bouquets and preserved in a plastic container. The bouquet consisted of 15 C. monnieri flowers with 12 cm stems wrapped with cotton at the bottom, placed into a 50 mL plastic container containing tap water. Before the experiments, the insects were carefully removed from the stems and flowers using a brush.C. monnieri flowers, were tested. Each treatment started with 150 first-instar larvae, which were then left to develop into adults. For the experiment, newly hatched larvae of H. axyridis were randomly selected from egg clusters in the colony (the fifth generation) and carefully transferred by a soft brush to individual 300 mL plastic cups  with punctured lids for air exchange. Each cup contained a single larva, and water was provided by a moist cotton ball fitted into a 3.5 mm plastic dish. R. padi were offered ad libitum and refreshed daily. Each larva of H. axyridis reared on the aphid plus flower diet was provided with one flower, and the flowers were replaced every day. The immature stage was the period from the first instar to adult emergence. The developmental duration and survival of immature stages were monitored daily. The color patterns and presence of exuviae were used to identify the larval instars [Two diets, aphids-only and aphid plus Pupae were weighed on the second day of pupation, and adult fresh body weight was measured during adult emergence. Pupae and newly emerged adults were weighed on a semi-microbalance Sartorius Genius SQP QUINTIX224-1CN  (±0.01 mg). The body length was measured at the time of death. Using an ocular micrometer , the distance between the anterior margin of the pronotum and the posterior margin of the elytra was measured.H. axyridis were provided with small pieces of folded brown paper towels as a substrate for oviposition. The number of daily oviposition eggs was determined by checking the plastic container and folded brown paper towels daily. All eggs were collected daily and placed in separate plastic Petri dishes lined with moist absorbent paper at the base. The plastic Petri dish was labeled with the date, number of females, and number of eggs. The number of hatched eggs was determined by daily observations, and newly emerged larvae were counted and removed over time.After eclosion, adults were reared on wheat aphids 48 h before the experiment. The sex of the newly emerged adults was determined. Thirty pairs were formed from individuals who had experienced the same diet. They were subjected to the same diet as during immature development and were left to reproduce. Each pair was then placed in a plastic container , and the top of the arena was covered with nylon mesh to allow ventilation. In each replicate, once the male died, a newly emerged male was placed in the container, and the experiment ended with the death of the female in each pair. During the experiment, wheat aphid was offered ad libitum and refreshed daily. Fresh flowers were replaced every five days, and water was provided by a moist cotton ball fitted into a 3.5 mm plastic dish and replaced every day. Females of Reproduction was characterized using four parameters: pre-oviposition period, oviposition period, fecundity, and hatching rate of eggs . The preH. axyridis adults feeding on C. monnieri flowers and wheat aphids, two diets of aphids-only and aphid plus C. monnieri flowers were tested. The two diets comprised thirty replicates per diet, and each replication started with one pair of H. axyridis adults (48 h). Each pair of H. axyridis adults was placed in the same-sized plastic container as in Experiment I (the containers used at the adult stage). The experiment procedure and conditions were the same as in Experiment I.To evaluate the fecundity and longevity of t-test to assess the treatment effect. Nonparametric tests  were performed on the developmental duration of the larva, pupa, the total immature period, and the preoviposition days. All statistical analyses were conducted using SPSS ver. 20 for Windows software package .Data were checked for normality using the Kolmogorov–Smirnov (K-S) test. Larval survival curves were analyzed using the log-rank (Mantel-Cox) test in GraphPad Prism 6.0 . The data for the pupa weights, freshly emerged adult weights, adult body lengths, female and male longevity, fecundity, and hatching rate of eggs were normally distributed and analyzed using an independent-sample C. monnieri flower is a suitable supplementary food for H. axyridis larvae and adults and positively affects the survival, development, and reproduction performance of H. axyridis. H. axyridis larvae with shorter developmental times, higher larval survival rates, heavier adult weights, and larger body sizes resulting from the provision of C. monnieri flowers in the aphids-only diet. Regardless of whether the flowers were provided at both larval and adult stages or only at the adult stage, the fecundity of H. axyridis was significantly enhanced.


Neoseiulus barkeri (Hughes) and the native enemy Scolothrips takahashii (Priesner) frequently occurs when target prey is scarce. It is hypothesized that intraguild predation is an adaptative strategy for predators, ensuring the persistence of the intraguild predators during periods of prey scarcity. Therefore, we undertook a study to evaluate the effects of the intraguild prey on the survival, development and reproduction of intraguild predators in a reciprocal predator guild with N. barkeri and S. takahashii. Our results support the hypothesis, showing that intraguild prey enhanced the survival, development and oviposition of intraguild predators, further confirming the effectiveness of intraguild predation as an adaptative strategy.Intraguild predation between the introduced enemy Neoseiulus barkeri (Hughes) and the predatory thrips Scolothrips takahashii (Priesner) are known as potential biocontrol agents for the two-spotted spider mite Tetranychus urticae (Koch). These two predator species occur simultaneously on crops in agricultural ecosystems and are proved to be involved in life-stage specific intraguild predation. The intraguild prey may play a role in securing the persistence of the intraguild predators during food shortage periods. To understand the potential of intraguild prey as food source for intraguild predators in the N. barkeri and S. takahashii guild at low T. urticae densities, the survival, development and reproduction of both predators was determined when fed on heterospecific predators. The choice tests were conducted to determine the preference of the intraguild predator between the intraguild prey and the shared prey. Results showed that 53.3% N. barkeri and 60% S. takahashii juveniles successfully developed when fed on heterospecific predators. Female intraguild predators of both species fed on intraguild prey survived and laid eggs throughout the experiment. In the choice test, both intraguild predator species preferred their extraguild prey T. urticae. This study suggested that intraguild prey served as an alternative prey for intraguild predators prolonged survival and ensured the reproduction of intraguild predators during food shortage, ultimately decreasing the need for the continual release of the predators.The predatory mites  Harmonia axyridis  to control aphids in the field have negatively affected the population of many indigenous natural enemies through intraguild predation [Pests in natural systems are always faced with multiple predators ,2. Theseredation . Conversredation , as intrredation . Moreoveredation .Phytoseiulus persimilis (Athias-Henriot), Neoseiulus cucumeris (Oudemans), Amblyseius andersoni (Chant) and Neoseiulus fallacis (Garman) have already been considered low nutrient food sources when used as intraguild prey. Although they allowed their intraguild predators to develop into adulthood, they did not enable them to reproduce [Amblyseius swirskii (Athias-Henriot) is an equally nutritious food compared with extraguild prey, as has been observed in the case of Frankliniella occidentalis (Pergande) [Numerous studies have reported that intraguild prey could be utilized as a food source for intraguild predators, even if the quality of intraguild prey was inferior to that of extraguild prey. For example, eproduce ,18. Howeergande) . ObtainiScolothrips takahashii (Priesner) (Thysanoptera: Thripidae) and Neoseiulus barkeri (Hughes) (Mesostigmata: Phytoseiidae), are natural enemies used for controlling populations of two-spotted spider mites, Tetranychus urticae (Koch) (Acari: Tetranychidae) [Scolothrips takahashii suppressed T. urticae populations at low levels in various crops, such as citrus, pear, apple and bean in China [N. barkeri, has been progressively released in orchards and greenhouses to control T. urticae populations before the spider mite damage