Placental chorioangioma is a rare disorder in pregnancy. We retrospectively reviewed the perinatal complications and long-term outcomes in pregnancies with placental chorioangioma and evaluated the factors affecting disease prognosis.We reviewed pregnant women who delivered at our hospital in the past decade and whose diagnosis of placental chorioangioma was confirmed by pathological diagnosis. Information on maternal demographics, prenatal sonographic findings and perinatal outcomes was obtained by reviewing the medical records. In the latter part of the study, follow-up of children was conducted by phone interview.In the 10 years from August 2008 to December 2018, 175 cases(0.17%) were identified as placental chorioangioma histologically and 44(0.04%) of them were large chorioangiomas. Nearly one-third of cases with large chorioangiomas were associated with severe maternal and fetal complications or required prenatal intervention. Although one-fifth of fetuses/newborns complicated with large chorioangiomas were lost perinatally, the long-term prognosis for surviving fetuses was generally good. Further statistical analysis revealed that tumor size and location affect prognosis.Placental chorioangioma may cause an unfavorable perinatal outcome. Regular ultrasound monitoring can provide the tumor characteristics which can be referred to for predicting the tendency of those complications and indicate when intervention may be necessary. It is not clear which factors lead to complications with fetal damage as the main manifestation or polyhydramnios as the main manifestation. Placental chorioangioma is the most common nontrophoblastic tumor of the placenta, occurring in 0.41-1.4% of examined placentas by microscopy . Most ofGray-scale sonography, color flow Doppler ultrasonography, and magnetic resonance imaging(MRI) can be used to diagnose placental chorioangioma prenatally. Ultrasound findings of chorioangioma commonly reveal a hypo -or hyperechoic well-circumscribed placental mass. Necrosis, degeneration, or calcification of the tumor may result in different ultrasonic echotexture. Application of color flow Doppler can confirm the vascular component of placental masses and differentiate chorioangiomas from other placental lesions. Placental chorioangioma appears as a heterogeneous mass on MRI, which can be used in conjunction with ultrasound findings .Many pregnancies with small placental chorioangiomas were asymptomatic and do not complicate the course of the pregnancy. Large placental chorioangiomas, by contrast, tend to be associated with perinatal complications and adverse fetal outcomes, such as polyhydramnios, preterm labor, fetal anemia, fetal growth restriction (FGR), fetal cardiomegaly or fetal heart failure, fetal hydrops, maternal mirror syndrome and even fetal demise . HoweverA retrospective study was designed from 102,399 pregnant women who were admitted to the Department of Obstetrics, West China Second University Hospital from Aug. 2008 to Dec. 2018. Specimens of 8704 placentas with 10% formalin fixation and their chips with paraffin embedding were cut into 4–5 μm thick tissue slices and stained with Hematoxylin-Eosin staining. By searching pathological results “= placental chorioangioma ” in the hospital electronic patient records database, 175 cases were identified histologically and 44 of them were large chorioangiomas. The medical records, as well as a series of color Doppler ultrasound examinations, of all the included cases, were reviewed for further analysis.The following data were retrieved from the pathological results and pathological pictures: the size of the tumor, and the distance from the tumor margin to the umbilical cord insertion point. The following data were retrieved from the series of color Doppler ultrasound examinations: the gestational age (GA) at first diagnosis, the size of the tumor, the blood supply of the tumor, amniotic fluid amount, middle cerebral artery peak systolic velocity(MCA-PSV), umbilical artery(UA) Doppler, presence of fetal effusions, heart size, fetal cardiothoracic ratio, cardiovascular profile score (CVPS). Maternal demographics and perinatal outcome were retrieved from the medical records: maternal age, gravidity, parity, complication, GA at delivery, mode of delivery, Apgar score of the newborn, birthweight, and the volume of postpartum hemorrhage.Primary maternal complications included polyhydramnios (defined as amniotic fluid depth(AFD) > 8 cm or amniotic fluid index(AFI) > 25 cm on ultrasound), preterm premature rupture of membrane , mirror syndrome , hypertensive disorders in pregnancy and postpartum hemorrhage (defined as the amount of blood loss exceed 500mL after delivery). Primary perinatal complications included preterm birth(PTB), fetal anemia , fetal growth restriction or small for gestational age(SGA) at birth, fetal cardiomegaly or fetal heart failure (CVPS < 7), fetal