Pseudacteon (Diptera: Phoridae) are potential biocontrol agents of the invasive fire ants Solenopsis invicta and S. richteri in the United States and other regions of the world due to their high host specificity and the direct and indirect damage to their host ants. Despite their importance and the fact that several flies have already been released in the US, little is known about the genetic variability and phylogenetic relationships of Pseudacteon flies parasitizing South American fire ants in the Solenopsis saevissima species-group. A species delimitation analysis was conducted using a distance-based method (ABGD) and two tree-based methods (GMYC and mPTP) using COI sequences of 103 specimens belonging to 20 of the 22 Pseudacteon species known from southern South America. Additionally, phylogenetic relationships between the already described and new candidate species were inferred using mitochondrial (COI) and nuclear (wingless) sequences. The species delimitation analysis suggests that species richness in these flies has been previously underestimated, due to the existence of putative cryptic species within nominal Pseudacteon obtusus, P. pradei, P. tricuspis, P. cultellatus, and P. nudicornis. Geographic distribution and host fire ant species seem to support cryptic lineages, though additional morphological data are needed to corroborate these results. All phylogenetic analyses reveal that South American fire ant decapitating flies are grouped into two main clades, with Pseudacteon convexicauda sister and well differentiated relative to these clades. Neither host nor geographic association appeared to be related to the differentiation of these two main clades within South American fire ant decapitating flies. This work provides information that will allow testing whether the putative cryptic phorid fly species show differences in their effectiveness as biocontrol agents against the highly invasive imported fire ants.South American fire ant decapitating flies in the genus  Pseudacteon Coquillett 1907 (Diptera: Phoridae) and derive their common name from their parasitoid life cycle in which their host ants are decapitated after the fly finishes its development inside the ant’s body [Fire ant decapitating flies belong to the genus t’s body . During t’s body , 3.Pseudacteon is currently comprised of 72 known species distributed worldwide with just over half of them inhabiting the Neotropical region [Solenopsis spp.; [Crematogaster Lund 1831, Dorymyrmex Mayr 1866, Formica Linnaeus 1758, Lasius Fabricius 1804, Linepithema Mayr 1866, Liometopum Mayr 1861, Myrmica Latreille 1804, Nylanderia Emery 1906, Pseudolasius Emery 1887, and Tapinoma Foerster 1850 [Pseudacteon species are known to parasitize the South American fire ants of the Solenopsis saevissima  species-group and 23 fly species attack fire ants in the Solenopsis geminata (Fabricius 1804) species-group [Pseudacteon species that parasitize these two fire ant groups species despite the overlapping host distribution both in their native and introduced ranges [Pseudacteon fowleri Pesquero 2000, which is known to parasitize S. saevissima species-group, but was collected in the range of S. geminata species-group (in French Guiana and Guyana) [Pseudacteon arcuatus Borgmeier 1969, P. kungae Plowes et al. 2009, and P. laticarinatus Plowes et al. 2009, which all parasitize fire ants of the S. geminata species-group. It is possible that the existence of cryptic species underlies this apparent host switch [The genus l region . Most ofis spp.; , 6), butd ranges . The onl Guyana) . Althoug Guyana)  due to tt switch .Solenopsis invicta Buren 1972 and S. richteri Forel 1909, in North America and other regions of the world [Pseudacteon curvatus Borgmeier 1925, P. litoralis Borgmeier 1925, P. tricuspis Borgmeier 1925, P. obtusus Borgmeier 1925, P. nocens Borgmeier 1926, and P. cultellatus Borgmeier 1925 [Fire ant decapitating flies are considered one of the most promising biological control agents of introduced fire ants, he world . To dateier 1925 –14, and ier 1925 . Howeverier 1925 , 16. Theier 1925 , 17. Morier 1925 , 18. SomP. obtusus presents variation in the adult body size with a large and a small biotype that form genetically distinct lineages [Pseudacteon obtusitus Plowes, Folgarait, and Gilbert 2015 [Pseudacteon flies in biological control programs for introduced fire ants, knowledge about the phylogenetic relationships between the different species within the genus is scarce [Pseudacteon) that parasitize the S. saevissima species-group in order to elucidate potential new cryptic species within these parasitoid flies and propose phylogenetic hypotheses. Through the use of three species delimitation approaches (a distance-based approach and two tree-based species delimitations) it will be possible to determine if the morphological variability observed in some Pseudacteon species corresponds to the presence of cryptic or sibling species, presumably isolated by geographic barriers or host species.Some fire ant decapitating fly species exhibit intraspecific morphological variation across their geographic ranges suggesting the existence of subspecies or cryptic species . For exalineages , the latert 2015 . Despites scarce , 23. Thein situ on the basis of the appearance of the colony and later in the laboratory examining major workers under stereomicroscope with available keys [Field surveys were conducted across southern South America , betweenble keys . Additioble keys , 25, 26.Pseudacteon species was sampled from nests of nine fire ant species  from Argentina were also included. P. convexicauda is the only phorid fly species reported using both the tawny crazy ant and the fire ant S. saevissima as host [A total of 103 individuals belonging to 20 nominal  species ; Fig 1.  as host  Morpholova Mayr 162 from A as host , 28–30. TM Tissue PCR Kit . Fragments of the cytochrome oxidase subunit I (COI)  and wingless (wg) (nuclear) genes were amplified by PCR (Polymerase Chain Reaction) using the primers C1-J-2183  and TL2-N-3014  for COI [wg [μl (1u) ExoI (exonuclease I) incubated at 37°C for 15 min, stopping the reaction by heating the mixture at 85°C for 15 min. Most of the purified PCR products were sequenced at the sequencing and genotyping unit of the Faculty of Exact and Natural Sciences of the University of Buenos Aires , using a 3130-XL Automatic Sequencer (Applied Biosystems), and some of the samples were sequenced at the UC Berkeley DNA Sequencing Facility. A total of 102 COI and 48 wg sequences were inspected, trimmed and aligned using Geneious Pro v4.8 (http://www.geneious.com/) and deposited at GenBank , Apocephalus paraponerae Borgmeier 1958, Argentinomyia longicornis , Drosophila acanthoptera Wheeler 1949, Stichillus Ederlein 1924). GenBank accession numbers of outgroups sequences: GU559934.1, AF217478.1, DQ448549.1, KM270850.1, KF632601.1, GU559945.1. All identical sequences were removed prior analyses, resulting in a 91 COI sequence dataset, including the outgroups. All species delimitation analyses were conducted using a pruned outgroup tree and a complete tree including the outgroups.The traditional phenotype-based species boundaries in South American fire ant decapitating flies was corroborated using three different analyses: a distance-based approach  and twohttp://wwwabi.snv.jussieu.fr/public/abgd/), testing different relative gap width (X) values between 0.8 and 1.0 and intraspecific divergence (P) values between 0.001 (0.1%) and 0.1 (10%).The ABGD assumes that the intraspecific divergence is smaller than the interspecific divergence . In otheThe tree-based methods employ a coalescent framework in order to independently identify evolving lineages without gene flow, each representing a putative species . They caThe GMYC method , 39, 40 The third species delimitation approach was implemented using a Poisson tree process (PTP), which models the speciation using the number of substitutions to infer putative species boundaries on a given phylogenetic input tree . It assuPseudacteon species parasitizing fire ants, we analyzed a subset of the COI sequences plus additional sequences of the nuclear gene wg from 27 samples that represents the best supported lineages/species identified during the species delimitation analyses. The dataset (COI + wg) was analyzed in Bayesian inference framework as implemented in BEAST v.2.5.1 [wg- partitions using bModelTest [Anevrina oligocoenica Brues 1939, known from Eocene/Oligocene Baltic amber  and5–20 Ma; ). The an5–20 Ma;  by runni5–20 Ma; . Both re5–20 Ma; .wg), and bootstrap support values were estimated from 1000 replicates.Additionally, to establish the congruence of the relationships found in the maximum credibility tree further phylogenetic reconstructions were assessed by maximum parsimony (MP) and maximum likelihood (ML) methods. MP analysis was carried out with TNT v.1.5  employinThe COI alignment consisted of 91 sequences of 699 base pairs (bp) and 316 segregating (polymorphic) sites. In most of the ABGD, GMYC and mPTP analyses, the number of lineages found was higher than those identified by morphology . The anaP. convexicauda as a well-defined sister clade to the rest of the Pseudacteon species studied. Also, all the topologies recover two well-supported clades within the South American fire ant decapitating flies: Clade A comprised of Pseudacteon nudicornis Borgmeier 1925, P. borgmeieri Schmitz 1923, P. wasmanni (Schmitz 1914), P. affinis Borgmeier 1962, P. cultellatus, Pseudacteon pradei Borgmeier 1925, Pseudacteon notocaudatus Plowes et al. 2015, and Pseudacteon disneyi Pesquero 2000, and Clade B includes Pseudacteon solenopsidis (Schmitz 1914), P. dentiger Borgmeier 1962, P. fowleri, Pseudacteon lenkoi Borgmeier & Prado 1975, P. tricuspis, Pseudacteon calderensis Calcaterra 2007, P. nocens, P. litoralis, P. curvatus, P. obtusus (large (L) and medium size (M) biotypes), and P. obtusitus . No evident host or geographic association was found to be related to the differentiation of these two main clades. Three species P. nudicornis, P. solenopsidis, and P. obtusus (M biotype) varied their position in the phylogeny between for the GMYC and mPTP input trees  from Bolivia was the most differentiated lineage of the group (in GMYC appears in a different clade) and could be related to an Amazon (basin) lineage of the genus (not included in this study). Other two lineages of P. nocens were found: one in Uruguay and another one in La Pampa, Argentina. The remaining specimens constitute a clade with two lineages, one composed by P. nocens from Brazil and Argentina and the other one composed of all the individuals morphologically identified as P. litoralis. The latter comprises a group of populations from Brazil and Argentina and another one from Paraguay and Bolivia.All methods split each one of the following species: t clades  and grou species . The P. P. cultellatus resolves as paraphyletic, being composed of two different clades: a specimen found in a S. invicta colony in Brazil  + P. notocaudatus + P. pradei + P. disneyi. Also, in this group, P. pradei appears into two different lineages, also paraphyletic, with one clade  being sister of P. disneyi  or three (GMYC and mPTP) putative species. In all analyses, the specimen from Goiás (Brazil) is placed as a distant lineage separated from all the other P. nudicornis , which appears to be a well-supported different species  that uses S. invicta and Solenopsis macdonaghi Santschi 1916 as hosts, and are distributed in northeastern Argentina; another clade is composed of by P. obtusus (M) from Chile that parasitizes Solenopsis gayi (Spinola 1851)  specimens that use S. interrupta as host and are mainly distributed in the Monte and Yungas ecoregions in western Argentina.analyses . One clala 1851) ; anotherla 1851)  which usP. tricuspis form a well-defined clade . However, two specimens that parasitize S. richteri in La Pampa ecoregion constitute another well-defined clade, closely related to P. lenkoi and P. fowleri  sites. All studied Pseudacteon species (26) were recovered as a single group, with the only exception of P. convexicauda that was recovered as sister to all the other species  and the split between the two major clades A and B occurred during the early Miocene . Moreover, the analysis suggests that most of the species diverged during the early Pliocene (~4 Ma).Both mitochondrial and nuclear markers were variable and informative for detecting variability within the species studied. The  species . The con species . HoweverOI trees , the COId clades . DivergePseudacteon species studied in this work that parasitize fire ants of the S. saevissima species-group belong in fact to at least 24 to 27 species. Alongside the morphological evidence, our study suggests that the number of South American fire ant decapitating fly species  has been previously underestimated Pseudacteon may be more diverse than what traditional morphology-based taxonomy suggests. One of the limitations of the approach employed here is related to the use of a single-locus dataset . Howevene trees  and the ne trees , 60. In ne trees  while thne trees , but witne trees , 61. Forne trees , 40. In ne trees , analytine trees . FurtherPseudacteon fly species that parasitize fire ants in the S. saevissima species-group. Also, our approach using multiple species delimitation methods allowed us to recognize possible spurious or overestimated results.The species delimitation methods that used more conservative (single) thresholds resulted in closer estimates to the number of morphological delimited species based on morphological characters as compared to multiple thresholds. This may be due to differences among the algorithms, the guide tree used and/or to sampling representation, as mentioned in the literature , 38. AlsP. tricupsis, P. nudicornis, and P. obtusus complex; S. saevissima species-group [Pseudacteon species may coexist and parasitize the same host species [This study suggests that some nominal species among the South American fire ant decapitating flies with broad geographic distributions, are actually composed by the nominal and one or more cryptic species ) or were reduced to a single sequence during the analyses because these were identical . Although, the support for these flies as distinct species is consistent with their morphology, genetic distance, and estimated times of divergence, these results should be taken with caution until a larger number of individuals and populations can be included, or additional morphological and molecular evidence can be analyzed.Some species in our analyses were represented by a single individual  were considered by all delimitation analyses as the same species as P. obtusitus (S. invicta as a host. The remaining P. obtusus (L) were split by the delimitation analyses in two putative species  from Chile belongs to a different species, sister of P. obtusitus. In agreement with this result, the female ovipositor of this biotype is more rounded than the Argentine large biotype and is the only known species of Pseudacteon that attacks S. gayi [P. obtusus complex needs to be revised and biotypes/lineages need to be redefined taking into account their geographic distribution, morphology, host preferences, and perhaps also body size.Our results confirm that the lyzed by , who foulyzed by  describeCOI + wg , at leasbtusitus , Fig 3. ies Figs  and 5. A S. gayi . Based oP. cultellatus, P. pradei, P. notocaudatus, and P. disneyi also shows the existence of cryptic and/or paraphyletic species  that parasitize S. richteri have an ovipositor with a more laterally pronounced central extension than that of populations from northeastern Argentina and São Paulo (Brazil), which parasitize S. invicta [P. cultellatus associated with S. invicta is more closely related to P. pradei + P. notocaudatus + P. disneyi than to the P. cultellatus of central eastern Argentina that parasitizes S. richteri. Possibly, the individuals referred as P. cultellatus “a”, in Figs S. invicta, because this sample is geographically closer to the type locality  than thP. nocens and P. litoralis populations that we genetically analyzed seem to belong to a single species according to the delimitation analyses. Both share similar annual and daily activity patterns [P. litoralis has a group of long setae in the base of the lateral lobes. These are absent in P. nocens [Most of the patterns , 67 and patterns . Alternapatterns ) shows t. nocens , in whic. nocens . WhateveP. nudicornis studied not only differ in their phylogenetic position, but also in the host fire ant. The population from Goiás, Brazil, parasitizes S. saevissima, while the populations from Bolivia and Argentina parasitize S. invicta or S. interrupta (P. nudicornis “a”) have differences in the female ovipositor compared to the rest of Brazilian populations (e.g. São Paulo flies that parasitizes S. invicta), which present a more concave plate of the lateral lobes [The populations of terrupta . Being tal lobes .P. tricuspis resolved into two separate, well-supported clades. Populations of P. tricuspis from Buenos Aires and Córdoba , which parasitize S. richteri, should be separated from populations of P. tricuspis that occur elsewhere. In the tree topologies obtained during the phylogenetic and species delimitation analyses, these samples were always separated from the rest of P. tricuspis and were placed as sister group to P. fowleri + P. lenkoi clade. Additional evidence supporting these fly populations as a different species are their geographic distributions in the Pampa ecoregion for the “P. tricuspis a”  varied between the different phylogenetic reconstruction methods, all of these species were recovered within the same major clade. It is likely that the uncertainty of their topological placement arises from recent divergence times and associated low levels of genetic differentiation, as in their host fire ants.Several hypotheses regarding the relationships between some  made by  who propSolenopsis originated in the Neotropical region in the early Oligocene  [Pseudacteon is close to the Oligocene–Miocene transition, just before the appearance of fire ants. This time period also seems to coincide with the moment when P. convexicauda and fly species that parasitize South American fire ants of the S. saevissima species-group were separated. This is also consistent with the origin of Nylanderia genus in Oligocene–Miocene transition (~23–24 Ma) [P. convexicauda is known to parasitize N. fulva.The genus .4–47.1) . Even th3–24 Ma) , 72; as Solenopsis quinquecuspis Forel 1913, Solenopsis weyrauchi Trager 1991, and Solenopsis megergates Trager 1991. Furthermore, the head size of parasitized S. geminata and S. invicta workers that have been parasitized is known to be correlated with the body size of their Pseudacteon parasitoid species [The existence of two main clades in the South American fire ant decapitating flies which diverged about ~19 mya, is intriguing because there are no supported ecological or behavioral patterns that characterize these two clades. Nevertheless, it can be observed that the main clade A tends to be composed of medium to small body size flies (<0.44 mm), while the main clade B has both small and large body size flies . Indeed, species , 73. TheP. convexicauda. This species has been reported using both the tawny crazy ant (N. fulva) and the fire ant S. saevissima as host [P. convexicauda that we surveyed in Argentina, flies were usually found hovering over nests of N. fulva and attacking their workers, supporting that the assumption this species does not use S. saevissima species-group as host. This result is congruent with the fact that P. convexicauda is well differentiated from the rest of the Pseudacteon species studied here.It is important to remark on the phylogenetic position of  as host , 27. How as host  and stilPseudacteon flies and their host fire ants it is necessary to have a well resolved phylogeny of the latter. Fortunately, the phylogenetic relationships within Solenopsis fire ants are currently under revision [S. invicta clade , which contains one of the most detrimental invasive ant species [To understand the existence of host switching and/or co-evolutionary relationships between revision  due to trevision , specifi species .P. curvatus, P. litoralis, P. tricuspis, P. obtusus, P. nocens, and P. cultellatus have been released to control S. invicta in the US [P. obtusitus was recognized as a new species , it was already defined as a different lineage based on molecular evidence at the time it was used for biocontrol [Historically, the species n the US –14. Our n the US . Thus, oocontrol . Additioocontrol . Thus, nocontrol .P. nocens from Santiago del Estero, Argentina, which have high levels of host specificity to the biotype of S. invicta present in that region [S. invicta and is placed as a sister group to the P. litoralis that has been previously used as biocontrol agent [Pseudacteon genus, including the small P. tricuspis biotype from the state of Goiás, Brazil , those few not included in our study  and those that occur on fire ant species in the S. geminata species-group in North America. The application of additional methodologies would be useful to determine the robustness of our findings. In particular, matching the lineages of the fire ant S. invicta species-complex with their corresponding parasitoid fly lineages will surely improve the selection of biocontrol agents against this highly invasive pest ant.This work provides information that will allow testing whether the different lineages (putative cryptic species) of the fire ant decapitating flies show differences in their effectiveness as biocontrol agents. Our results should be considered when assessing potential new taxa as possible candidates. Such is the case for populations of t region . In our ol agent . Also, wol agent . Future S1 Table(XLSX)Click here for additional data file.S2 TableAs input a Kimura two parameters (K2P) distance matrix was used and four different relative gap width values (X) and intraspecific divergence (P) values between 0.001 (0.1%) and 0.1 (10%). .(XLSX)Click here for additional data file.S3 TableAll the analyzes were done using a BEAST ultrametric tree obtained using a strict molecular clock and coalescent constant tree prior. .(XLSX)Click here for additional data file.S4 TableAll the analyzes were done using a RAxML best fit tree. .(XLSX)Click here for additional data file.S5 TableP. obtusus biotypes are noted as follows: M = medium size, L = large size. Ant host genus abbreviated as: S. = Solenopsis; N. = Nylanderia.(XLSX)Click here for additional data file.S1 FigA) including and B) excluding the outgroups. Trees shown correspond to a relative gap width value of 0.9. Numbers at the end of the names correspond to the groups delimited.(TIF)Click here for additional data file.S2 FigA) single thresholds, B) multiple thresholds. Colored lines indicate the cladogenetic events (groups considered as a single species), in the case of multiple thresholds different colors are used in correspondence to each threshold.(TIF)Click here for additional data file.S3 FigA) single thresholds, B) multiple thresholds. Colored lines indicate the cladogenetic events (groups considered as a single species), in the case of multiple thresholds different colors are used in correspondence to each threshold.(TIF)Click here for additional data file.S4 FigA) single thresholds, B) multiple thresholds. Colored red lines indicate the cladogenetic events (groups considered as a single species).(TIF)Click here for additional data file.S5 FigA) single thresholds, B) multiple thresholds. Colored red lines indicate the cladogenetic events (groups considered as a single species).(TIF)Click here for additional data file.S6 FigMaximum parsimony strict consensus tree obtained with TNT. Values in branches are boostrap values. A) Maximum parsimony strict consensus trees obtained with TNT, B) Maximum likelihood best trees obtained with RAxML. Values on branches are boostrap values.(TIF)Click here for additional data file.