becomes visible [S. takahashii and released enemies N. barkeri frequently co-occur in the same ecosystem at low target prey densities, potentially leading to intraguild predation among the different life stages of the species in question. Previous studies have shown that intraguild interaction between S. takahashii and N. barkeri was bidirectional, with each species fed on the vulnerable stage of the other [Predatory thrips and mites, including ychidae) ,21,22. Sin China ,24. The  visible ,26. Bothhe other . HoweverN. barkeri eggs and first instar larvae of S. takahashii, which are the vulnerable stages of these intraguild prey, on the survival, juvenile development and female reproduction of their intraguild predator S. takahashii and N. barkeri, respectively. Furthermore, the preference of the intraguild predators for intraguild prey or T. urticae was also evaluated.In this study, we investigated the effects of T. urticae) was reared on cowpea bean plants Vigna unguiculata (L.). Neoseiulus barkeri were reared on a black polyethylene plastic sheet placed upside down on a water-soaked sponge inside a plastic container (15 × 20 × 10 cm), which was sealed by a lid with a fine mesh (150 mm mesh opening) as window in the middle to allow for ventilation. Sufficient numbers of mixed stages of T. urticae were brushed into the predatory mite cultures as food every other day. Colonies of S. takahashii were maintained on bean plants V. unguiculata infested by T. urticae. Rearing units were maintained at 28 ± 1 °C, 80 ± 5% R.H. and 16L: 8D photoperiod.The two-spotted spider mite . Thus, the protonymphs of T. urticae served as extraguild prey. For the development experiment, we used protonymphs of N. barkeri and first instar larvae of S. takahashii as intraguild predators. Adult females of both predators were used as intraguild predators for the oviposition experiment and choice test.According to our previous study, eggs of n guilds . NeoseiuN. barkeri were transferred to each experimental cell (as described). As soon as the obligatory feeding stage (protonymphs stage) was reached, they were fed by randomly assigning them one of the three following treatments: sufficient first instar larvae of S. takahashii as intraguild prey; T. urticae protonymphs as extraguild prey; or no food. Similarly, newly emerged first instar larvae of S. takahashii in each cell were offered with sufficient N. barkeri eggs as intraguild prey, T. urticae protonymphs as extraguild prey, or no food. These experimental cells were monitored twice a day to record the survival and development of the intraguild predators in each cell. To comprehensively evaluate the survival and development time of the intraguild predator’s immature stage, we also recorded the duration of the unfed stage  in each treatment. The observation was terminated when the intraguild predators N. barkeri and S. takahashii reached adulthood or died. Every day the experimental cells were renewed and the prey replenished. For both intraguild predator species, 15 replicates were set up for each treatment.Newly laid eggs of T. urticae, and one conspecific male was introduced into each leaf disc for mating. After 24 h (until mating was observed), each female was transferred into a fresh leaf disc with no food to standardize their level of hunger. Only females that deposited at least one egg during the starvation period were used as intraguild predators. Subsequently, starved intraguild predators were introduced to experimental cells, and the intraguild prey or natural prey were separately added into each arena. The survival and oviposition of each female was recorded daily for up to 10 days. The experimental cell was replaced daily with sufficient prey as before for each tested individual. Oviposition of females was also measured when they were offered no prey. Each treatment was replicated 15 times for each prey.One female (9–11 days old since egg hatching) of each predator species was transferred into a detached bean leaf disc with ample prey of mixed stages of N. barkeri randomly selected from the rearing units described above were subjected to starvation for 24 h. Subsequently, gravid females of N. barkeri were singly introduced into an experimental cell supplied with intraguild prey and extraguild prey at a ratio of 1:1 (ten first instar larvae of S. takahashii and ten active stage protonymphs of T. urticae). The same procedure was employed for S. takahashii females, except that prey provision was doubled (20 eggs of N. barkeri and 20 active stage protonymphs of T. urticae). The experimental cell was observed every 10 min for 6 h or until the intraguild prey or extraguild prey was attacked by the intraguild predator. Prey preference was determined based on the observation of the first three attacks [Gravid females of  attacks . After eN. barkeri eggs, total duration of development from egg to adult, and—for S. takahashii—first instar larvae, second instar larvae and total duration of development from first instar larvae to adult. A Mann–Whitney U-test was performed to compare the development time of N. barkeri larvae, protonymphs and deutonymphs, and S. takahashii prepupa and pupa when provided with two types of prey, because the data could not be normalized. The oviposition rates of adult females of intraguild predators provided with either extraguild prey or intraguild prey were also compared using the T-test for independent samples. Data from the first day were omitted from the calculations of oviposition capacity to reduce the influence of previous feeding history. Results of the choice experiment were analyzed through the chi-square test.All analyses were performed using SPSS 21.0. To compare the cumulative survival rates of intraguild predators under different types of diet , Kaplan–Meier survival analysis was performed using Log-rank tests. T-test for independent samples was used to compare the two types of prey (intraguild prey and extraguild prey) on the developmental parameters of the intraguild predators: including N. barkeri as intraguild predator overall test, as shown on χ2 = 14.979, df = 2, p = 0.001; T. urticae protonymph as extraguild prey vs. no food: χ2 = 11.911, p = 0.001; S. takahashii first instar larva as intraguild prey vs. no food: χ2 = 4.851, p = 0.028; S. takahashii as intraguild predator overall test, as shown on χ2 = 49.999, df = 2, p < 0.001; T. urticae protonymph as extraguild prey vs. no food: χ2 = 29.578, p < 0.001; N. barkeri egg as intraguild prey vs. no food: χ2 = 26.729, p < 0.001). Moreover, the intraguild predators that were provided with intraguild prey had a lower survival rate than the intraguild predators fed on extraguild prey .The survival rate of both intraguild predators when fed on prey was significantly higher than when they were deprived of food , deutonymph  and the overall development time  among N. barkeri resulting from the different food treatments , second instar larva  and the overall development time . Similarly, S. takahashii had a higher oviposition rate when fed on extraguild prey than on intraguild prey .Oviposition rates of both intraguild predator species were higher when they were provided with extraguild prey . During Neoseiulus barkeri exhibited a preference for extraguild prey over intraguild prey . The attack frequency of S. takahashii was significantly higher on extraguild prey T. urticae than on intraguild prey N. barkeri egg  of mixed stages [N. barkeri was capable of maintaining oviposition  when fed on intraguild prey first instar larvae of S. takahashii; and their oviposition rate was similar to that when they were fed on other prey such as Tyrophagus putrescentiae (Schrank) [Aleuroglyphus ovatus (Troupeau) [Luffa cylindrical (Roem) pollen [N. barkeri when their food sources such as tetranychid and Acaridae prey or pollen are scarce. The present results confirm the finding of Momen [N. barkeri juveniles completed their development, and female produced 1.35 eggs per female per day when fed on the intraguild prey Typhlodromus negevi (Swirski and Amitai) eggs. Other phytoseiid species—including Typhlodromus athiasae (Porath and Swirski) [N. cucumeris [Neoseiulus longispinosus (Evans) and Galendromus occidentalis (Nesbitt), failed to develop into adults on intraguild prey. The nutritional benefits of intraguild prey for the predators e nymphs  and Eoted stages . In addiSchrank) , Aleurogroupeau)  and Luff) pollen . Thus, iof Momen , who repSwirski)  and N. cucumeris —were alsucumeris  reportedS. takahashii was shorter when they were fed on T. urticae (5.50 days) [Panonychus citri (McGregor) (5.57 days) [N. barkeri eggs (8.167 days). However, S. takahashii fed on N. barkeri eggs laid 3.56 eggs per female per day, which was slightly less than that when fed