hydrops , fetal distress, intrauterine death (IUD) or neonatal death (NND).At the end of 2020, phone interviews were conducted with 36 cases (43 live newborns) of large chorioangiomas. Information on mental development and psychomotor development, as well as developmental complications, were collected based on the parents` description. Three patients were lost to follow-up.The prevalence of placental chorioangioma in our centre is 0.17% . The result may have been underestimated because most of the microtumors were diagnosed by accident after biopsy, by inference, similar lesions may also exist in placentas not sent for pathological examination. However, large chorioangioma was difficult to miss in the general examination, and the prevalence of large chorioangioma  seems reliable.case 1 to case 44 in descending order of tumor size). The largest tumor (case 1) was 15 × 12 × 12 cm.Most of placental chorioangiomas 102/177) were microscopically small (< 1 cm) and discovered only during histological inspection of the placenta, while in 29 cases were 1-3.5 cm in size, and in 44 cases the maximum diameter of tumors were larger than 4 cm weeks.case 23, the tumor was pedunculated, that is, protruding into the amniotic cavity and connected to the placenta by a slender vascular pedicle of the large chorioangiomas were paracentral located in the placenta parenchyma; 38.6%(17/44) were localized on the outer edge. In 6/44 of t84.1%(37/44) occurred in singletons, and 15.9% (7/44) occurred in twin pregnancies. The 44 patients were between the age of 20 and 40, with a median age of 29.6 y/o. Most of the patients  were under 35 years old, while eight patients were in the 35 to 40 age bracket. Only 6 patients had previous childbirth history.case 21) had a high fever and the possibility of intrauterine infection. Only one case(case 14) of preeclampsia was found.Severe maternal and fetal complications occurred in 15 cases. Details were shown in Table case 1, the patient was referred at 32 + 3 weeks because of threatened premature labor and maternal mirror syndrome, which was characterized as hypoxemia (lowest oxygen saturation of 72%), cyanosis, edema, abdominal distention, proteinuria, hypoproteinemia, moist rales of both lungs, and patchy opacity of lower lungs on X-ray scan. At referral, the fetus had nonimmune hydrops, thickened placenta(7.6 cm), and polyhydramnios. Given the severe maternal situation, none of the tocolytic agents was used, and after the demand of parents, craniotomy during labor proceeded because of the possible poor prognosis of the newborn. 24 h after the delivery, the maternal symptoms were obviously relieved. In case 4, lethal induction of labor after the demand of parents was also operated on account of the fetal cardiomegaly, fetal pericardia effusion, and polyhydramnios. In case 28, the fetus was the only one who underwent amniotic fluid culture for whole-exome sequencing prenatal diagnosis in 44 cases of large chorioangiomas, for concomitant complex congenital heart disease(aortic coarctation and hypertrophic cardiomyopathy). Lethal induction of labor was also demanded because of RAF-1 gene mutation to indicate the possibility of Leopard spot syndrome and Noonan syndrome. Because of central placenta praevia with placental implantation, the bleeding during delivery was about 1330 ml, and this was the only case of postpartum hemorrhage in the large placental chorioangioma group. In case 5, the patient was admitted for an increased MCA-PSV value of 2.13MoM. Chorioangioma radiofrequency ablation was planned. Sadly, fetal demise happened before the scheduled surgery. In case 6, despite the timely use of ritodrine hydrochloride, premature breech delivery still occurred in 10 h after PPROM, which was associated with an intrapartum stillbirth. Similarly, in case 15, stillbirth during breech delivery was also recorded despite the use of ritodrine hydrochloride and amnioreduction against the AFD of 15.2 cm. In case 24 and case 42, fetal demise happened in the late third trimester. Chorioangiomas failed to be detected by ultrasound in the two cases so ultrasonic monitoring was not so frequent. Before fetal demise, ultrasound was not taken for more than 4 weeks in case 24 and more than 6 weeks in case 42, which makes the analysis of maternal and fetal complications difficult.9 fetal/neonatal losses of large placental chorioangiomas were observed in our retrospective study, including 3 lethal induction of labor, 5 IUDs, and 1 NNDs. In case 21, the baby was delivered at 29 + 3 week`s gestation, suffering from neonate respiratory distress syndrome, neonatal shock, neonatal sepsis, severe neonatal anemia(hemoglobin 85 g/l), metabolic acidosis and dysfunction of coagulation that led to the newborn death. Other severe neonatal complications included neonatal intracranial hemorrhage(NIH), hydrocephalus, bronchopulmonary dysplasia(BPD), and renal function