Plasmodium knowlesi is a potential cause of severe and fatal malaria, but comprehensive studies of its pooled prevalence and risk factors are lacking. This study aimed to explore the prevalence and risk factors related to severe P. knowlesi infection.P. knowlesi available in Web of Science (ISI), Scopus, and PubMed (MEDLINE). Titles, abstracts, and full-text articles were screened, and any irrelevant studies were excluded. The random-effects model was used to compute the pooled prevalence estimate of severe P. knowlesi infection by a metaprop command provided in STATA software. Differences in demographic characteristics, clinical characteristics, and laboratory data were analysed using Review Manager Version 5.3 software for patients in the following groups: 1) patients with severe and non-severe P. knowlesi infection and 2) patients with severe P. knowlesi and severe P. falciparum infection.A systematic review was conducted by retrieving all published articles on severe P. knowlesi infections were eligible to be included in this systematic review and meta-analysis. The pooled prevalence estimate of severe P. knowlesi infection was 19% . Severe acute kidney injuries (AKI) , jaundice , and hyperparasitaemia  were the common clinical manifestations found among patients with severe complications. In comparison to non-severe P. knowlesi infections, patients with severe P. knowlesi infections had significantly higher age, leucocyte count, and parasitaemia levels (P < 0.05). In comparison to patients with severe P. falciparum infections, patients with severe P. knowlesi infections had significantly higher age, neutrophil count, and creatinine levels (P < 0.05).Out of the 2382 studies retrieved from the three databases, seven studies with a total enrolment of 1124 patients with P. knowlesi infections. Patients with severe P. knowlesi infections had higher age, leucocyte count, and parasitaemia levels than those with non-severe P. knowlesi infections. In addition, patients with severe P. knowlesi infections had higher age, neutrophil count, and creatinine levels than those with severe P. falciparum infections.This systematic review and meta-analysis demonstrated a high proportion of severe  Plasmodium knowlesi is a naturally occurring malarial parasite in monkeys [P. knowlesi are the long-tailed macaque (Macaca fascicularis) and the pig-tailed macaque  [P. knowlesi is found only in Southeast Asian countries because its natural hosts and the female Anopheles mosquito vectors are very limited and specifically exist in these areas [Leucosphyrus Complex transmitted P. knowlesi in southern Asia, while the Dirus Complex transmitted P. knowlesi in Vietnam, Myanmar, Laos, Cambodia and Thailand [P. knowlesi is transmitted from human to human but rather through mosquito bites [P. knowlesi occur through quotidian (24-hour schizogonic cycles) malarial parasites and lead to severe and lethal manifestations. monkeys  and is a monkeys . The natleonine) –6. The gse areas . A previThailand . At presto bites . InfectiP. knowlesi infection in humans was reported by Singh et al. in Sarawak, Malaysia, in patients previously misdiagnosed with P. malariae infections [P. knowlesi is similar to that of P. malariae, a quartan malarial parasite causing non-severe human malaria [P. knowlesi confirmed by polymerase chain reaction (PCR) were miss-detected as P. malariae by microscopic methods (76%) [P. knowlesi, but they were insensitive in cases of low parasitaemia and were unable to distinguish P. knowlesi from P. vivax [P. knowlesi requires a molecular technique, such as the nested PCR assay. A previous study demonstrated that P. knowlesi misdiagnosis as P. malariae caused severe malaria and resulted in death because of inappropriate treatment [P. vivax and to prevent the fatal outcome of patients by administering intravenous therapy with artesunate or quinine as the first drug of choice for severe malaria, especially caused by P. knowlesi. Although P. knowlesi is a potential cause of severe and fatal malaria, comprehensive studies of its pooled prevalence and risk factors are lacking. In addition, a meta-analysis determining the difference in the prevalence estimate of severe P. knowlesi infection among studies conducted on P. knowlesi is needed. Therefore, this study aimed to estimate the prevalence and risk factors related to severe P. knowlesi malaria. In addition, the comparison of demographic characteristics, clinical data, and laboratory data between severe P. knowlesi and severe P. falciparum infection was evaluated to predict disease severity in co-endemic primary care settings of P. knowlesi.The first case of fections . The mor malaria . This sids (76%) . The rapP. vivax , 12. Henreatment . It is vPlasmodium knowlesi”) OR knowlesi)”  guidelines (PRISMA 2009 Checklist S” Table S. This seTwo reviewers (MK and KUK) selected and extracted the data from the included studies independently. We used the Endnote software X7  to manage the references. First, we removed any duplicate papers retrieved from the three databases. Then, we screened the titles and abstracts according to the inclusion criteria. Finally, we assessed all full-text articles for detailed assessments for further data extraction. The data extraction was conducted using the Microsoft Excel 2010  and included data on the primary author, year of publication, study area, study design, demographic characteristics of patients (age and sex), and clinical characteristics . Laboratory profiles including parasite count, haemoglobin, leucocyte count, platelet count, creatinine, sodium, bilirubin, glucose, albumin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were additionally collected. The malaria endemicity of the years when the included studies were conducted by their authors was demonstrated by the slide positivity rate as reported by the World Malaria Report 2010–2014 . DisagreP. knowlesi malaria were defined by the World Health Organization (WHO) [The criteria of severe on (WHO) .A quality assessment was performed following the quality assessment tool developed by the Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analysis . It provP. knowlesi infection. The data analysis for the pooled prevalence began with entry of the extracted data into the Excel sheet and to the STATA Statistical Software version 15.0 . The number of patients with severe P. knowlesi and total cases of P. knowlesi were imported and analysed using a command “metaprop case population, random/fixed” provided in the STATA software. A metaprop command allowed Freeman-Tukey double arcsine transformation to stabilize the variances. The random-effects or fixed-effect model could be used to compute the pooled estimate, depending on the heterogeneity among the included studies [CI).The primary outcome of the present study was the pooled prevalence of severe  studies . The resP. knowlesi malaria using the demographic characteristics, clinical characteristics, and laboratory profiles of patients with severe and non-severe P. knowlesi malaria. The third outcome of the present study was to compare the demographic characteristics, clinical characteristics, and laboratory profiles between patients with severe P. knowlesi and severe P. falciparum malaria. The results of the second and third outcomes were presented as the pooled mean difference (MD) with 95% CI for continuous outcomes and odds ratio along with 95% CI for categorical outcomes. The data heterogeneity was assessed using Cochrane Q (chi-square) and Moran’s I2 (inconsistency). The I2 was quantified as low, moderate, and high, with upper limits of 25, 50, and 75% for I2, respectively. If moderate or high heterogeneity was presented by Moran’s I2, the random-effects model was used to calculate the pooled estimates. If a low heterogeneity (I2 < 25%) was presented, the fixed-effect model was used to calculate the pooled estimates. The subgroup analysis of study sites was performed to demonstrate whether the transmission intensity between Sarawak and Sabah, the area with the highest transmission intensity of all malaria cases and the area with the highest cases of P. knowlesi in Malaysia during 2013–2017, affected the pooled prevalence estimate or affected the risk of severe P. knowlesi infection in the meta-analysis. The analysis of the second and third outcomes was performed using the Review Manager 5.3 . The meta-regression analysis of a covariate, namely, malaria endemicity (slide positive rate), between severe P. knowlesi and non-severe P. knowlesi was performed using STATA Statistical Software version 15.0.The secondary outcome of the present study was to identify factors associated with severe P. knowlesi infection. The evaluation of publication bias among the included studies helped explore the underlying cause of data heterogeneity if it was present in the included studies.The funnel plot was used to demonstrate any symmetrical funnel plot among patients with severe n = 117), Scopus (n = 2139), and Web of Science (n = 126) databases  had severe complications. Most of the severe complications found in patients included severe acute kidney injury (AKI) , jaundice , and hyperparasitaemia . The number of patients with P. falciparum infections included a total of 394 patients in five studies [P. knowlesi and P. falciparum infections are shown in Table The characteristics of the seven studies included in this study are presented in Table  studies , 28, 29  studies , 24, 27, studies , 28, 29, studies , 24, a r studies , and a cP. knowlesi malaria was 19% , calculated by a random-effects model since high heterogeneity (Moran’s I2: 93.7%) was found  [CI: 25–40, I2 = 93.7%) [P. knowlesi malaria was reported in a study by Grigg et al.  [CI: 8–44%, I2 = 96.7%) was higher than that of the studies conducted in Sarawak .The pooled prevalence of severe  28–52%)  and by B= 93.7%) , while tI: 4–8%) . The subI2 analysis of age (I2 = 66%), leucocyte count (I2 = 54%), and parasitaemia level (I2 = 91%) between patients with severe and non-severe P. knowlesi infections demonstrated that medium and high heterogeneities were found among the included studies; therefore, the random-effects model was used to calculate the pooled MD along with 95% CI. In comparison to patients with non-severe P. knowlesi infections, the results demonstrated that patients with severe P. knowlesi infections were significantly older than patients with non-severe P. knowlesi infections   and those studies conducted in Sarawak . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the age difference .Cochrane Q and Moran’s 6%) Fig. . The subP. knowlesi infections had a significantly higher leucocyte count than patients with non-severe P. knowlesi infections   but not in the included studies conducted in Sabah . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the leucocyte count difference .Patients with severe P. knowlesi infections had a significantly higher parasitaemia level than patients with non-severe P. knowlesi infections  , and the subgroup analysis demonstrated that no significantly higher parasitaemia level was found in the included studies conducted in Sarawak  compared to the included studies conducted in Sabah . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the parasitaemia level difference .Patients with severe I2 analysis of platelet counts between patients with severe and non-severe P. knowlesi infections demonstrated a high level of heterogeneity (I2 = 89%); therefore, the random-effects model was used to calculate the pooled MD along with 95% CI. In comparison to patients with non-severe P. knowlesi infections, patients with severe P. knowlesi infections had a significantly lower platelet count  , and a significantly lower platelet count was found in both the included studies conducted in Sabah  and those studies conducted in Sarawak . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the platelet count difference .Cochrane Q and Moran’s I2 analysis of age (I2 = 92%), neutrophil count (I2 = 66%), and creatinine level (I2 = 89%) between patients with severe P. knowlesi and severe P. falciparum infections demonstrated that medium and high heterogeneities were found among the included studies; therefore, the random-effects model was used to calculate the pooled MD along with 95% CI. In comparison to patients with severe P. falciparum infections, patients with severe P. knowlesi infections were older    , neutrophil count , or creatinine level .Cochrane Q and Moran’s vel I2 = 9% betweeP. knowlesi was not different from those infected with P. falciparum    was higher than that of the studies conducted in Sarawak (10%). Previous studies reported that P. knowlesi, not P. falciparum or P. vivax, was the most common cause of malaria in Malaysia, particularly in Sabah and Sarawak [P. knowlesi malaria was observed in wild macaques (87%) and long-tailed macaques (50%) living in Sarawak, Malaysian Borneo [P. knowlesi was higher in Sarawak, the highest proportion of severe malaria caused by P. knowlesi was found in another state, Sabah. The result of our study was supported by the average mortality rate per 100 000 patients with P. knowlesi being higher in Sarawak (0.074) than in Sabah (0.064) [This study provided prevalence data and risk factors related to severe  Sarawak , 32. Anon Borneo , 33. Then Borneo . Our res (0.064) .P. knowlesi in wild long-tailed macaques (19%) was also reported from the island of Palawan, the Philippines [P. knowlesi parasites were also found in individuals living close to forested areas in Cambodia (0.59%) [P. malariae should be reported as P. knowlesi to increase recognition and be treated with intravenous artesunate for severe P. knowlesi malaria [P. knowlesi malaria management guidelines recommend an initial treatment with intravenous artesunate for any patients > 50 years of age or with a parasitaemia level > 20 000/μl [In addition to being endemic in Malaysia, the presence of lippines . In huma (0.59%) . A previ malaria . In Mala0 000/μl , 37.P. knowlesi malaria found in the present study were severe AKI, jaundice, and hyperparasitaemia. The results were similar to the largest study of P. knowlesi malaria conducted in three hospitals in Malaysia, where AKI was frequently observed [P. knowlesi in humans are not well understood. However, even a low parasite density from P. knowlesi infections can lead to severe diseases [P. knowlesi malaria, hyperparasitaemia has been defined as a parasite count of 100000 parasites/μl or higher [P. knowlesi in the human host is a 24-hour life cycle in which it rapidly reproduces at the asexual stage at a high parasitaemia level more than P. falciparum [P. knowlesi is still unknown; however, microvascular sequestration similar to that in P. falciparum infections might be the cause that plays a role in kidney injury [The most common complications of severe observed . The mecdiseases . In sever higher , 38. Thelciparum . For AKIy injury .P. knowlesi infections than younger patients, which was related to the low risk of anaemia and high risk of thrombocytopenia [P. knowlesi infected a wide range of ages from young teenagers to mature adults, which was in accordance with a study in Sarawak, Malaysia [P. knowlesi infections occurred more frequently among older patients was the strong correlation of increasing age with increasing parasitaemia [P. knowlesi infections [P. falciparum malaria, where endothelial activation increases with age [P. knowlesi infections among these age groups [P. knowlesi over a wide age range might be related to the presence of family clusters [P. knowlesi and P. falciparum infections. This demonstrated that patients who worked as farmers and plantation workers could be infected with either P. knowlesi or P. falciparum at the same proportion. However, a previous study indicated that P. knowlesi parasitaemia was significantly higher in farmers and plantation workers than in other occupations [P. knowlesi malaria but not for non-P. knowlesi malaria [P. knowlesi infection was related to human behaviours, such as higher forest exposure or working in agricultural areas [P knowlesi infection and that the risk of infection by P. knowlesi and non-P. knowlesi was comparable in males [P. knowlesi seropositivity in a study by Fornace et al., 2018, who conducted a study in northern Sabah, Malaysia, and Palawan, the Philippines [P. knowlesi infection were work-related, including Armed Forces and security personnel, boatmen and researchers, Department of Forestry officers, border patrol officers, and non-work-related, including peri-domestic transmission [The meta-analysis demonstrated that older patients had more severe complications from ytopenia , 28, 29.Malaysia . Two preMalaysia , 28. A pfections . This mewith age . In addie groups . A previclusters . The resupations . Moreove malaria . Previoual areas , 28. A pin males . In contlippines . The mossmission .P. knowlesi malaria, the present study demonstrated that patients with severe P. knowlesi malaria had a higher leucocyte count and parasitaemia level than those with non-severe P. knowlesi malaria. The subgroup analysis of study sites demonstrated that a significantly higher leucocyte count was found in the included studies conducted in Sawarak but not in the included studies conducted in Sabah. These results demonstrated that P. knowlesi patients in Sawarak had an immune response that was strongly correlated to P. knowlesi infection, as the leucocyte differential count and neutrophil counts were strongly correlated with P. knowlesi parasitaemia [P. knowlesi malaria had a parasitaemia level greater than 100 000 parasites/μl [P. knowlesi parasitaemia levels, suggesting that severe P. knowlesi malaria can lead to parasite-induced inflammation such as lung injury [P. knowlesi malaria than in those with non-severe P. knowlesi malaria. A previous study reported thrombocytopenia (84.9%) among patients with P. falciparum and P. vivax infections [P. knowlesi malaria demonstrated a decrease in coagulation system markers [P. knowlesi infection demonstrated in Sabah, Malaysia, were female sex, age ≥ 45 years, cardiovascular disease, microscopic misdiagnosis, and delay in administrating intravenous treatment [Comparing patients with severe and non-severe sitaemia . The ressites/μl . They alg injury . The prefections . The thrfections . A previ markers  or an in markers . The risreatment .P. knowlesi malaria were higher than those with P. falciparum malaria. This result supported the conclusion of a previous study in Sabah, Malaysia, which demonstrated severe P. knowlesi malaria at 29% with a greater than 3-fold risk of severity when compared to P. falciparum malaria [P. knowlesi and severe P. falciparum malaria, patients with severe P. knowlesi infections were older than patients with severe P. falciparum malaria. This result was similar to a previous study demonstrating that patients with P. knowlesi malaria were older than those with P. falciparum or P. vivax malaria [P. knowlesi infections and death have not been reported in children, unlike severe P. falciparum infections [The results of the present study demonstrated that severe complications in patients with  malaria . Compari malaria . Previoufections .P. knowlesi malaria had a higher neutrophil count than patients with severe P. falciparum malaria. These results were supported by a previous study that revealed that neutrophil counts strongly correlated with P. knowlesi parasitaemia [P. knowlesi parasitaemia and neutrophils involved the cytokines IL-1ra and IL-10, which were related to anti-inflammatory effects on the patient’s response to infection [P. knowlesi malaria might be due to a consistent correlation between high neutrophil counts and high parasite densities in P. falciparum-infected patients when compared to those with low and moderate parasitaemia groups [P. knowlesi than in non-severe P. knowlesi infections [P. knowlesi malaria had a higher creatinine level than those with severe P. falciparum malaria. These results demonstrated the association between a higher creatinine level and a higher proportion of severe AKI among patients with severe P. knowlesi malaria. As the risk of severity occurred in a high proportion compared to P. falciparum malaria in Malaysia, all patients recognized with severe malaria due to P. knowlesi were given intravenous artesunate as a first-line treatment upon admission to reduce the potential of mortality [The present study demonstrated that patients with severe sitaemia . They sunfection . A previnfection . A high a groups , 49. Prefections , 27. Theortality .P. knowlesi infections could be selected and extracted, so the pooled analysis needs to be carefully interpreted. Second, some clinical and laboratory data were missing among the seven included studies, causing a low statistical power to infer the differences between severe P. knowlesi and severe P. falciparum malaria. Third, subgroup analysis of study sites between Sabah and Sarawak in the meta-analysis of severe P. knowlesi and severe P. falciparum infections could not be performed, as three studies included in the analysis were conducted in Sabah and only one included study was conducted in Sarawak. Fourth, some confounding factors might have affected the haematological analysis of the present study, such as demographic factors, malaria immunity, underlying diseases, and the haemoglobinopathy of patients with malaria. Due to the limitations of the data on haemoglobinopathies and G6PD deficiency of the study populations reported in the included studies, the effect of haemoglobinopathies and G6PD deficiency on the severity or laboratory parameters between patients with severe P. knowlesi and non-severe P. knowlesi infections could not be examined in the present study.The present study had limitations. First, only a few articles on severe P. knowlesi infections. Patients with severe P. knowlesi malaria were older, had a higher leucocyte count, and had a lower platelet count than those with non-severe P. knowlesi malaria. Patients with severe P. knowlesi infections were older, had a higher neutrophil count, and had a higher creatinine level than those with severe P. falciparum infections. Future work must aim to explore the risk factors with a higher number of enrolled studies to aid in the understanding of severe P. knowlesi infections in malaria-endemic settings.This systematic review demonstrated a high rate of severe Additional file 1. PRISMA 2009 Checklist S1.Additional file 2: Table S1. Search term.Additional file 3: Table S2. Characteristic of severe and non-severe complications of Plasmodium knowlesi.Additional file 4: Table S3. Characteristic of severe Plasmodium knowlesi and P. falciparum.Additional file 5: Figure S1. The proportion of farmers infected with Plasmodium knowlesi and P. falciparum.Additional file 6: Figure S2. The proportion of plantation workers infected with Plasmodium knowlesi and P. falciparum.