on P. citri  [T. urticae  [S. takahashii with other phytoseiid mites or predatory insects. However, Scolothrips longicornis (Priesner), which belongs to the same genus as S. takahashii, showed a daily oviposition rate of 0.08 and 0.13 when fed on eggs of phytoseiid mites Typhlodromus bagdasarjani (Wainstein & Arutunjan) and Neoseiulus californicus (McGregor), respectively [S. takahashii laid 3.56 eggs per female per day when fed on N. barkeri eggs. As a result, S. takahashii demonstrated its capacity to sustain a population over a long term by consuming N. barkeri eggs when the target prey was scarce.Previous studies have reported that the development time of 50 days)  and Pano57 days)  compared, 28 °C)  and T. u, 25 °C) . Currentectively , whereasN. barkeri did not show a preference between extraguild prey and intraguild prey in their first attack. This may be attributed to the fact that S. takahashii larva is probably a suitable prey for the N. barkeri. However, S. takahashii larvae exhibited defensive behavior such as jerking the end of the abdomen rapidly or producing excrement during the experiment [N. barkeri to avoid them and then alter their preferences for thrips larvae in the second attack. This defensive behavior has also been observed in other insects such as F. occidentalis and Thrips tabaci (Lindeman) [S. takahashii may have high nutritional value for N. barkeri, the difficulty in catching thrips makes them a less suitable food source.Overall, both intraguild predator species preferred extraguild prey, which may be due to its high quality. Both predators showed higher survival rates, shorter development times and higher oviposition rates on extraguild prey than on intraguild prey. However, periment , which cindeman) ,42. ThisN. barkeri and S. takahashii, increasing their persistence period with prey scarcity, although extraguild prey was of higher quality for both predators. However, evaluating persistence and coexistence of predators in the presence of all the species and in arenas differing in spatial structure in the field warrants using experiments based on population/community dynamics, since these would allow more realistic predation rates to be compared under scenarios of intraguild predation. In addition, a polytypic diet would allow for a balanced nutritional intake for the predators [The present study revealed that intraguild prey could ensure the survival, development, and oviposition of predators redators . Thus, i


The pathogenic viruses transmitted by mosquitoes cause a variety of animal and human diseases and public health concerns. Virome surveillance is important for the discovery, and control of mosquito-borne pathogenic viruses, as well as early warning systems. Virome composition in mosquitoes is affected by mosquito species, food source, and geographic region. However, the complex associations of virome composition remain largely unknown.Culex, Aedes, Anopheles, and Armigeres in Hainan Island from 2018 to 2020. We detected 57 known and 39 novel viruses belonging to 15 families. We established the associations of the RNA viruses with mosquito species and their foods, indicating the importance of feeding acquisition of RNA viruses in determining virome composition. A large fraction of RNA viruses were persistent in the same mosquito species across the 3 years and different locations, showing the species-specific stability of viromes in Hainan Island. In contrast, the virome compositions of single mosquito species in different geographic regions worldwide are visibly distinct. This is consistent with the differences in food sources of mosquitoes distributed broadly across continents.Here, we profiled the high-depth RNA viromes of 15 species of field-caught adult mosquitoes, especially from Thus, species-specific viromes in a relatively small area are limited by viral interspecific competition and food sources, whereas the viromes of mosquito species in large geographic regions may be governed by ecological interactions between mosquitoes and local environmental factors.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01556-4. Mosquito-borne pathogens pose an immense threat to public health threat in nearly half the human population and many nonhuman vertebrates . In factAedes aegypti and Culex (Cx) quinquefasciatus was revealed in a shared region less than 15 km wide [Armigeres subalbatus, Cx. fuscocephala, and Mansonia uniformis are defined primarily by the host mosquito species rather than by geographical location at a 20-km scale [Culex species, with overlapping geographical distributions within 600 km, share numerous viruses [Many virome studies have demonstrated that the virome composition in mosquitoes is principally structured by host mosquito taxon in a relatively limited area –10. A makm scale . However viruses . In fact viruses . HoweverFood is one of the important ways for mosquitoes to acquire RNA viruses in addition to their symbiotic viruses. Mosquitoes harbor pathogenic viruses, which can replicate and be transmitted by mosquito vectors to vertebrate hosts via the bloodmeal. They also carry plant viruses that are not capable of replicating in mosquitoes and may be present in plant liquids they feed upon , 13. SinVirome dynamics are dependent on the interactions between mosquitoes and their environments. Except for vertically transmitted viruses, the main components of the virome in mosquitoes are transmitted horizontally in the environment . BecauseWe conducted large-scale mosquito collection and virome identification in 13 counties covering the main ranges of Hainan Island from 2018 to 2020. The viromes in 15 mosquito species across different locations and years were quantitatively analyzed. In particular, we explored the co-occurrent associations among mosquito species, food sources, and RNA viruses. Furthermore, we compared the virome composition and food source of the same species in different hierarchical geographic regions. Together, our results reveal that environmental factors contribute much more than mosquito species in shaping the viromes in mosquitoes.Culex (Cx), four Aedes (Ae), one Anopheles (An), and one Armigeres (Ar), were used for virome sequencing, which was carried out on 200 pools of mosquitoes , emphasizing the capacity for virus detection based on the high-depth sequencing data in this study.A total of 6.76 terabytes of virome sequencing reads  were generated and assembled de novo into 120,834,188 contigs  for virus discovery and characterization  comprise putative members of five unclassified viruses and 15 different viral families , Hainan mosquito orbi virus 5 (59.9%), and Hainan mosquito reo virus 7 (65.0%), suggest that they are novel viruses at the species rank  to the kingdom Metazoa showed that the bloodmeal hosts belonged to three classes, Mammalia, Aves, and Actinopteri Fig. D. At theLupinus, Glycine, and Oryza species , an endemic plant species in Hainan Island, was detected in the assemblies and was further verified using PCR Sanger sequencing  were the major plant-derived diet component of mosquitoes Fig. D. Althouies Fig. E. The saies Fig. . Becauseies Fig. . In addiCx. gelidus. Most likely, the Cx. gelidus mosquitoes took bloodmeals from Bovidae species. In Cx. pallidothorax and Cx. pipiens quinquefasciatus, we found two plant viruses, Peony yellowing associated secovirus and Himetobi P virus, which were significantly co-occurred with the Salicaceae species, the willow family of flowering plants. Peony yellowing-associated secovirus and Himetobi P virus were previously detected only in peony (Paeoniaceae) plants and planthopper species (Hemiptera), respectively [To assess the significant associations between RNA viruses and their food sources, we performed hypergeometric tests between each family of food sources and each RNA virus across all the mosquito sequencing libraries Fig. F. We staectively . Our resAe. albopictus, Cx. pipiens quinquefasciatus, and Ar. subalbatus were the most prevalent mosquito species in Hainan Island [Ae. vexans and the lowest in Ar. subalbatus  of the virome did not differ across the locations in Hainan Island.To examine the effects of mosquito species and location on virome composition, multidimensional scaling was performed to determine their correlation. We found that the virome composition of the three species of mosquitoes, ion Fig. . In the ion Fig. . Furtherion Fig. C. Three Ae. albopictus, Cx. pipiens quinquefasciatus, and Ar. subalbatus. Species-specific persistence was observed in a few virus species across the years, suggesting that these viruses are maintained by vertical transmission. These vertically transmitted viruses include (1) Guapiacu virus, Guangzhou sobemo-like virus, Aedes albopictus negev-like virus, Hainan mosquito