damage.The median GA at delivery for the other 36 cases with alive newborns was 37  weeks. Preterm deliveries before 37 weeks, 34 weeks, and 32 weeks were 30.6%(11 cases), 13.9%(5 cases), and 11.1%(4 cases), respectively. Neonatal intensive care unit (NICU) Admission of newborns was 23.3%.In case 8, case 10, and case 13) failed to follow up. The most common newborn complication was neonatal benign hemangiomatosis, with five cases affected but different prognoses. Case 33 developed a single 4 cm-in-diameter hemangioma on the head skin, which had neither expanded nor shrunk at the follow-up time when the child was 15 months old. Likewise in case 22, hemangioma on the bottom of newborn feet remained when the child grew to 18 months old. By contrast, In case 35, a coin-size hemangioma on the baby`s head skin gradually disappeared within 1 year. Similar trend was observable in case 18, hemangioma on the right thigh vanished quickly after discharge from NICU. And in case 34, most of the 16 scattered hemangiomas disappeared within 1 year, finally leaving a 5 mm-in-diameter hemangioma on the child`s left palmar skin, and the size of the primary lesion was substantially decreased. No intrahepatic lesions were observed in all newborns in our research. Fetuses of case 2 and case 3 were complicated with cardiomegaly. Termination of pregnancy was decided for the former based on the negative normal stress test and larger gestational weeks. Case 3, who suffer from fetal cardiomegaly since the early stage of 3rd trimester, received maternal transplacental administration of digoxin at 32 + 3 week`s gestation. Echocardiography of newborns of the 2 cases manifested cardiac cavity with normal size before discharge from NICU. None of the children complained of cardiac symptoms at 2–3 years follow-up evaluation. All 3 premature infants with NIH received rehabilitation training. The baby of case 3 grew and developed well at 2 years follow-up. In case 11, hydrocephalus secondary to gradeII NIH and infant fine motor delay was observed in the premature baby. In case 18 with grade IV NIH, who had a regular follow-up in the children’s rehabilitation department in our hospital, normal language development and slight claudication of left lower extremity at 2 years follow-up was observed. The motor development of the upper limb, right lower limb, and fingers was good. Delay of gross motor function development and short stature occurred in case 29 with normal pregnancy process.Nearly all alive babies had good outcomes. The follow-up period ranged from 1 to 12 years, 3 cases was adopted with the application of the statistical package of SPSS 26.0 to carry out data processing analysis. As shown in Tables A retrospective review of 102 cases of small chorioangioma (< 1 cm) and 29 cases of medium chorioangiomas (1-3.5 cm) was also performed. 2 fetal/neonatal losses were observed in the small tumor group, due to twin-twin transfusion syndrome and early onset severe preeclampsia. One fetal demise occurred at 38 + 3 gestational weeks with a tumor size of 2 cm, and umbilical vein thrombosis was confirmed by pathological examination. The median GA at delivery was 35 weeks in the small chorioangioma group and 36 weeks in the medium tumor group, respectively.According to previous literature, large chorioangioma is associated with a high rate and variety of perinatal complications. Zanardini et al. surveyed 19 cases of large placental chorioangioma, in which the natural history, intrauterine treatment, and outcome of pregnancy were evaluated. Two-thirds developed complications that required either elective delivery for fetal growth restriction or intervention for tumor-related effects . Dong etBut we also found, in previous literature and our study, partial cases had a benign process. What are the key factors in the prognosis? In a systematic review and meta-analysis including twenty-eight studies and 161 pregnancies, the size of the mass and presence of fetal hydrops were found to be the main determinants of perinatal outcome in pregnancies complicated by chorioangioma . In our Based on the concept of blood shunt, some researchers hypothesized that the vascularity of the tumor may be another independent factor of fetal complications regardless of the tumor size. Avascular chorioangiomas with less blood supply may have little effect on fetal blood circulation . It suggSince the overall blood supply of the tumor is not easy to assess, some indirectly evaluating indicator was assumed to reflect chorioangioma vascularity. Increasing umbilical cord-tumor distance lead to less blood flow and smaller vessel diameter, so the fetus will be less affected by the chorioangioma. Sepulveda et al. reported 3 cases of large placental chorioangiomas managed with endoscopic laser coagulation of the feeding vessels; in two cases with poor pregnancy outcomes, the chorioangioma was close to the umbilical cord