The effects of CA and OCA feeding on maternal and fetal morphometry, bile acid and lipid levels, and cecal microbiota were investigated. OCA administration during gestation did not alter the maternal or fetal body weight or organ morphometry. OCA treatment during hypercholanemic pregnancy reduced bile acid levels in the fetal compartment. However, fetal dyslipidemia was not reversed, and OCA did not impact maternal bile acid levels or dyslipidemia. In conclusion, OCA administration during gestation had no apparent detrimental impact on maternal or fetal morphometry and improved fetal hypercholanemia. Because high serum bile acid concentrations in ICP are associated with increased rates of adverse fetal outcomes, further investigations into the potential use of OCA during cholestatic gestation are warranted.Intrahepatic cholestasis of pregnancy (ICP) is characterized by elevated maternal circulating bile acid levels and associated dyslipidemia. ICP leads to accumulation of bile acids in the fetal compartment, and the elevated bile acid concentrations are associated with an increased risk of adverse fetal outcomes. The farnesoid X receptor agonist obeticholic acid (OCA) is efficient in the treatment of cholestatic conditions such as primary biliary cholangitis. We hypothesized that OCA administration during hypercholanemic pregnancy will improve maternal and fetal bile acid and lipid profiles. Female C57BL/6J mice were fed either a normal chow diet, a 0.5% cholic acid (CA)-supplemented diet, a 0.03% OCA-supplemented diet, or a 0.5% CA + 0.03% OCA-supplemented diet for 1 wk before mating and throughout pregnancy until euthanization on NEW & NOTEWORTHY We used a mouse model of gestational hypercholanemia to investigate the use of obeticholic acid (OCA), a potent FXR agonist, as a treatment for the hypercholanemia of intrahepatic cholestasis of pregnancy (ICP). The results demonstrate that OCA can improve the fetal bile acid profile. This is relevant not only to women with ICP but also for women who become pregnant while receiving OCA treatment for other conditions such as primary biliary cholangitis and nonalcoholic steatohepatitis. Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic condition that affects 0.4–2.2% of pregnancies in North America and Western Europe but is more common in Chile and Bolivia, where it can affect 1.5–4% of pregnancies , 13, 44.The adverse fetal outcomes that occur in ICP include preterm birth, fetal hypoxia, meconium-stained amniotic fluid, stillbirth, and prolonged admission to the neonatal unit . MaternaIt has previously been described that during normal pregnancy the activity of farnesoid X receptor (FXR), the master nuclear receptor regulating bile acid homeostasis, is decreased, allowing for a maternal procholestatic profile even during normal gestation , 33, 39.In recent years, synthetic FXR agonists have been developed. In particular, the semisynthetic bile acid obeticholic acid (OCA) has more than 100 times higher affinity for FXR than its most potent natural ligand, chenodeoxycholic acid (CDCA), and has been shown to promote bile acid efflux and reduce bile acid synthesis . ClinicaIn this study, we used a previously established model of 0.5% cholic acid (CA) feeding in pregnancy to mimic the hypercholanemia of ICP , 41. BecSix- to eight-week-old C57BL/6J mice were purchased from Envigo and allowed to acclimatize for 1 wk before any experimental procedures were carried out. All mice were kept on a 12:12-h light-dark cycle with access to food and water ad libitum. All procedures were approved by the Animal Welfare and Ethical Review Body at King’s College London and carried out according to the UK Animals (Scientific Procedures) Act 1986. All diets were supplied by Special Diet Services.−1·day−1. Females were mated to control males and checked daily for the presence of a copulatory plug. The day of identification of the copulatory plug was considered day 1 of pregnancy (D1). Body weight of pregnant females was measured on days 7 (D7), 14 (D14), and 18 (D18) of pregnancy. On D18, females were fasted for 4 h and euthanized by CO2 inhalation. Maternal and fetal sera were collected, and pup number per litter was assessed. Maternal liver, subcutaneous white adipose tissue (sWAT), gonadal white adipose tissue (gWAT), brown adipose tissue (BAT), and fetal and placental weight were measured. Maternal liver, terminal ileum, fetal liver, and placenta were collected and snap-frozen. Nonpregnant control female mice were maintained on the same diets as pregnant females for an equivalent length of time and were assessed for the same parameters.We have previously shown that cholic acid (CA) feeding can induce maternal hypercholanemia in mice , 41. Femcyclophilin b was used as an internal reference for quantification of relative gene expression. Primer sequences of genes of interest are provided in Supplemental Table S1 .Total RNA was extracted from frozen tissue samples using the RNeasy Mini kit (Qiagen) according to the manufacturer’s guidance. After RNA extraction, 1 μg of total RNA was reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen). RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) was used as an RNase inhibition step. Assessment of the expression of target genes of interest was assessed using quantitative RT-PCR with a ViiA 7 Real Time PCR System (Thermo Fisher Scientific) by adding cDNA in duplicate to a 384-well plate, followed by a reaction mix of 1× SYBR Green Jumpstart Readymix (Sigma-Aldrich) and 1 μM of forward/reverse primers. The housekeeping gene Serum and tissue lipid content were extracted and measured as previously described . In brieMeasurements of serum and cecal bile acids were performed on an ultraperformance liquid chromatography Alliance 2695 system coupled to a Xevo TQ mass spectrometer using a SunFire C18 column, as previously described . Analytewww.mothur.org) was used for data analysis, following the MiSeq SOP Pipeline (www.arb-silva.de/) was used for sequence alignments, and sequences were classified according to the RDP database reference sequence files using the Wang method . Bacterial relative abundance was expressed as extended error bar plots using the Statistical Analysis of Metagenomic Profiles software package and analyzed by White’s nonparametric t test with Benjamini-Hochberg False Discovery Rate (FDR). The α-diversity  was calculated using Mothur, and Tukey’s Honestly Significant Difference test was performed using IBM SPSS Statistics Software version 23. P and q values of 0.05 were considered to be significant.Cecal samples were homogenized, and DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen) according to the manufacturers’ protocol. Sample libraries were prepared as previously described  using thPipeline . The Silg method . The Uni library  within tP ≤ 0.05. Statistical analysis of 16S rRNA gene sequencing data is detailed in the relevant section above.All values are shown as means ± SE. Statistical analysis was performed using GraphPad Prism 7 software. One-way ANOVA followed by a Newman-Keuls post hoc test was used, with a significance cutoff of We first aimed to establish the effect of hypercholanemia and OCA supplementation during pregnancy on body weight and organ morphometry. During pregnancy, no body weight differences were seen between groups, except on D7 when CA and CA + OCA-fed females were significantly lighter than OCA-fed females A. AlthouDespite the changes in maternal liver and gWAT morphometry in the CA and CA + OCA-fed groups, no changes in pup number, pup weight, or placental weight were registered C.Outside of pregnancy, both CA and CA + OCA nonpregnant females were lighter than NC- and OCA-supplemented females on D18 . This weight difference likely reflected a decrease in gWAT, sWAT, and BAT depot weight despite an increase in liver weight .These results demonstrate that OCA administration either alone or to hypercholanemic pregnant females did not negatively impact maternal or fetal body or organ morphometry.P > 0.05) and DCA (P ≤ 0.05) , taurocholic acid (TCA), and taurodeoxycholic acid (TDCA) compared with NC controls, confirming that CA-feeding induces maternal hypercholanemia, as has previously been described , 41. CA  ≤ 0.05) A.In nonpregnant females, total bile acids and DCA, TCA, and TDCA levels were significantly elevated by CA feeding and were not reduced by CA + OCA cofeeding .In the fetal compartment, maternal hypercholanemia led to a significant rise in fetal serum total bile acids B. HoweveOverall, OCA administration to hypercholanemic females did not significantly ameliorate maternal hypercholanemia but improved the fetal bile acid profile.A and B). Muricholic acids levels were markedly reduced in both CA and CA + OCA groups (B and C). OCA administration alone significantly reduced total cecal bile acid levels compared with all other groups, which was due to an overall reduction in bile acids (B and C).Cecal bile acid concentrations were also measured. As expected in the cecum, bile acids were largely unconjugated . Total cA groups , B and Cle acids A. Interele acids , B and CProteobacteria in the cecum of pregnant mice compared with NC groups . Alpha diversity (Shannon diversity index) plots showed that CA supplementation alone or cofed with OCA resulted in decreased bacterial diversity . Pregnancy caused a significant increase in an unclassified class of Bacteroidetes in NC controls . In nonpregnant mice, NMDS analysis and alpha diversity plots were similar to pregnant mice . However, changes between the dietary groups differed at phylum level; in particular, significant differences were observed in Bacteroidetes, Firmicutes, and Proteobacteria .Conversion of primary to secondary bile acids, as well as bile acid deconjugation, is performed by intestinal bacteria. Because changes in bile-metabolizing bacteria will affect the host bile acid pool, the cecal bacterial community was investigated by 16S rRNA gene sequencing. Nonmetric multidimensional scaling (NMDS) analysis of weights UniFrac distances, which shows how the microbial communities vary between the groups, demonstrates significant differences between all the dietary groups in pregnant mice B; specifC groups C. OCA fe females C. Signifr groups D. This wTo further assess the effects of hypercholanemia and OCA administration on bile acid homeostasis during pregnancy, the expression of key genes for bile acid homeostasis in the liver and terminal ileum was investigated.Shp was significantly upregulated in pregnant females fed a CA or a CA + OCA diet, and this change was concomitant with the repression of hepatic Cyp7a1 . Of note is that lower hepatic gene expression of several FXR targets was observed in pregnant mice compared with nonpregnant mice, regardless of diet . Expressregnancy . Shp exppregnant .Cyp7a1 expression.Overall, we conclude that despite decreased expression of FXR target genes during pregnancy, activation of intestinal rather than hepatic FXR can mediate OCA-induced suppression of hepatic Shp expression and a concomitant reduction in Cyp7a1 and Ntcp in the fetal liver  and Oatp1b2 , which suggests enhanced elimination of fetal bile acids via the placenta. Increased placental expression of MRP2 has previously been associated with reduced bile acids in the fetal compartment in ICP pregnancies following UDCA treatment , alanine transaminase (ALT), and alkaline phosphatase (AP) levels as well as hepatocyte size, mitosis, and necrosis  in the cecum of CA + OCA-fed females. We recently reported in pregnant mice that bile salt hydrolase, which was involved in deconjugation of bile acids, was exclusively detected in Bacteroidetes, with Proteobacteria also enriched in pregnancy, likely secondary to increased taurine made available after bile acid deconjugation  Imperial Biomedical Research Centre (BRC) based at Imperial College Healthcare National Health Service (NHS) Trust and Imperial College London. C.W. is an NIHR Senior Investigator and receives support from the NIHR BRC based at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London.The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. According to Wellcome Trust’s Policy on data, software and materials management and sharing, all data supporting this study are available on request.Intercept Pharmaceuticals provided the obeticholic acid used in the experiments described herein. L. Adorini and D. Shapiro are employees of Intercept Pharmaceuticals. H.-U. Marschall receives grants, personal fees, and nonfinancial support from Intercept Pharmaceuticals.V.P., G.P., and C.W. conceived and designed research; V.P., J.A.K.M., A.W., E.J., and H.-U.M. performed experiments; V.P., S.M., and J.A.K.M. analyzed data; V.P. and S.M. interpreted results of experiments; V.P., S.M., and J.A.K.M. prepared figures; V.P., S.M., and C.W. drafted manuscript; V.P., S.M., G.P., C.O., J.A.K.M., A.W., E.J., L.A., D.S., J.R.M., H.-U.M., and C.W. edited and revised manuscript; S.M. and C.W. approved final version of manuscript.


Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions . Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127  Ever-increasing levels of antimicrobial resistance (AMR) are considered a top global health concern and pose a “threat to the success and continuation of clinical medicine as we know it.” ,2. The igyrA, gyrB) and topoisomerase IV  genes. Mutations within the quinolone resistance determining regions (QRDRs) of these genes can occur spontaneously or with intentional application of selective pressure [Standard therapeutic guidelines for treatment of infections with Category A agents are well defined . Of partpressure ,6,7,8,9.Yersinia pestis strains isolated in Madagascar in the 1990s harbored plasmids with up to eight AMR determinants, at least four of which confer resistance to recommended therapeutics or prophylactic treatments [Y. pestis and other Category A agents, such as Bacillus anthracis and Francisella tularensis [Horizontally transferred resistance determinants also pose a significant threat. MDR eatments ,15,16,17eatments ,19. Eveneatments ,20,21. Flarensis ,24,25,26Bacillus spp., 23 Francisella spp., and 42 Yersinia spp. strains for both horizontally and vertically transferred AMR determinants; included amongst these strains were 46 Category A Select Agents. Microarrays were used for broad spectrum screening of all 127 strains for the presence/absence of 500+ AMR genes and gene families, while high resolution melt analysis (HRMA) was used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. To our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.In spite of the large number of publications describing the pathophysiology of these Category A agents, there still remains a dearth of information regarding the full spectrum and prevalence of AMR and their molecular mechanisms within these agents and their near neighbors. For this reason, we performed a survey of 62 Phenotypic antimicrobial susceptibility testing (AST) is still considered the gold standard for determination of resistance profiles. However, ASTs cannot be used to trace the spread of AMR strains nor the mobile genetic elements responsible for transmission of resistance. Phenotypic testing further cannot distinguish between various resistance mechanisms with overlapping specificities and are generally inadequate for epidemiological and forensic tracking. While not always predictive of phenotype, molecular tests can nonetheless determine the genetic basis and overall mechanisms for resistance, as well as the potential for transmission to other strains or species via horizontal gene transfer.gyrA, parC, and parE that confer resistance to fluoroquinolones used as first-line therapeutics. By combining the broad screening capability of microarrays with the fine discriminatory capabilities of HRMA, we detected markers acquired through both horizontal transfer and mutation. The dataset generated here provides a baseline measurement of the spectrum and prevalence of various resistance mechanisms in both Category A select agents and their near neighbors. These baseline data may be helpful in development of predictive algorithms designed to assess risk associated with outbreaks with naturally occurring or engineered threats, as well as in guiding development of new therapeutics.Here, we genetically characterized 127 archived Category A agents and their near neighbors with respect to AMR. To accomplish this, we determined the presence/absence of >500 AMR determinants using the ARDM v.3.1. In parallel, we used a combination of targeted PCR and HRMA to identify mutations in blaNDM), as well as those derived from several Select Agent species , their near neighbors, and more distantly related species in the same genera. In total, the ARDM v.3.1 content (4480 probes per two sub-arrays) covers 512 AMR determinants, conferring resistance to 19 categories of antimicrobials ; 6 genes from Yersinia spp. ; and 30 genes from a wide variety of Bacillus species .Based on the ARDM v.2  and v.3 crobials a. Microaia phyla b. PertinF. tularensis and three F. philomiragia strains were characterized on the ARDM v.3.1. Not surprisingly, all F. tularensis strains were positive for the six F. tularensis-derived genes and the three F. philomiragia strains were positive for the single F. philomiragia-derived gene, bla  and the other two F. philomiragia strains via BLAST. The bcr/cflA1 reference sequence had 91.6% sequence identity with Jensen O#319-029 while sequence identity with Jensen O#319L and Jensen O#319-036 was only ~65%. Similar BLAST comparisons showed ~80% or lower sequence identities between the three F. philomiragia strains and the other F. tularensis-derived genes on the microarray. Though not PCR-confirmed, these results support previous observations [Twenty ene, bla . One of rvations ,29 that Yersinia spp. strains was characterized on the ARDM v.3.1. Strains included 26 isolates from the closely related Y. pestis/Y. pseudotuberculosis group [Y. enterocolitica strains, and nine strains from six species related more distantly to both Y. pestis/Y. pseudotuberculosis and Y. enterocolitica. Not surprisingly, all of the Y. pestis (17 isolates) and Y. pseudotuberculosis (9 isolates) strains were positive for the same five Y. pestis-derived genes: bacA, qacE, emrA, emrB, and yegB. Importantly, Y. pestis KIM(pCD1Ap+) was also positive for the presence of blaTEM; KIM(pCD1Ap+) is a KIM5 strain harboring plasmid pCD1 into which blaTEM was inserted into the yadA gene  [Y. pestis.DNA from 42 is group , seven Y marker) . TherefoY. enterocolitica strains except strain 8081 were positive for the Y. enterocolitica-derived gene, vatF/sat , was also detected in a number of B. anthracis samples. The cat(pSCS5) gene has no identical allele in B. anthracis. However, six of the ARDM’s 10 probes for this gene have a 25-nt sequence with 92% identity to the B. anthracis-derived cat gene represented on the ARDM v.3.1 -positive calls therefore represent false-positive results. To minimize the potential for future false-positives, redesign of the ARDM—or more specifically, the group of cat(pSCS5) probes—is needed to improve specificity and eliminate redundant sequences.DNA preparations from 62 different strains from the ed genes . A plasmDM v.3.1 ; this laB. cereus and five B. thuringiensis were also positive for the same 16 genes observed in all of the B. anthracis strains. Of the remaining samples that did not harbor all 16 genes, the number of positive genes ranged from two (B. thuringiensis var. israelensis) to 15 genes (B. cereus D17 and 03BB108). Perhaps not surprisingly, the number of genes detected was generally correlated to the phylogenetic similarity between strains . The microarray was, however, able to detect a non-native AMR determinant (blaTEM) in a single Y. pestis strain (KIM (pCD1Ap)); this gene was engineered into a plasmid in the parent strain as a selectable marker in knock-out experiments. The ability to detect such a non-native gene in a Select Agent species—within <24-h and without a priori knowledge of its potential presence—illustrates the value of a broad-based screening method such as microarray analysis.In general, the microarray-based survey of AMR determinants did not reveal any significant surprises, with Select Agent species harboring AMR genes expected to be present. Similarly, near-neighbor species harbored the expected genes  combines the finely detailed discriminatory capabilities of sequence analysis with the rapidity, relevance, and user-friendliness of PCR. Originally developed for genotyping studies, HRMA determines the melting profile of double stranded DNA sequences using a DNA intercalating dye. The shape and position of the melting curve yield information about the composition of a given DNA sequence from which mutational data can be derived; substitution of a G/C with A/T will reduce the TgyrA, gyrB, parC, parE. Loveless [F. tularensis, Y. pestis, and B. anthracis. Here, we applied their strategy to assess sequence differences in 10 Francisella spp. strains , 34 Yersinia spp. strains (gyrA only), and 60 Bacillus spp. strains  to determine the practicality of this method for rapid screening.High level resistance to ciprofloxacin—a first- or second-line therapeutic for tularemia, plague, and anthrax—often arises from point mutations in the QRDRs of Loveless  previousgyrA and parC were assessed by HRMA in seven F. tularensis and three F. philomiragia strains using PCR primers designed to bracket mutations observed to result in ciprofloxacin resistance [gyrA assay, all F. tularensis strains clustered together .QRDRs in sistance ,34,35. Itogether ; these rparE assay were designed to detect a 5-bp deletion previously noted by Loveless [F. tularensis and F. philomiragia—clustered together, in spite of second Tm peak observed with F. philomiragia O#319-036. Clustering together of the F. tularensis and F. philomiragia strains was not unexpected, though the clustering was not as tight as observed with gyrA. Though the 5-bp region interrogated had a single nucleotide difference (T→A) not predicted to cause a change in Tm, there were multiple differences within the primer regions that might affect the Tms of half the amplicons in each PCR reaction , likely due to these changes in the primed regions.The HRMA primers used here for the Loveless . All of Y. pestis from mutations in the gyrA QRDR [parC or parE have not been documented. For this reason, only gyrA-specific HRMA assays were used with Yersinia spp. The site bracketed in the Y. pestis assay encompassed four mutations causing phenotypic ciprofloxacin resistance, with an additional two mutations found within the primer sequences [Multiple studies have reported ciprofloxacin resistance arising in yrA QRDR ,38,39,40equences ,38.Yersinia spp. strains, including 10 Y. pestis, eight Y. pseudotuberculosis, and seven Y. enterocolitica isolates. In many cases, two peaks (or a peak and a shoulder) were observed , Y. enterocolitica DATR and WA, Y. intermedia, Y. kristensenii, and Y. rohdei strains. A single Y. enterocolitica isolate (strain 8081) was an outlier.HRMA was performed on DNA from thirty-five observed ; Tms of  strains . A separYersinia spp. strains  were split evenly between clusters 1 and 2. The predictability in clustering could potentially be improved through analysis of additional replicates. Furthermore, inclusion of quinolone-resistant strains with documented QRDR mutations (to which we did not have access) would help demonstrate the true efficacy in using HRMA to detect both species-specific and sequence-specific changes predictive of phenotype. As is, based on the current knowledge of known point mutations causing fluoroquinolone resistance and the location of sequence differences, none of the tested strains would be predicted to be resistant. Indeed, for those strains for which phenotypic susceptibility has been determined , none are resistant [Based on the published sequences available for the  strains , the basB. anthracis strains. This sample set included DNA preparations from both 18 wild-type strains and 19 plasmid-cured strains selected for development of high-level ciprofloxacin resistance [gyrA and parC QRDRs could be detected.HRMA was performed on DNA preparations from 37 sistance  to assesgyrA, all B. anthracis wild-type strains produced Tms in the range of 75.9 °C to 76.2 °C, whereas Tms for B. anthracis gyrA mutants had Tms at least 0.5 °C lower . Interestingly, strain S3-16 has two mutations in gyrA (C254→T and A266→C) that should thermodynamically balance each other; Loveless previously