reo virus 6, Wenzhou sobemo-like virus 4, Usinis virus, and Riboviria sp. in Ae. albopictus; (2) Wuhan Mosquito Virus 6 and Hubei partiti-like virus 22 in Cx. pipiens quinquefasciatus; and (3) Hubei partiti−like virus 22, Hubei sobemo−like virus 41, Hainan mosquito virus 1, and Riboviria sp. in Ar. subalbatus. The mosaic distribution pattern for a large portion of viruses within species-specific viromes implies that these viruses were derived from the specific interactions between mosquito feeding and environments  and Cx. pipiens quinquefasciatus (Yunnan of China and Sweden) were analyzed . The results were also robust at 50% and 25% random downsampling rates. The lower diversity of viromes in Hainan Island resulted from the lower observed richness and abundance of viruses compared to those in the regions of America and Europe  and Cx. pipiens quinquefasciatus  in three continental regions were analyzed  and 6.67% (1 in 15) of the viruses detected in Hainan Island were intersected with the viruses detected in other three continental regions, respectively. For Ae. albopictus, only one (Barstukas virus) of ten environment-derived viruses in Hainan Island was detected in the mosquito species of the other three continental regions. Three  of the seven vertically transmitted viruses in Hainan Island were shared by the Ae. albopictus mosquito from the other three continental regions. For Cx. pipiens quinquefasciatus, we did not detect any intersection of the 13 environment-derived viruses between Hainan Island and the other three continental regions  of 15 families in Viridiplantae were shared between the two mosquito species. We detected a total of 7 Metazoan families, but only 4  of them were shared between Ae. albopictus and Cx. pipiens quinquefasciatus. These results showed a considerable difference in the food sources of mosquito species in Hainan Island. We further expanded this analysis to mosquito species in different geographic regions  of 11 Metazoan families and one (Muridae) of 14 Metazoan families were shared by the four continental regions studied. We did not detect any intersection of the families of Viridiplantae in the two mosquito species from the four continental regions, suggesting that the food source derived from green plants was more variable than that from animals.Because the acquisition of RNA viruses in mosquitoes is largely from foods in local environments, we compared the food source differences in single mosquito species in Hainan Island , was an unpublished Secoviridae virus (GenBank: QNN26213.1) as of 2021. The presence of peony yellowing-associated secovirus in Cx. pallidothorax implies the potential acquisition of plant viruses via plant feeding. Our study demonstrated a 96.7% nucleotide identity and 97.3% amino acid identity of RdRp in peony yellowing-associated secovirus compared to that previously detected in peonies. More likely, the high sequence similarity reflected a recent historical transmission event of peony yellowing-associated secovirus between willows and peonies. Plant nectar resources are intermediate for virus spreading via a food-borne transmission pathway [Since several virus lineages infecting vertebrates and plants evolved from insect-specific viruses through host shifts , the ass disease . Thus, t pathway . Because pathway .Our virome analysis in a mosquito species deepens the understanding of the virome composition in mosquito species across different-scale regions. In Hainan Island, we found that a large fraction of RNA viruses were persistent in the same mosquito species across 3 years and collection locations, indicating that the virome composition was largely structured by mosquito species. The virome is mainly composed of inherent vertically transmitted RNA viruses and environment-derived RNA viruses . Our resThe distinct virome composition across mosquito species in the shared environment of Hainan Island indicates that food source preference may be another important factor affecting virome composition. This emphasizes the important contribution of environmental food availability and mosquito-feeding behavior to shape the virome dynamics in a natural ecosystem. In a relatively independent region such as Hainan Island, the feeding preferences of different mosquito species can allow them to avoid interspecific competition via the partitioning of natural resources, including foods and habitats, thereby resulting in distinct, species-specific viromes.Ae. albopictus and Cx. pipiens quinquefasciatus possess diverse and abundant viromes, only a few vertically transmitted RNA viruses in Hainan Island could be detected in the three continental regions. These shared RNA viruses belonged to the species-specific core virome, which refers to a set of viruses consistently associated with a host group [Ae. albopictus and Cx. pipiens quinquefasciatus. The high differentiation of viromes between mosquito species in Hainan Island showed that a large fraction of endemic RNA viruses is circulated or spread in a mosquito species in local environments. In a cross-continental context, the virome in a mosquito species may be mainly shaped by environment-related food availability. Overall, the acquisition and spread of RNA viruses in a natural ecosystem are dependent on local environmental associations and mosquito species barriers. Further investigations should explore how large the range must be for the destabilization of species-specific viromes.The diversity of mosquito viromes in Hainan Island was significantly lower than that in the three continental regions, probably because the isolated island environment and geographic barrier hindered the spread of viruses. The three continental regions may be more effective in facilitating the long-distance dispersal of viruses . In our st group . Feedingst group . Indeed,c oxidase subunit I (COI) gene was performed using Sanger RT‒PCR sequencing. The resulting barcode sequences were searched against the Culicidae barcode records in the NCBI nonredundant protein database (nr) and the BOLD database (www.boldsystems.org).Mosquitoes were sampled in the summer for three consecutive years (2018 to 2020) across 13 geographic districts in Hainan Island in China, including Baisha County (BS), Dongfang City (DF), Danzhou City (DZ), Haikou City (HK), Ledong County (LD), Lingao County (LG), Lingshui County (LS), Qinghai City (QH), Sanya City (SY), Tunchang County (TC), Wenchang City (WC), Wanning City (WN), and Wuzhishan City (WZS). All mosquito samples were initially identified based on morphological characters initially, washed using PBS buffer, and then placed in RNAlater immediately. Genomic DNA was extracted from one leg of each mosquito using the HotSHOT method . To furtAe. albopictus, Cx. pipiens quinquefasciatus, and Ar. subalbatus were the most prevalent mosquito species in Hainan Island [The total RNA was isolated from three mosquito individuals of each species using TRIzol Reagent (Invitrogen). The rRNA-depleted RNA was fragmented and converted into a strand-specific cDNA library using a TruSeq Stranded Total RNA Library Prep Kit (Illumina). The 150 bp paired-end sequencing was performed on an Illumina HiSeq 4000 System. The sequencing libraries of mosquito species were generally proportional to their prevalence in field estimates from two previous studies, which showed that n Island , 37. Adan Island . De novon Island . The ambn Island . The besn Island , 54. Phyi) against the virus protein database from GenBank (downloaded on 8/18/2021) with a 1e-5 value cutoff; (ii) against the RNA virus sequences in the Virus Metadata Repository  with a 1e-5 value cutoff; and (iii) against the RNA-dependent RNA polymerase (RdRp)-related position-specific score matrices (PSSMs) in Conserved Domain Database (CDD) version 3.19 using domain-based RPS-BLAST with a 1e−2 value cutoff. False-positive contigs were removed based on BLAST searches against three databases: the nr database, the nonredundant nucleotide database (nt), and the genome sequences within the family Culicidae. The redundant duplicates were clustered using CD-HIT-EST v4.8.1 if their nucleotide identities were higher than 90% . The contigs showing the best hits against the viral proteins under Riboviria  and whose nucleotide lengths were greater than 300 were kept for further analysis. The clustered putative viral RdRp contigs were considered different viral taxonomic units and thus could be used to explore virome community structure and diversity. The RdRp protein is considered “complete or nearly complete” if it aligns with >80% coverage of the known virus species deposited in the public databases mentioned above. Novel viruses are those in which sequences match with <80% aa identity and <90% nucleotide identity. For multiple-segment