insertion and the feeding vessels were voluminous . Similarcase 23, although the size of the tumor was not particularly large and the tumor was far enough away from the umbilical cord, complications still occurred. It is speculated that the pedunculated tumor may have a large contact area with the amniotic cavity, which caused more transudation of fluid and eventually excessive amniotic fluid.However, it can still be found that some cases may not follow the above rules. As in Complications can manifest in various forms and, in general, fall into two categories, one with fetal intrauterine damage as the main manifestation, including fetal cardiomegaly, hydrops, anemia, distress, etc. In the other group of patients, no intrauterine fetal damage is found, but only polyhydramnios, PPROM, and PTB are the main manifestations, and the effects in children often come from the PTB itself. It is unclear which factors determine the exact form of complications and more research are needed.Treatment of complications depends on gestational age and mater-fetal symptoms. Termination of pregnancy should be considered for worsening maternal condition, or late-onset complications with viable fetuses. If severe complications occur before the fetus is viable, prenatal intervention should be carried out to extend the gestation, which is classified as supportive and definitive treatment. The choice of supportive treatment is based on the specific type of complication, such as intrauterine transfusions for anemic fetuses and hydropic fetuses, amnioreduction and oral indomethacin for polyhydramnios with respiratory embarrassment and/or preterm labor, transplacental digoxin intervention for fetal cardiomegaly. Definitive treatment refers to the treatments to block arteriovenous shunting including alcohol injection, microcoil embolization, endoscopic laser coagulation, radiofrequency ablation, and interstitial laser therapy. In this study, intrauterine supportive treatment was performed in a limited number of cases with mostly satisfactory outcomes. Unfortunately, no cases in this study underwent definitive treatment. One patient was scheduled for radiofrequency ablation for fetal anemia. However, the fetus died preoperatively due to the rapid progression of the disease. To date, there is still no consensus on how to choose the appropriate treatment for a given complication. More research is needed on whether to choose supportive or definitive treatment, and whether to choose a single treatment modality or combined therapy.To our knowledge, this study is one of the research with the largest number of cases and longest period of follow-up of large placental chorioangioma. Using this advantage, we can better understand the full spectrum of the illness and its complications. Nearly one-third with large placental chorioangioma suffer from severe perinatal complications. Regular ultrasound monitoring can provide the tumor characteristics which can be referred to for predicting the tendency of those complications and indicate when intervention may be necessary.


Pseudomonas aeruginosa. Further, the strength of this phenotype is higher when nfxB mutants, over-producing the efflux pump MexCD-OprJ, are selected. Here, we induce transient nfxB-mediated ciprofloxacin resistance by using the antiseptic dequalinium chloride. Notably, non-inherited induction of AR renders transient tobramycin CS in the analyzed antibiotic-resistant mutants and clinical isolates, including tobramycin-resistant isolates. Further, by combining tobramycin with dequalinium chloride we drive these strains to extinction. Our results support that transient CS could allow the design of new evolutionary strategies to tackle antibiotic-resistant infections, avoiding the acquisition of AR mutations on which inherited CS depends.Collateral sensitivity (CS) is an evolutionary trade-off traditionally linked to the mutational acquisition of antibiotic resistance (AR). However, AR can be temporally induced, and the possibility that this causes transient, non-inherited CS, has not been addressed. Mutational acquisition of ciprofloxacin resistance leads to robust CS to tobramycin in pre-existing antibiotic-resistant mutants of  Pseudomonas aeruginosa to investigate transient collateral sensitivity to tobramycin.In this work, the authors induce ciprofloxacin resistance in clinical isolates of  Pseudomonas aeruginosa stands out2. This bacterium is an opportunistic pathogen causative of nosocomial infections, as well as of chronic infections in patients suffering from chronic obstructive pulmonary disease or cystic fibrosis5. P. aeruginosa presents a low intrinsic susceptibility to different antibiotics7 and, in addition, it can increase its level of resistance through the acquisition of mutational changes9. Among the different AR determinants of P. aeruginosa, multidrug efflux pumps should be highlighted, since they