observed that this strain clustered with wild-type B. anthracis [B. anthracis strains  specific control samples  support their exclusion from the wild-type cluster .Most of the rvations , two strand S1-2 ) clusterB. cereus and eight B. thuringiensis strains were also analyzed by HRMA to determine whether their gyrA and parC sequences were sufficiently similar to B. anthracis to allow robust amplification, for species discrimination, and for detection of QRDR mutants.Twelve B. anthracis (cluster 1) in gyrA assays  and B. cereus E33L. Although closely related to B. anthracis, [m. The published sequence for B. cereus 4342 shows two T→C substitutions within the interrogated sequence, which may explain its larger increase in Tm and formation of its own outlying cluster.Almost two-thirds (14/19) of the near neighbor strains clustered closely with wild-type A assays . Two add cluster . Clusterthracis, , E33L’s parC HRMA assays, roughly half of both B. cereus and B. thuringiensis strains clustered with wild-type B. anthracis controls (cluster 1) and most of the remainders formed a separate cluster (cluster 2). There were two outliers: B. cereus 03BB108 clustered with B. anthracis mutant S3-5 (cluster 3) and B. thuringiensis kurstaki formed its own cluster (cluster 4) . It is unclear, however, why B. cereus 03BB108 (predicted T→C substitution) produced an amplicon with lower Tm and did not fall within cluster 2. For this strain—and all of these near neighbors—it is possible that various substitutions within the primed regions affected the melt curves in ways that could not always be predicted.In uster 4) . Compariuster 4)  support Y. pestis or F. tularensis strains with known mutations causing ciprofloxacin resistance in this study. Therefore, we were unable to assess whether HRMA could be used to discriminate between wild-type and resistant strains within these two genera. Fortunately, we were clearly able to distinguish between wild-type and mutant strains of B. anthracis in a readily observed and relatively predictable fashion.We did not have access to parC amplifications, clustering of most near neighbor species for gyrA, parC, and parE in all three genera could be predicted through comparisons of the sequences targeted. F. tularensis could be easily discriminated from F. philomiragia based on gyrA, but not parE melt curves, potentially due to the short (5-nt) parE sequence targeted. Similarly, as expected, all Y. pestis and wild-type B. anthracis clustered with most, but not all, near neighbor strains having identical target sequences, though some anomalous clustering was observed . It is certainly possible that the unpredictable clustering may simply have been due to noisy data [Furthermore, in spite of numerous failures in isy data . More liFrancisella spp., Yersinia spp., and Bacillus spp. strains used in this study are shown in All genomic materials were obtained from Unified Culture Collection (UCC) of the Defense Biological Product Assurance Office (DBPAO), Frederick, MD USA and were provided as purified, certified inactive preparations previously extracted using Epicentre MasterPure DNA Purification kits . Illustra GenomiPhi HY kits  were used to amplify preparations of genomic DNA from each strain per the manufacturer’s instructions, using 10 ng of starting material. An amplification time of 90 min at 30 °C was used, followed by a 10 min enzyme inactivation step at 95 °C. After quantification using Qubit 2.0 fluorometer , amplicons were subjected a combined fragmentation/biotin-labeling step using the Bionick DNA-Labeling System (ThermoFisher); this method has proven superior to sequential fragmentation and biotinylation for subsequent microarray analysis on the ARDM . Each reThe ARDM v.3.1 has a total of four subarrays . The subarrays are based on two previous versions—ARDM v.2 (238 AMR determinants) ,53,54, at > 35 cycles or without sigmoidal amplification curves were called negative or invalid, respectively. Melt curves of valid amplifications were analyzed and clustering performed using ScreenClust software with unsupervised cluster analysis [The HRMA protocol, primers, and conditions used here were based on those described by Loveless  and are analysis .Both PCR and microarrays are valuable tools for the tracking the genetic underpinnings of AMR resistance. Here, we used two complementary technologies—microarray analysis and HRMA—to survey 127 Select Agents, exempt strains, and near-neighbor species for a broad variety of resistance mechanisms acquired through both horizontal transfer and gene mutations. To our knowledge, this is the largest survey of Category A agents, exempt strains, and near-neighbor species for genes covering multiple mechanisms of AMR.B. anthracis strains with point mutations within QRDRs of gyrA and parC, though several strains clustered anomalously . One limitation of HRMA is that each reaction must be designed for each target sequence and reactions are not easily multiplexable. Furthermore, changes in DNA sequence detectable by HRMA do not always represent a change in amino acid sequence, and near-neighbor species may be erroneously identified as resistant mutants.Both of the methods used here had benefits and limitations. The broad detection capabilities of the microarray analysis enabled detection of the genes that we expected to find within the Select Agent species for all three genera. We further detected a non-native gene within a Select Agent strain (KIM (pCD1Ap)+) and detected families of similar genes in near-neighbor species; detection of these related genes was highly dependent on the phylogenetic distance  between the near neighbor species and the species from which the reference sequence was derived. However, each microarray analysis is only as good as its content, and while its content is relatively comprehensive (500+ AMR genes), the ARDM v.3.1 is certainly not all-encompassing and is unable to detect small mutations that can affect specificity. HRMA, on the other hand, was able to rapidly discriminate between ciprofloxacin-resistant Provided suitable sensitivity can be achieved and data processing simplified, this is where rapidly evolving technologies such as next generation sequencing (NGS) may find tremendous application . Several


Paphiopedilum malipoense S.C.Chen & Z.H.Tsi is a new orchid species found in Malipo county, Yunnan. Here we report the complete chloroplast (cp) genome sequence and the features of P. malipoense. Its cp genome sequence of P. malipoense is 158,708 bp, including one large single-copy region , one small single-copy region , and two inverted repeat regions . The cp genome encoded 132 genes, of which 112 were unique genes . The phylogenetic relationships show that P. malipoense is sister with P. armeniacum-P. wardii. Paphiopedilum was established by Pfitzer (1886:11), and now ∼90 species are recognized within the genus, and 27 species are distributed in China. Some new species of this genus have been published (Chen and Tsi Paphiopedilum belongs to the subfamily Cypripedioideae (Orchidaceae), and distributes from tropical Asia to the Pacific islands, with some species extending to subtropical areas . Complete chloroplast genome sequence of P. malipoense was assembled in this study. Total genomic DNA was extracted from fresh material using the modified CTAB procedure of Doyle and Doyle  is 158,708 bp length and presented a typical quadripartite structure including one large single-copy region , one small single-copy region , and two inverted repeat regions . The cp genome encoded 132 genes, of which 112 were unique genes , The overall GC content was 36%.The cp genome sequence of P. malipoense, a molecular phylogenetic tree was constructed based on the maximum-likelihood (ML) methods with eight species from Paphiopedilum and two species as outgroup. The ML analysis was performed using the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE 8.2.10) with 1000 bootstrap replicates and settings as described by Stamatakis et al. (P. malipoense is mostly related taxa with Paphiopedilum armeniacum and Paphiopedilum wardii. (Paphiopedilum species.To confirm the phylogenetic position of s et al. . The res wardii. . This ne


Myzus persicae aphids by Aphidius gifuensis (Ashmead) on the entire two-sex life table and predation rates of Aphidoletes aphidimyza (Rondani). Our results showed that feeding on parasitized aphids did not influence the survival rates of immature A. aphidimyza individuals but significantly increased the development time of A. aphidimyza individuals and markedly reduced their longevity. The predation rate of immature A. aphidimyza individuals was also adversely affected by feeding on parasitized aphids. These results provide basic data for the potential use of A. aphidimyza in combination with A. gifuensis in M. persicae control programs.Intraguild predation is a common phenomenon between predators and parasitoids. Despite numerous studies on the performance of intraguild predators by consuming on intraguild prey, the entire two-sex life table and predation rates of intraguild predators fed on intraguild prey remain poorly known. In this study, we investigated the effect of parasitized Aphidoletes aphidimyza (Rondani) to compare when its populations fed on aphids (Myzus persicae Sulzer) (Hemiptera: Aphididae) that were either unparasitized or parasitized by Aphidius gifuensis (Ashmead) (Hymenoptera: Braconidae). Our results showed that individuals of A. aphidimyza were capable of completing their development and attaining maturity when they fed on parasitized aphids. Although feeding on parasitized aphids did not influence the survival rates of immature A. aphidimyza, it did significantly slow their development and extended their longevity, thereby reducing the fecundity and predation rates of A. aphidimyza. These findings may be pivotal for better understanding the sustained coexistence of predators with parasitoids in the biological control of aphids.Intraguild predation interactions have substantial theoretical and practical implications for the dynamics of natural competitor populations used for biological control. Intraguild predation on parasitized aphids not only has a direct, negative effect on the parasitoid species, but it may indirectly influence the predator’s development, survival, reproduction and predation rates. In this study, we used two-sex life table theory, life table parameters and predation rates of  Micromus variegatus (Fabricius) and lady beetle Coccinella septempunctata L. prefers to consume parasitized aphids over unparasitized aphids [M. variegatus and Aphidius ervi Haliday did not result in intraguild predator densities and shared prey compared with intraguild predator alone [Intraguild predation (IGP) arises when natural competitors of a shared resource also engage in predation or parasitism in food webs of prey and their natural enemies at their same trophic level ,2. A comd aphids ,5. Howevd aphids ,7,8,9,10or alone . The simor alone ,12. Yet,Myzus persicae (Sulzer) (Hemiptera: Aphididae), is the most devastating pest aphid worldwide, capable of attacking more than 400 plant species, including key Solanaceae, Cruciferae, and Leguminosae crops [Myzus persicae can directly extract sap from young leaves and excretes honeydew, which contaminates foliage and fruit and induces sooty mold [Aphidoletes aphidimyza (Rondani), aphidophagous gall midge that can attack more than 80 aphid species [A. aphidimyza can kill 4–50 M. persicae nymphs per day [A. aphidimyza pupae in a predator:aphid ratio (1:10 and 1:15) causes exceeded 