RNA viruses, segments from the same virus species were identified based on co-occurrence frequency and codon usage in different mosquito samples. We further verified the viral genome sequences provided here by mapping reads to their corresponding contigs and by assessing the continuity of read coverage manually using the IGV 2.7.2 program [All assembled contigs were translated into six frames using the Transeq program embedded in the EMBOSS 6.6.0 software package  and were program . RT‒PCR In phylogenetic inference, multiple sequence alignments of RdRp proteins were performed using MAFFT v7.215 , employiIf the contigs were aligned to the genomic sequences from arthropods (NCBI Taxid 6656 and its subordinate Taxid) in the nr/nt database , they were considered “presumptive host contigs” and thus were removed from the analyses. The remaining contigs were taxonomically assigned to the Eukaryota taxon (except for fungi) to identify the putative bloodmeal sources using BLAST searches against the nr/nt databases. Putative food sources were determined based on the following criteria: a 1e−5 value cutoff, an aligned fragment with a size greater than 200 bp, and alignment with >80% coverage of the contigs. If the contigs showed the best hit to multiple species with equivalent BLAST bitscores, they were annotated to the last common ancestor (LCA). Due to the absence of genome sequences in environments, our analysis was set to the family level and genus level. The hypergeometric test was used to determine the statistical significance of co-occurrence events between food sources and RNA viruses.H) of the virome in the sequencing libraries was determined using the following formula:S is the number of RNA viruses identified in each sequencing library and pi is the proportional abundance of the ith RNA virus relative to the overall RNA viruses.All RdRp ORFs, including those downloaded from Genebank 8/18/2021 and those from newly identified viruses in this study, clustered into divergent virus OTUs on the basis of a protein identity of 90%. Non-rRNA reads from each sample were mapped against the above virus OTUs using BBMap v38.96 (sourceforge.net/projects/bbmap/), and only the virus OTUs with read coverage >50% were considered as expressed viruses for further analysis. The viral richness of each sample was estimated using the number of viral OTUs. The virus abundance of each sample was evaluated using the total number of all viral reads per million (RPM) using the formula “total mapped viral read/total non-rRNA reads * 1,000,000”. The Shannon index  and RNA virus abundance (B) in different sequencing libraries across mosquito species. *P < 0.05; **P < 0.001. Ae.alb, Aedes albopictus; Ae.aeg, Aedes aegypti; Cx.pip, Culex pipiens quinquefasciatus; Ar.sub, Armigeres subalbatus; Cx.tri, Culex tritaeniorhynchus; Cx.pal, Culex pallidothorax; Ae.vex, Aedes vexans; Cx.vis, Culex vishnui; Cx.fus, Culex fuscocephala; Cx.sit, Culex sitiens; Cx.pse, Culex pseudovishnui; An.vag, Anopheles vagus; Cx.lut, Culex (Lutzia) fuscanus; Ae.mal, Aedes malayensis; Cx.gel, Culex gelidus. Fig. S23. Multiple scaling analysis for RNA virome composition across mosquito species using a Euclidean distance matrix. BS, Baisha. DF, Dongfang. DZ, Danzhou. HK, Haikou. LD, Ledong. LG, Lingao. LS, Lingshui. QH, Qionghai. SY, Sanya. TC, Tunchang. WC, Wenchang. WN, Wanning. WZS, Wuzhishan. Fig. S24. Heatmap plot showing the presence of virus OTUs over the three consecutive years from 2018 to 2020 in three mosquito species. Ae.alb, Aedes albopictus; Ar.sub, Armigeres subalbatus; Cx.pip, Culex pipiens quinquefasciatus. Fig. S25. Shannon index of operational taxonomic units of RNA viruses across collection locations. Statistically significant differences were detected using a Kruskal‒Wallis test in cases of multiple comparisons. In pairwise comparisons, significant differences were detected by pairwise Wilcoxon rank sum tests. *P < 0.05; **P < 0.001. BS, Baisha; DF, Dongfang; DZ, Danzhou; HK, Haikou; LD, Ledong; LG, LinGao; LS, Lingshui; QH, Qionghai; SY, Sanya; TC, Tunchang; WC, Wenchang; WN, Wanning; WZS, Wuzhishan. Fig. S26. The observed richness of operational taxonomic units of RNA viruses  and RNA virus abundance  between Hainan Island and other collection locations in Yunnan , California  and Sweden (Europe). In pairwise comparisons, Wilcoxon rank sum tests were applied to detect significant differences. *P < 0.05; **P < 0.01. Ae.alb, Aedes albopictus; Cx.pip, Culex pipiens quinquefasciatus. Fig. S27. Bubble plot showing the distribution of food sources derived from animals and plants in Aedes albopictus and Culex pipiens quinquefasciatus in Hainan Island. The shared families are linked using blue lines. Ae.alb, Aedes albopictus;Cx.pip, Culex pipiens quinquefasciatus. Fig. S28. Bubble plot showing the distribution of food sources derived from animals and plants in Aedes albopictus (left) and Culex pipiens quinquefasciatus (right) in the four continental regions. The shared families are linked using blue lines.Additional file 2: Table S1. Sequencing statistics of mosquito samples in this study.Additional file 3: Table S2. Summary of the RNA viruses detected in this study.Additional file 4: Table S3. Number of RNA viruses containing full-length RdRps detected in each mosquito species.Additional file 5: Table S4. Virome sequencing data retrieved from the NCBI database.


Staphylococcus capitis in samples from hospitalised infants. Staphylococcus capitis has been known to cause outbreaks in neonatal units across the globe, but the extent of the UK spread was unclear. A literature review was undertaken to support case identification, clinical management and environmental infection control. A literature search was undertaken on multiple databases from inception to 24 May 2021, using keywords such as “Staphylococcus capitis”, “NRCS-A”, “S. capitis”, “neonate”, “newborn” and “neonatal intensive care unit” (NICU). After screening, 223 articles of relevance were included. Results show incidences of S. capitis outbreaks have frequently been associated with the outbreak clone (NRCS-A) and environmental sources. The NRCS-A harbours a multidrug resistance profile that includes resistance to beta-lactam antibiotics and aminoglycosides, with several papers noting resistance or heteroresistance to vancomycin. The NRCS-A clone also harbours a novel SCCmec-SCCcad/ars/cop composite island and increased vancomycin resistance. The S. capitis NRCS-A clone has been detected for decades, but the reasons for the potentially increased frequency are unclear, as are the most effective interventions to manage outbreaks associated with this clone. This supports the need for improvements in environmental control and decontamination strategies to prevent transmission.In June 2021, a national incident team was formed due to an increased detection of  Staphylococcus capitis. One particular clone of S. capitis, the NCRS-A clone, demonstrates resistance to aminoglycosides  and beta-lactam antibiotics, which are agents frequently used in empiric treatment of late-onset neonatal sepsis. The UK Health Security Agency noted a possible rise in S. capitis-related invasive infections in hospitalised infants during the summer of 2020. In response, a national incident was set up, and a review of the literature was carried out.A frequent cause of morbidity and mortality in hospitalised infants, particularly in those with very low birth weights, is late-onset neonatal sepsis. The most frequent cause of late-onset sepsis is coagulase-negative staphylococci, including n = 56) and book chapters (n = 28) were excluded, along with articles that were unavailable (n = 7) or in non-English languages (n = 31).We performed literature searches in Ovid Medline, Embase and EmCare, the Cochrane Library, Web of Science, Science Direct and Google Scholar, from database inception to 24 May 2021. Abstracts , or did not cover clinical or human infection (n = 50). This included S. capitis papers that focused on veterinary care (n = 33) or other nonhealthcare settings, such as the environmental industry.Of the remaining records (S. capitis (n = 79), with 67 papers covering clinical management in both adults (n = 37) and neonates (n = 30).After screening and exclusion, there were 223 studies remaining. The findings of the 223 studies were thematically grouped and are presented below . The majS. capitis was first isolated from human skin in 1975 [S. capitis subspecies capitis and S. capitis subspecies ureolyticus. The main differentiating feature between the two is the ability of S. capitis subspecies ureolyticus to produce urease, to produce acid aerobically from maltose and its fatty acid profile [ profile ,3.S. capitis isolates can be distinguished from other