contribute to intrinsic, acquired, and transient AR13.Antibiotic resistance (AR) of bacterial pathogens constitutes a major threat to human health. Among bacteria causing the greatest concern, 19. In the case of MexCD-OprJ of P. aeruginosa, the operon encoding this efflux pump is overexpressed when loss-of-function mutations in the gene that encodes its local repressor, nfxB20, are acquired. In addition, mexCD-oprJ is overexpressed when inducer compounds, such as benzalkonium chloride, dequalinium chloride, tetraphenylphosphonium chloride, chlorhexidine, ethidium bromide, rhodamine 6G, as well as the antimicrobial human peptides LL-37, procaine or atropine, temporally induce its expression, possibly by binding NfxB24.Expression of efflux pumps encoding genes is usually low under regular growing conditions. However, antibiotic-resistant isolates overexpressing them, as the consequence of mutations in genes encoding their regulators, are frequently isolated from patients under treatment. In addition, the expression of multidrug efflux pumps encoding genes can be transiently triggered in the presence of specific effectors that bind said regulators26. Hence, the presence of inducers might compromise the efficacy of the antibiotics that are substrates of this efflux pump. In particular, the potential risk associated with the utilization of antiseptics, disinfectants, and anesthetics, commonly used in hospitals, due to the transient induction of ciprofloxacin resistance in P. aeruginosa they render, has been highlighted13. However, it is also well known that nfxB loss-of-function mutants over-expressing mexCD-oprJ present an inherited collateral sensitivity (CS) to aminoglycosides  and β-lactams 28. It has been proposed that the molecular cause behind CS to aminoglycosides in mexCD-oprJ-overexpressing mutants is a reduction of the amount of the outer membrane protein OprM, which forms part of the aminoglycosides’ efflux pump MexXY20. Since CS to aminoglycosides robustly emerges after ciprofloxacin exposure, it has been proposed that this phenotype could be exploitable for the improvement of treatments against P. aeruginosa infections29. Therefore, here we analyze if it would be possible to temporarily induce and exploit transient CS to tobramycin associated with the transient overproduction of MexCD-OprJ.MexCD-OprJ overproduction provides resistance to tetracyclines, chloramphenicol, and quinolones 31. While some cases of CS associated with the acquisition of AR genes by horizontal gene transfer have been described32, most works in the field have focused on mutational resistance34. Different adaptive laboratory evolution (ALE) assays have been performed to analyze the possible combination37 or alternation of drug pairs40. However, the fact that different patterns of CS emerge in replicate populations of a single genetic background submitted to ALE in presence of the same drug43, complicates the applicability of this evolutionary trade-off. Further, different isolates of P. aeruginosa, including antibiotic-resistant mutants, may be present in cystic fibrosis patients after exposure to antimicrobial therapies44; each one could present a different CS pattern when challenged with the same drug45. This is because pleiotropic and epistatic phenomena determine fitness costs associated with the acquisition of AR and therefore, the possible mutations—and the associated trade-offs—that can be selected in a specific genetic background49. Therefore, the existence of different genetic backgrounds limits the clinical exploitation of CS to those cases in which a robust pattern of CS emerges when a drug is used. This conservation may occur due to parallel evolution50, although resistance to a specific antibiotic in different genetic backgrounds more often occurs by the acquisition of different genetic variations. Therefore, it would be unlikely—although very desirable—that all of them present the same pattern of CS.CS is a trade-off of AR evolution by which the acquisition of resistance to an antibiotic leads to an increased susceptibility to anotherP. aeruginosa28. The same robust CS pattern was also found in clinical isolates of P. aeruginosa presenting different genomic backgrounds and mutational resistomes29. The mutations involved in this phenotype were acquired in ciprofloxacin target encoding genes gyrAB (encoding the DNA gyrase)55 and in nfxB or mexS genes 58. Notably, the strength of CS to tobramycin, an antibiotic that, as ciprofloxacin, forms part of usual therapies against P. aeruginosa infections59, was higher in the genetic backgrounds that acquired mutations in nfxB28. In fact, it has been suggested that the phenotypic convergence towards CS to aminoglycosides observed in clinical isolates of P. aeruginosa may be mainly associated with the acquisition of genetic variations in nfxB60.In this regard, we have recently identified a robust tobramycin CS associated