95% loss rates of M. persicae nymphs on tobacco in the field [A. aphidimyza on aphids appears high when implemented with parasitoids [Aphidius gifuensis (Ashmead) (Hymenoptera: Braconidae), has been extensively used as a biocontrol agent against M. persicae in China [A. gifuensis has been successfully mass-reared and incrementally released to control M. persicae populations in fields of tobacco and vegetables [A. gifuensis and A. aphidimyza often co-occur in host-crop fields, and the latter can easily replace colonies of A. gifuensis on tobacco in greenhouses in Leshan Town of Zunyi City in Guizhou Province, China . The IGP interaction between this predatory midge and parasitoid is unidirectional, with A. aphidimyza killing parasitoids [Aphidoletes aphidimyza ate aphids parasitized by Aphidius colemani Viereck and thereby reduced the production of parasitoids [A. aphidimyza populations.The green peach aphid, ae crops ,14. Thisae crops ,16. Myzuoty mold . Aphidol species . Third i per day . Releasihe field . The conin China ,22,23. Nasitoids . Aphidolasitoids . Intraguasitoids . HoweverLife table analysis can provide comprehensive information on population growth rates, and life table parameters are important tools for comparing the fitness of populations and understanding the effects of external factors on fitness  ,27. Chi A. aphidimyza has a negative preference for parasitoid mummies [A. aphidimyza’s survival, development, reproduction, and predation rate. Therefore, in this study, we chose young parasitized aphids to understand their effects on life history and rate of predation by A. aphidimyza to provide information for the simultaneous application of two biocontrol agents in the greenhouse.Previous studies showed that  mummies . We hypoM. persicae aphids were collected from tobacco (Nicotiana tabacum) leaves at the Natural Enemy Breeding Center, in Jinhua Town of Fenggang County , on 1 May 2017. The reared colonies were kept on tobacco (“K326”) plants in mesh cages (45 cm × 45 cm × 30 cm) in the greenhouse belonging to the Institute of Entomology, Guizhou University.The A. gifuensis parasitoids were originally obtained from M. persicae in a tobacco farm located in Leshan Town , and then, in a greenhouse, reared on M. persicae as a host for more than five generations. We transferred more than 400 M. persicae onto tobacco plants enclosed by synthetic fine-nylon mesh cages, after which we introduced 30–40 pairs of A. gifuensis adults into each cage at 25 °C ± 1 °C; 24 h later, we removed these adults from the mesh cages, and young parasitized fourth instar M. persicae nymphs were prepared for experiments 2 days later. The young parasitized aphids moved very slowly, and their abdomen backs were lightly colored. The percentage of parasitized aphids exceeded over 98% in the experiment.The A. aphidimyza larval predators from the same location as A. gifuensis. Aphidoletes aphidimyza was reared with M. persicae aphids on tobacco for three generations in an artificial climate chamber. Thirty pairs of A. aphidimyza adults were allowed to oviposit on fresh and healthy tobacco plants with enough M. persicae aphids for one day in a mesh cage (45 cm × 45 cm × 30 cm). After all A. aphidimyza adults were removed from the plant, A. aphidimyza eggs were prepared for the experiment. All experiments were conducted in an artificial climate chamber at 25 °C ± 1 °C, 65% ± 5% relative humidity (RH), with a 14-L:10-D photoperiod.We obtained the M. persicae on life-history parameters and predation rates of A. aphidimyza, we maintained a total of 52 freshly laid eggs (<12 h) by A. aphidimyza females in modified Petri dishes (35 mm-diameter) as described above, for each treatment (parasitized vs. unparasitized aphids as food). We recorded the incubation rate of eggs daily until the larvae hatched.To evaluate the effect of parasitized A. aphidimyza on M. persicae [M. persicae. We observed the larvae every day and determined developmental time and survival rate. We also counted the number of unparasitized and parasitized M. persicae consumed by A. aphidimyza daily before pupation. When A. aphidimyza reached the third instar stage of development, we placed balls of moist cotton on each Petri dish; mature larvae were able to access this moist absorbent cotton for their pupation. We sprayed distilled water daily onto cotton balls where the pupae were developing to keep the cotton moist until the emergence of adults. Newly emerged male and female A. aphidimyza were paired and moved into an artificial fine-nylon mesh cage (45 cm × 45 cm× 30 cm), which contained a tobacco seedling with 120 unparasitized or parasitized fourth instar M. persicae nymphs. New tobacco seedlings with infected aphids (as described above) and absorbent cotton soaked in a 10% honey solution were provided daily for these A. aphidimyza adults. We recorded the preoviposition period, oviposition period, fecundity, and longevity of A. aphidimyza individuals until all of them had died.Based on a previous study of the predation ability of persicae , each haxjS), female age-specific fecundity (x6f), age-specific maternity (xmxl), age-specific survival rate (xl) and age-specific fecundity of the total population (xm) of A. aphidimyza were calculated using the program TWOSEX-MSChart [A. aphidimyza, as well its predation rates, were estimated with 10,000 bootstrap replicates. A paired bootstrap test was used to calculate life table parameters of A. aphidimyza when it fed on unparasitized or parasitized M. persicae [xjc), age-specific predation rate (xk), age-specific net predation rate (xq), net predation rate (C0), stable predation rate (ψ), finite predation rate (λ) and transformation rate (pQ) of A. aphidimyza were analyzed according to the CONSUME-MSChart program [A. aphidimyza reared on parasitized or unparasitized M. persicae. We used Sigma Plot v12.5 to construct the curves for A. aphidimyza’s survival rates, reproductive value, and predation rates.For the age-staged life table, age-stage specific survival rate  of individual immature stages of A. aphidimyza overlapped between unparasitized and parasitized M. persicae (xl) of A. aphidimyza showed a similar trend when it fed on unparasitized and parasitized M. persicae  of A. aphidimyza feeding on parasitized and unparasitized M. persicae. The total preoviposition period (TPOP) of A. aphidimyza was significantly longer for those that fed on parasitized than unparasitized M. persicae. Recorded oviposition periods were longer, and fecundity was markedly higher of A. aphidimyza that fed on unparasitized M. persicae than the A. aphidimyza that consumed parasitized M. persicae (x6 =f 41.58) and maximal age-specific maternity (xmx =l 19.19) of A. aphidimyza occurred at 16 days of age when feeding on unparasitized M. persicae; however, age-specific fecundity showed a later maximal peak (xm), at 18 days, when parasitized M. persicae were the prey (Feeding on ongevity . Third ipersicae . The maxthe prey .R0), intrinsic rate of natural increase (mr), and finite rate of increase (λ) of A. aphidimyza was similar between predator populations feeding on unparasitized and parasitized M. persicae. Nevertheless, the mean generation time (T) of A. aphidimyza was significantly higher when it fed on parasitized than unparasitized M. persicae aphids  of A. aphidimyza was strongly affected by parasitized M. persicae as prey, especially in the second and third larval stages (xjC) of A. aphidimyza occurred at the age of 4 d when the predator fed on unparasitized M. persicae. Age-specific predation rate (xk) and age-specific net predation rate (qx) curves of A. aphidimyza were both higher when unparasitized aphids were eaten instead of parasitized ones , stable predation rate (ψ), and finite predation rate (λ) when compared with its feeding on unparasitized M. persicae. However, eating parasitized M. persicae did not affect the transformation rate (pQ) of A. aphidimyza  for A. aphidimyza that fed on parasitized aphids (A. aphidimyza individuals feeding on unparasitized aphids also featured higher predation ability with those feeding on parasitized aphids at 56 days (To evaluate the effect of parasitized f aphids . The totd aphids a. Moreov 56 days b. Since A. aphidimyza individuals, but it did significantly delay their overall developmental time and also reduced the predator’s longevity, fecundity and predation rates. To our best knowledge, this study is the first to have comprehensively evaluated the effect of parasitized aphids on the entire life history and predation rates of aphid predators.Consumption of parasitized aphids and mummies could adversely affect the development, body size, survival, and behavior of aphid predators ,10,34,35A. aphidimyza were not significantly influenced by feeding on parasitized M. persicae, and over 90% of the predator egg survived to adult emergence. Similar results were reported for Harmonia axyridis and Propylea japonica when each fed on Aphid craccivora parasitized and mummified by A. colemani [Coccinella undecimpunctata that fed on “injured” M. persicae mummies parasitized by A. colemani [C. septempunctata or H. convergens into adulthood [The survival rates of immature colemani , and alscolemani . Comparedulthood ,35.A. aphidimyza that fed on parasitized M. persicae were longer than those of predator larvae feeding on unparasitized M. persicae. Our results are consistent with studies of C. septempunctata, H. convergens, H. axyridis, and P. japonica that subsisted on mummified aphids, demonstrating that predators could slow their ontogenetic development [A. aphidimyza revealed that its longevity, oviposition period, and fecundity were all significantly affected by its feeding on parasitized aphids. Little research has been conducted on the effects of parasitized aphid species on the reproduction parameters of their predators. When fed A. gifuensis mummies, aphid enemy H. axyridis females had a longer pre-oviposition period and did not lay eggs within 30 days of observation [Episyrphus balteatus females was affected by mummified aphids and the exuvia of mummies of Acyrthosiphon pisum, but the number of eggs laid by E. balteatus was nonetheless similar when the female fed with healthy aphids and parasitized aphids [E. balteatus females laid significantly fewer eggs on mummified M. persicae colonies parasitized by A. colemani than they do on colonies with unparasitized aphids [Development times for each stage and total immature stage of elopment ,10,34. Od aphids . Under ad aphids . Mummiesd aphids . Furtherd aphids . Compared aphids ,10. The H. axyridis try to consume more unparasitized aphids than mummies parasitized by A. asychis in 1 h and 3 h [A. craccivora, the larvae of H. axyridis, P. japonica and C. septempunctata consume less parasitized and mummified aphids [A. aphidimyza, leading to a weaker predation ability than for those feeding on unparasitized aphids across population projections.The predation potential of aphid predators is markedly influenced when they feed on parasitized aphids ,7. Fourt and 3 h . Compared aphids . HoweverA. aphidimyza could complete its development if feeding solely on parasitized aphids, but it had a longer larval stage development and shorter longevity, longer preoviposition period, and lower reproduction than when feeding on unparasitized aphids. The completed development and reproduction of the predator when feeding on unparasitized and parasitized aphids are both pivotal for maintaining its coexistence with the parasitoid and effective suppression of the aphid in the ecosystem.In conclusion,