coagulase-negative staphylococci (CoNS) by their large colony size and colonial appearance when incubated in moist conditions for 5 days at 37 °C on MRSA Brilliance 2 agar . S. capitis NRCS-A isolates grow as mauve colonies with a cream colour halo, whereas MRSA isolates form blue colonies and CoNS form small white colonies [When undertaking phenotypic-based identification, colonies . AnecdotS. capitis and S. epidermidis using Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS PAGE) analysis. The authors further illustrated that the standard biochemical test panel in API Staph  was unable to robustly differentiate between the species, and this level of similarity led to some reports suggesting that S. capitis similarity with S. hominis and S. warneri meant that they have been overclassified into too many species [When comparing molecular and phenotypic identification techniques, Pennington et al.  demonstr species . Enhance species . This wa species , which d species .S. capitis isolates. RT-PCR with a tuf gene target and MALDI-ToF each identified all 134 correctly: Vitek identified 121 (90.3%) and biochemical phenotyping testing 103 (76.8%). The 10 S. capitis isolates were misidentified by both the Vitek and biochemical characterisations [tuf gene PCR and MALDI-ToF was also demonstrated by Carpaij et al. [S. capitis [More recently, molecular tools, such as real-time PCR, and phenotypic tools, such as Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-ToF), have increased in availability and demonstrated potential for rapid identification of isolates. RT-PCR offers faster identification than conventional PCR; however, mixed bacterial cultures may decrease assay reliability . A recenfied 121 0.3% and rpoB gene provided correct identification of 332/335 CoNS isolates [S. capitis, 14 were subspecies of ureolyticus and 3 were subspecies of capitis. Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR had low discriminatory power, except for S. hominis, and was not able to distinguish between subspecies of S. capitis, where all 17 isolates were considered clonal [Other molecular techniques requiring sequencing post-target amplification have been evaluated for staphylococcal species differentiation. Spanu et al.  demonstrisolates . Enterobd clonal .S. capitis and S. caprae, leading to concern about the method for CoNS characterisation [S. epidermidis and S. capitis cannot be distinguished between either the V1-V2 or V3-V4 16S subregions, and therefore, species identification for S. capitis utilising 16S rRNA sequencing should be interpreted with caution [S. capitis, meaning it was possible to differentiate; however, it was considered an epidemiological rather than an identification technique [A study by Song et al.  demonstrrisation . Ederveerisation  further  caution . High-re caution  to analyechnique .S. capitis are believed to be required for functions including virulence, adherence and biofilm formation [S. capitis genome does not have the same variety of exotoxins that are found in Staphylococcus aureus [Proteins identified in certain strains of ormation . Howevers aureus .S. capitis TE8 strain has a number of genes postulated to play an important role in adherence and skin colonisation. The genome contains both the icaADBC gene for intracellular adhesion and the icaR regulatory gene. Kumar et al. [S. capitis TE8 strain contains 14 adhesins to improve adherence, and they support the effectiveness of the strain in colonising human skin [In terms of colonisation, the r et al.  report tman skin .S. capitis isolates demonstrated the strongest urease activity of all non-H. pylori bacteria isolated from the stomach of patients. S. capitis subspecies ureolyticus were able to survive for at least an hour in stomach acid pH conditions, exhibiting greater resistance than Helicobacter pylori, supporting colonisation of the digestive system [S. capitis isolates exhibited lower invasive ability than S. auricularis (1–10%); however, all bloodstream isolates included in this study were clonally related and therefore may not be representative of other strains [In a study by Brandi et al. , S. capie system . Despite strains .S. capitis species was the ability to form biofilm. The percentage of S. capitis strains that were able to produce biofilm varied greatly between studies. Szczuka et al. [S. capitis strains demonstrated the ability to form a biofilm [S. capitis strains isolated from bacteraemia patients in Turkey as biofilm-positive, compared with 71% in S. epidermis and 43% across all CoNS strains [S. capitis strains compared with those of other CoNS. A comparison of S. capitis AYP1020 with S. epidermidis RP62a demonstrated a six-fold decrease in biofilm production in the S. capitis strain [ica operon has been demonstrated to play a key role in biofilm production in S. capitis, as well as in other CoNS, with all biofilm-producing species identified as ica-gene-positive [The primary pathogenicity factor identified for a et al.  recorded biofilm . In cont biofilm  recorded strains . The rels strain . The prepositive .S. capitis to form biofilm has been linked with particular experimental conditions and environmental cues, for example, growth in media with high osmolarity [S. capitis strains produced higher density biofilms when platelet concentrates were present [The ability of molarity . Detecti present . In anot present .S. capitis [S. capitis strains that had been previously categorised as biofilm nonproducers, although no specific mechanism was identified [Antibiotic-resistant phenotypes have been frequently associated with biofilm-producing phenotypes, with Koksal et al.  determin capitis . In eryt capitis  demonstr capitis . In anotentified . Furtherentified . TherefoS. capitis holds a number of genetic determinants of resistance, which can make infections recalcitrant to therapy.In addition to the antimicrobial-resistant phenotypes linked to biofilm production, S. capitis. S. capitis isolates are frequently associated with SCCmec mobile genetic elements, which are a vehicle for exchanging resistance genes between staphylococcal species and are widely distributed among CoNS and Staphylococcus aureus [mec cassette, which is structurally similar to the type V SCCmec resistance cassette that has been characterised as providing resistance to β -lactams and demonstrating reduced susceptibility to aminoglycosides and vancomycin [There are published data identifying plasmids or mobile genetic elements that encode for multidrug resistance in s aureus . The NRCncomycin ,29. NRCSncomycin .S. capitis infection, but treatment failures have frequently been associated with vancomycin heteroresistance, leading to scenarios such as persistent bacteraemia [S. capitis, particularly the NRCS-A clone, where it leads to the acquisition of vancomycin resistance at a 1.9-fold faster rate than the control S. aureus Mu3 strain [mec (SCCmec) associated with the NRCS-A clone leads to reduced susceptibility or resistance, resulting in significantly reduced management options, especially within the NICU setting [Vancomycin is a frequently utilised antimicrobial in teraemia . Althoug3 strain . The typ setting .S. capitis, linezolid is an important therapeutic option. Resistance is primarily derived from point mutations in domain V of the 23S rRNA drug target site  and/or the presence of cfr (chloramphenicol–florfenicol resistance) genes, which encode for a 23S rRNA methyltransferase that confers resistance to linezolid [For invasive infections caused by vancomycin-resistant strains of inezolid . Within inezolid ,35. All inezolid ,37,38. IS. capitis is common, with between 27.2% and 86% of isolates containing the mecA gene [S. capitis include the multidrug-resistant plasmid pSC16875, which leads to isolates that are not only resistant for fusidic acid but are also positive for the qacA gene, which encodes for an efflux pump which can extrude chlorhexidine [Oxacillin resistance in ecA gene . ResistaecA gene , the bacecA gene , tetracyecA gene  and daptecA gene . Other rhexidine .nsr gene, which confers nisin resistance. The nisin-resistant phenotype of the NRCS-A S. capitis clone is thought to impact the establishment of the neonatal gut microbiota following birth, as it is a bacteriocin with activity against other Gram-positive microorganisms [The NRCS-A clone has also been found to harbour the rganisms .S. capitis, including apolipophorin III, an insect-derived protein with moderate activity [Novel antimicrobials are being investigated which have demonstrated activity against activity ; nemonoxactivity ; and Lauactivity .S. capitis infection has resulted in a number of different clinical presentations. These include the following:Native valve endocarditis ,50,51,52Prosthetic valve endocarditis ,56,57,58Osteomyelitis ,60,61,62Polymicrobial infections ,64,65,66Surgical implant infections ,69,70,71Within adults, Other conditions have included case reports of pyomyositis, peritonitis, septic arthritis and community-acquired meningitis. ,73,74,75n = 4), where 50% of patients died (n = 2) and the remaining had long-term complications (n = 2) [S. capitis infection in other sites resolved using the described management.A variety of antimicrobial treatment options were used in conjunction with surgical interventions and removal of implants/prosthetic devices where appropriate. For endocarditis, vancomycin plus gentamicin in the presence of a prosthetic valve or rifampicin with a native valve was common ,56,57,58 (n = 2) ,52, S. cS. capitis infection is neonatal sepsis  [S. capitis bacteraemia include prior surgical treatment, receiving broad-spectrum antibiotics in the previous two weeks and longer hospital stays [S. capitis, especially with the NRCS-A clone, has also been associated with an increased risk of both bacteraemia and requirement for ventilation support in neonates [S. capitis (55.4%) than the non-S. capitis group (32.0%) [S. capitis has overtaken S. epidermidis as the leading cause of late-onset sepsis [The most common presentation for er (CVC) . In one er (CVC) . In a 20er (CVC) , it was er (CVC) . Risk faal stays . Colonisneonates . In a stneonates , severe  (32.0%) . In somet sepsis . There wS. capitis within healthcare environments are predominantly associated with the S. capitis subspecies urealyticus, particularly linked to the NRCS-A clone, which is known to have caused outbreaks within NICUs [Outbreaks of in NICUs . The NRCin NICUs . Isolatein NICUs ,90. Howein NICUs ,76,87,91While most infections linked to the NRCS-A clone have been associated with bacteraemia within NICUs, there have been reports of the NRCS-A clone identified in prosthetic joint infections in adults in three areas of Sweden. However, no adult outbreaks have been reported in the peer-reviewed literature . Most ceS. capitis within hospital settings. Salimi et al. [S. capitis in their nasal cavity as a commensal bacterium [S. capitis study, NRCS-A was identified by swabbing the hands of caregivers in an NICU, and it was hypothesised that this could be a cause of interpatient transmission [Multiple studies have investigated the sources and reservoirs of i et al.  showed tacterium . In a sesmission .S. capitis outbreak in a French NICU was traced back to contaminated almond oil bottles assigned to individual beds [S. capitis from the surfaces of incubators in NICUs, and a study by Cadot et al. [n = 26), with S. capitis isolated on 7.1% [S. capitis on 100% of incubators (n = 2) in an NICU both before and after disinfection, while Butin et al. [S. capitis NRCS-A in 57% (n = 56/99) of samples from NICU incubators [Within NICU outbreak settings, environmental contamination has been noted to be of particular significance. The source of a ual beds . Multiplt et al.  recovere on 7.1% . Work by on 7.1%  also foun et al.  isolatedcubators ,95.S. capitis has been identified on reusable electroencephalography cup electrodes and lead wires [S. capitis in 8 of 40 needleless connectors connected to peripheral IV catheters in adult medical patients in hospitals, with the authors highlighting the urgency of developing an effective needleless connector decontamination procedure [Medical equipment has been demonstrated to be a source for bacterial surface contamination both in NICU outbreaks and in adult settings. ad wires , thyroidad wires  and postad wires . Slater ad wires  also reprocedure .S. capitis infection is a potential complication of surgery, and therefore surgical environments and equipment have been considered as a source of S. capitis. Clesham et al. [n = 40), finding S. capitis contamination on 5% (n = 2) [S. capitis. This included contamination of 9% of IV extension lines, leading to an increased risk of bacteraemia [m et al.  analysed (n = 2) . A study (n = 2)  showed bteraemia .S. capitis within clinical settings, especially within NICUs, surface decontamination is a crucial intervention. S. capitis is linked with biofilm production, which can be induced by environmental stressors , both pre and post processing [S. capitis catheter-related bacteraemia linked to a twice-daily scrubbing routine with packed alcohol gauze of intravascular lines and hubs, saturation monitor lines and oximetry tips, electrocardiography connecting jacks, electrocardiography leads, stethoscopes and thermometers [S. capitis.Steam cleaning was implemented in one study as a way to disinfect heating tables and incubators and demonstrated a statistically significant reduction in loads  has contributed to its increased detection in NICUs and outbreak settings. The mechanism of its spread and the best interventions to mitigate it remain unclear. Additional work would help understand the prevalence of carriage in neonates outside of outbreak scenarios and the best intervention package in order to control outbreaks once they occur.


Bubo virginianus) inhabits myriad habitats throughout the Americas and shows complex patterns of individual and geographic morphological variation. The owl family Strigidae is known to follow ecogeographic rules, such as Gloger’s rule. Although untested at the species level, these ecogeographic rules may affect B. virginianus plumage coloration and body size. Previous studies have indicated that, despite this species’ morphological variability, little genetic differentiation exists across parts of their range. This study uses reduced representation genome-wide nuclear and complete mitochondrial DNA sequence data to assess range-wide relationships among B. virginianus populations and the disputed species status of B. v. magellanicus (Magellanic or Lesser Horned Owl) of the central and southern Andes. We found shallow phylogenetic relationships generally structured latitudinally to the north of the central Andes, and a deep divergence between a southern and northern clade close to the Marañón Valley in the central Andes, a common biogeographic barrier. We identify evidence of gene flow between B. v. magellanicus and other subspecies based on mitonuclear discordance and F-branch statistics. Overall differences in morphology, plumage coloration, voice, and genomic divergence support species status for B. v. magellanicus.The Great Horned Owl ( Bubo virginianus) is a common and widespread species throughout the Americas . Information on patterns of intra-specific genetic variation is scant but suggests low genetic variation and no genetic structure across the southwestern United States , and (2) evaluate the putative species status of the populations of southwestern South America (subspecies magellanicus). We used reduced-representation nuclear genomic and mitochondrial sequence data to reconstruct a phylogeny and address the question of species status in the broader context of variation across the range of the B. virginianus complex.Here, we present the first genome-wide estimate of genetic differentiation in Great Horned Owls across their entire distribution. Specifically, we (1) characterize range-wide genetic variation and differentiation within the species by sampling thirteen of the sixteen recognized subspecies  were downloaded from the Sequence Read Archive , based on Two DNA extraction methods were used, depending on tissue type. DNA was extracted from toepad samples using a Promega Maxwell RSC automated instrument and the Maxwell RSC Tissue DNA Kit. This instrument minimizes potential for contamination, which is important for degraded DNA samples. Fresh tissue samples were extracted using a manual bead-extraction protocol . DNA from toepad samples was already relatively degraded, and therefore did not need to be sonicated . We thenhttps://github.com/emilyostrow/ReplaceUninformativeSites) due to potential data degradation in toepad samples. In addition to missing data, toepad samples tended to have erroneous bases sequenced near cut sites. This script replaced any base that is represented only once at a particular site with an N to address branch length concerns caused by degraded DNA.All UCE data were processed using the standard Phyluce pipeline with the mapping and correction workflows . Brieflyhttp://broadinstitute.github.io/picard) and indexed the files using SAMtools  using the consensus UCE sequences as a reference with the GATK pipeline. We used Geneious Prime  to generate consensus sequences on all UCEs found in at least 75% of the individuals using a 50% strict cutoff for each site. To call SNPs, we first trimmed raw read files using AdapterRemoval  from NCBI as a reference mitochondrial genome for alignment. Briefly, we interleaved reads one and two from the