with the acquisition of different ciprofloxacin resistance mutations in varied pre-existing antibiotic-resistant mutants of 30. A seminal work has identified promising combination therapies making use of antimicrobial peptides, as they enhance the activity of other antibiotics and slow down the de novo evolution of resistance61. However, selection of resistant mutants—even presenting CS to a second drug—carries a risk if these mutants are not efficiently eradicated by the second antibiotic of the pair. This risk could be eliminated by temporarily inactivating the proteins encoded by those genes whose mutations lead to acquired AR and CS. The reason is that, in this situation, selection of resistant mutants would not be a requirement to achieve CS , since it presents many pharmacological and therapeutic benefits, including its antiseptic and disinfectant capacities64, its anticancer properties69, as well as its antiparasitic and antiviral activities71. We test whether a temporary and robust CS to tobramycin could emerge in different P. aeruginosa strains, including antibiotic-resistant mutants, when DC was present. Our findings show the existence of transient CS to antibiotics as the result of non-inherited AR, which is not associated with the fixation of genetic variations. In addition, we demonstrate that transient CS may drive antibiotic-resistant mutants and clinical strains of P. aeruginosa to extinction.In this work we transiently force the mentioned P. aeruginosa PA14 containing single  or multiple mutations (MDR6 and MDR12)  by specifically inducing a nfxB-mediated ciprofloxacin resistance transient status13 in the mentioned set of mutants, using the antiseptic DC.Using a set of pre-existing antibiotic-resistant mutants of 13, we determined the ciprofloxacin and tobramycin minimal inhibitory concentrations (MICs) in the presence or absence of this compound, in the seven genetic backgrounds. MIC variations  above or below an increase or a decrease of 2-fold, respectively, indicated biologically relevant variations of resistance, as previously described72. As expected, DC caused a transient induction of ciprofloxacin resistance in all the genetic backgrounds analyzed, with exception of the already ciprofloxacin-resistant mutant nfxB177, which stably overexpresses mexCD-oprJ  and 3 genetic backgrounds were close to resistant to tobramycin (MIC of 2 µg/ml), following the criteria of the European Committee on Antimicrobial Susceptibility Testing. Moreover, we observed a decrease of tobramycin MICs similar, or even higher, than those selected in ALE assays in the presence of ciprofloxacin28  but that the strength of this phenotype is improved in some genetic backgrounds, such as mexZ43, parR87, or MDR12, as compared with the one observed in ciprofloxacin-resistant mutants. It is also important to highlight that while the acquisition of CS to tobramycin associated with the use of ciprofloxacin depends on the acquisition of high-level stable, inherited, ciprofloxacin resistance, induction of CS to tobramycin associated with the use of DC depends on more moderate levels of transient, non-inherited, ciprofloxacin resistance. These results also indicate that, while stable CS to tobramycin in some mutational backgrounds would be linked to important levels of stable, inherited, resistance to ciprofloxacin, depending on the possible mutations they may acquire, the induction of transient and fairly mild nfxB-mediated resistance to ciprofloxacin in all of them renders an improved CS to tobramycin  or whether it can increase susceptibility to drugs from different structural families, we analyzed the MICs of a battery of antibiotics in presence or absence of this compound, in the seven aforementioned genetic backgrounds.It has been described that several pharmacological properties and therapeutic benefits of DC are due to its capacity to interfere with the activity of diverse proteinsparR87 and mexZ43, 4-fold in orfN50 and MDR6, and 2-fold in PA14, while no changes of MIC were observed in nfxB177. These data support that the induction caused by DC of CS to amikacin, as previously observed for tobramycin, is nfxB-mediated. It is important noticing that the highest decrease of amikacin or tobramycin MICs associated with the presence of DC occurred in the genetic backgrounds MDR12  and parR87 , indicating the relevant role of genetic background in shaping the strength of transient CS. Gentamycin MIC decreased up to 10.5-fold in PA14, 8-fold in MDR12, 7.9-fold in parR87, 4-fold in mexZ43, 3-fold in orfN50 and 2.6-fold in MDR6. However, gentamycin MIC decreased up to 4-fold of in nfxB177, suggesting that the induced CS to this aminoglycoside caused by DC in the different genetic backgrounds may be due to other causes beyond those directly related to NfxB.Firstly, we observed that DC also induces CS to other aminoglycosides, as amikacin and gentamycin Fig. ; Table 1mexZ43, orfN50, and MDR6, but no changes in parR87, MDR12, or nfxB177  in any of the genetic backgrounds analyzed Fig. ; Table 173, it might be possible that this potential alteration, if it existed, impacted the susceptibility to cationic peptides, as colistin and polymyxin B. To address this possibility, we determined the MICs of those compounds in the presence and absence of DC, in the seven genetic backgrounds. No changes in MIC to these antibiotics were observed  and, in some cases, to β-lactams , as it has been described in the case of stable, inherited, nfxB mutations28.All together, these results indicate that the temporary P. aeruginosa PA14  and with nfxB177, which did not show DC-induced CS to tobramycin, as a control  during three days. In parallel, we grew 28 populations in DC or tobramycin, using the same concentrations present in the drug pair, and 28 populations in the absence of any drug (84 control populations), as described in Methods. As shown in Fig. To further confirm that there are no other molecular mechanisms, beyond transient CS, responsible for the extinction observed in the antibiotic-resistant mutants analyzed, we tested the outcome of the combined ALE assay using ceftazidime, an antibiotic for which DC does not induce CS Fig. ; Table 1parR87, nfxB177, orfN50, or MDR6 , but some synergy was observed in the mutant mexZ43 and MDR12, a multiple mutant containing the mexZ43 mutation (FIC Index of 0.35 in both cases) (Supplementary Table mexZ43 mutation (mexZ43 and MDR12), might be increased by synergistic interactions.To further analyze if the efficiency of the DC-tobramycin combination could be the result of a synergistic interaction between both compounds, we performed checkerboard analyses (Methods) with PA14 and the 6 mutational backgrounds here analyzed. We did not observe synergy (neither antagonism) between DC and tobramycin in PA14 or in the mutants nfxB28. These results are in agreement with previous findings from our laboratory in which we demonstrated a decline of the expression of mexCD-oprJ, previously induced by DC, two hours after the removal of this compound, to levels similar to those of non-induced cells13. In fact, in our previous work we sequenced nfxB and the promoter region of mexCD-oprJ of P. aeruginosa cells treated during 20 h with DC, finding no ciprofloxacin resistance mutations. To further analyze if DC could have selected other mutations, leading to cross-resistance to other antibiotics, we measured MICs to antibiotics from other structural families  in the populations grown during 4 days in presence of DC, finding no differences respect to the parental strains . We found that this compound did not lead to an increase of ciprofloxacin or tobramycin MICs in the different genetic backgrounds of P. aeruginosa analyzed  and some of them belonging to high-risk epidemic clones (ST111 and ST244)  and some of them belonging to high-risk epidemic clones (ST111 and ST244).76. One trade-off of AR evolution that could allow the design of efficient therapeutic strategies is CS. However, there are barriers that hinder the implementation of these CS-based treatment strategies. One of the most important is the lack of conservation of CS in different genetic backgrounds, including pre-existing antibiotic-resistant mutants, which cause infections9. Supporting this statement, the conclusion of a recent analysis of around 450,000 susceptibility tests, spanning a 4-year period at different University of Pittsburgh’s hospitals, is that therapeutic strategies based on alternating antibiotics may be mostly ineffective due to differences in patterns of CS77. Therefore, the identification of robust CS patterns is critical for the application of CS-based treatment strategies.The existing crisis of AR requires the design of novel therapeutic strategies that improve the efficacy of available antibiotics50 or the result of different genetic events28. Concerning the second case, we have recently identified a robust CS to tobramycin in different antibiotic-resistant mutants and clinical strains of P. aeruginosa presenting different genomic backgrounds, associated with the use of ciprofloxacin, caused by the selection of mutations in different genes depending on the genetic background29. However, although this phenotypic convergence made possible the design of therapeutic strategies to drive the analyzed resistant mutants to extinction, the strength of CS to tobramycin was higher when mutations in nfxB, and not in gyrAB or mexS, were acquired. Therefore, in this work, we tried to “circumvent” constrains imposed by historical contingency and specifically, and temporally, canalize CS by inducing the nfxB-mediated ciprofloxacin resistance (and tobramycin hyper-susceptibility) mechanism, using DC. This approach has two benefits. One is to reduce the effect of historical contingency that makes difficult the emergence of