cleaned Illumiprocessor data. We created an initial read map using MIRA : XC428421 from Minnesota, USA, XC511167 from New York, USA, XC548133 from California, USA, XC76397 from Napo, Ecuador, XC212734 from Salta, Argentina, and XC494437 from Santiago, Chile. Audio recordings were chosen based on audio quality and geographic breadth. We imported these songs into Adobe Audition (Build 14.1.0.43) and iteratively used the denoise tool to reduce background noise in the sonograms until the song was clearly differentiated from the background noise. We then exported audio files from Adobe Audition and imported them into Raven Lite  included 4299 UCE loci, with a 1.26 Mbp overall alignment length and an average UCE length of 796 bp for fresh tissues and 299 bp for the toepad samples. Our unfiltered SNP dataset included 7584 SNPs. After filtering for SNP completeness, phred score, and biallelic sites, our final dataset included 6556 SNPs.We generated a mean of 4248 UCE loci per individual. Fresh tissue samples yielded a mean of 4318 UCE loci per individual, whereas toepad samples yielded a mean of 4127 loci per individual. The 75% matrix (B. v. virginianus (FMNH 396867), had 23.05% missing data and disproportionately contributed to the overall missing data in the mitogenome matrix. The effects of these missing data and degradation, contamination, or sequencing error at the edges of gaps are apparent in the longer branch in the mitochondrial tree.The mitochondrial baiting method was successful for all individuals in this study. MITObim contigs had a mean read depth of 101X and a mean contig length of 18,967 bp. Fresh tissue samples had a mean read depth of 95X, whereas toepad samples had a mean read depth of 111X, presumably due to increased sequencing effort dedicated to toepad samples. After trimming to genic regions only, we generated an 11,347 bp alignment, with 1.09% missing data. One individual in particular, B. v. nigrescens (FMNH 102981), which was sister to B. v. nacurutu (KU 90879) + B. v. nigrescens (USNM 368745) in the SVDquartets tree whereas in all other full sampling trees, B. v. nigrescens (FMNH 102981) was sister to B. v. nigrescens (LSUMZ 30041). The one topological change between the maximum-likelihood tree and neighbor-joining tree was the placement of B. v. nacurutu (KU 90879) + B. v. nigrescens (USNM 368745), which was sister to B. v. nigrescens (FMNH 102981) + B. v. nigrescens (LSUMZ 30041) in the neighbor-joining tree . Within the northern clade, lineages branching from earlier nodes belonged to more southern taxa (B. v. nacurutu and B. v. nigrescens) with less geographically concordant branching order in other northern subspecies.Maximum-likelihood, quartet-based, and neighbor-joining methods using the nuclear data produced trees that were congruent at most moderate to highly supported nodes (bootstrap support above 90%); the trees mainly differed in topology at nodes with low support. The one topological difference with bootstrap support in the quartet-based tree  was the ing tree  but is wing tree . The quaing tree . HoweverB. v. mayensis (FMNH 187122) and B. v. pinorum (UWBM 74120) switched positions, and B. v. elachistus (DMNS 21432) and B. v. pacificus (LACM 115875) switched positions. Support values from both analyses are mapped onto the RAxML tree at concordant nodes  and B. v. nigrescens (LSUMZ 30041). In the fresh tissues only UCE tree, they are sequential sister taxa to the remainder of the northern clade, whereas in the all samples UCE tree, B. v. nacurutu (KU 90879) is found within the larger North American clade (B. v. nigrescens (LSUMZ 30041) and B. v. nacurutu (KU 90879) switch branching order as compared to the UCE trees  from Ecuador and Peruvian B. v. magellanicus support Nuclear gene flow between B. v. magellanicus and non-magellanicus B. virginianus populations in the Andes  and much of the northern clade  and all other samples (12 sampled subspecies). The Marañón Valley is a common biogeographic barrier for higher-elevation birds in South America due to a steep transition between wet mountain regions and a dry valley  is well over the 2% divergence that has been suggested as indicative of species-level divergence in birds , Cordoba, Argentina (XC51622), and Salta, Argentina (XC212734), whereas recordings of individuals giving typical northern songs  occur within southern South America as far west as Buenos Aires, Argentina (XC645511) and Beni, Bolivia (XC149520). The non-overlap of B. v. magellanicus and other B. virginianus subspecies may be due to habitat partitioning, in which non-magellanicus B. virginianus subspecies may be more forest-associated (Audio recordings on Xeno-Canto suggest that sociated .magellanicus by range and nuclear data) had the mitochondrial genome of the non-magellanicus group. This mitonuclear discordance is most commonly produced by either incomplete lineage sorting  or gene flow between populations (versus gene flow) using the nuclear sequence data and identified significant gene flow between B. v. nigrescens in Ecuador and B. v. magellanicus in Peru. The mitochondrial and UCE data were bioinformatically extracted from the same DNA sequencing reaction, so the mitonuclear discordance should not be caused by contamination. Contamination from other samples during DNA extraction or library preparation is possible, but unlikely given the consistently southern nuclear data recovered from the individual with the northern mitochondrial sequence. The combination of mitonuclear discordance and significant signal of nuclear gene flow between B. v. nigrescens in Ecuador and B. v. magellanicus in Peru suggests some level of ongoing or historical gene flow between these two distinctive populations. The nature and extent of this mitonuclear discordance and the gene flow in northern Peru can only be determined with denser sampling.We found one instance of incongruence between the topologies of the UCE and mitochondrial trees regarding the deep divergence between northern and central Andes. Although the UCE data showed a clear divide between populations, the mitochondrial data showed that one individual from Lima, Peru (subspecies ulations . F-brancB. virginianus clade but display shallow population structure. Individuals sampled from across North America in particular have low genetic divergence and little support for phylogenetic relationships, indicating prevailing gene flow, range expansion, or recent divergence. Bubo virginianus are known to be philopatric, but mixed evidence for yearly migration or dispersal has been documented (Our results also confirm the shallow structure seen in the southwestern United States  and showcumented . Althougcumented , these mB. virginianus. We found little genetic differentiation among populations throughout North America, Central America, and northern South America north of the central Andes. In contrast to that shallow differentiatioin, we identified substantial divergence, in both nuclear and mitochondrial DNA sequences, between populations of the central and southern Andes and all others. These southern populations, currently assigned to the subspecies B. v. magellanicus, show morphological, vocal, and genetic differences consistent with species-level differentiation. Despite deep mitochondrial and nuclear divergence between B. v. magellanicus and other B. virginianus, we found one case of mitonuclear discordance and we observe evidence of nuclear gene flow across the north-south break in northern Peru. Although our data do not identify the geographic distribution of the transition area between these putative species, the vocal and genetic data presented here provide support for species-level differentiation. Additional genetic and vocal data are needed to assess the extent of interactions between the two populations and yield a more thorough understanding of the dynamics between these two putative species.In this article we present the first genome-scale, range-wide molecular analysis of genetic variation in 10.7717/peerj.15787/supp-1Figure S1Support values are shown in the shaded circles on nodes. All nodes without support values are not highly supported.Click here for additional data file.10.7717/peerj.15787/supp-2Figure S2Bootstrap values are listed to the right of nodes. Topologies with less than 50% bootstrap support are collapsed.Click here for additional data file.10.7717/peerj.15787/supp-3Figure S3Support values shown in the shaded circles on nodes. Nodes without a circle have the lowest category of support.Click here for additional data file.10.7717/peerj.15787/supp-4Figure S4F-branch values are shown in varying shades of red. P-values are shown according to levels of significance: *** p < 0.001, ** p < 0.01, * p < 0.05.Click here for additional data file.