robust, clinically exploitable, CS patterns, by funneling bacterial adaptation through the induction of a single, well-defined, mechanism of AR. The other is to achieve CS without the risky selection of antibiotic-resistant mutants.Phenotypic convergence towards CS may be the consequence of parallel evolutionnfxB28. In addition, the overexpression of mexCD-oprJ may occur due to the presence of inducer compounds19, which may affect the regulatory capacity of NfxB on the expression of this operon15. In particular, DC directly binds the transcriptional repressor of the TetR family RamR, which regulates the expression of the operon encoding the efflux pump AcrAB-TolC, reducing its DNA-binding affinity78. Similarly, DC may also bind and inhibit the regulatory capacity of NfxB leading to both, ciprofloxacin resistance and CS to tobramycin, phenotypes associated with loss-of-function mutations in nfxB28. In fact, the nfxB177 mutant is the only genetic background that did not present variations in the levels of resistance to ciprofloxacin or aminoglycosides  in the presence of DC, further supporting that the effect of DC is specific and principally falls on NfxB.Ciprofloxacin resistance associated with the overproduction of the MexCD-OprJ efflux pump may occur by the acquisition of loss-of-function mutations in the gene that encodes its local repressor, 71. Further, and although we have previously described its capacity to reduce the efficacy of ciprofloxacin through transient AR13, here we demonstrate that it is able to transiently induce CS to tobramycin, avoiding the acquisition of inherited ciprofloxacin resistance , to extinction. Therefore, it would be very interesting to analyze if a combined formulation of tobramycin, frequently inhaled during P. aeruginosa therapy59, with DC could be effective for the treatment of pulmonary infections caused by this bacterium. In this line, an inhaled formulation of curcumin-DC for pulmonary administration of encapsulated curcumin molecules with anticancer capacities has been described79. However, deeper studies that determine the concentrations of DC that could be safely applied by inhalation, alone or in combination with other drugs , are still required.DC is an antiseptic and disinfectant frequently used in clinics that presents many useful therapeutic properties30, and deeply studied by many scientists43, is even broader, including cases where susceptibility to a second drug is neither stable nor inherited, but transient. In fact, our results constitute a proof of concept supporting the hypothesis that it is possible to exploit transient and robust CS phenotypes associated with the induction of AR. Thinking about the clinical exploitation of CS, inducing a susceptible state of transient AR is less risky than forcing the selection of stable AR. Further, the latter does not always lead to a conserved pattern of CS in different genetic backgrounds, while our approach would allow inducing the same molecular mechanism of CS, regardless of the genetic background.Overall, we show that the concept of CS coined in the 1950s, understood as susceptibility to a drug associated with the acquisition of resistance mutations to anotherOverall, our results support that the identification of new compounds able to induce CS patterns might be valuable for the design of evolution-based strategies to tackle antibiotic-resistant infections. In addition, we suggest that looking for other inducible CS mechanisms in this and in other bacterial species could constitute a relevant advance in the rational design of therapeutic approaches to manage bacterial infections.This research complies with all relevant ethical regulations.P. aeruginosa PA14, PA14-derived isogenic mutants   at 37 °C with shaking at 250 rpm. MICs of ciprofloxacin, tobramycin, amikacin, gentamycin, aztreonam, ceftazidime, imipenem, piperacillin, fosfomycin, tetracycline and erythromycin or colistin and polimixin B were determined at 37 °C, in Mueller Hinton (MH) agar or MHII agar respectively, using E-test strips  in the presence or absence of 10 μg/ml of DC, as previously described13, using bacteria previously grown in 1 ml of LB supplemented or not with 10 μg/ml of DC.Unless stated otherwise, parR87, orfN50, nfxB177, mexZ43, MDR6, and MDR12), diluting (1/100) cultures in fresh LB every day and starting from glycerol stocks. Assays consisted in one day of ALE in the presence of 10 μg/ml of DC and 3 days of ALE in the presence of tobramycin, at the concentration that hinders—but allows—the growth of each P. aeruginosa mutational background under these culture conditions: 0.5 µg/ml for nfxB177, 0.75 µg/ml for PA14, 1.5 µg/ml for MDR6 and mexZ43, 2 µg/ml for orfN50, 2.5 µg/ml for parR87, and 12 µg/ml for MDR12. The 28 challenged populations (DC followed by tobramycin) and the 84 control populations  were grown during 4 days.For the alternated strategy, short-term ALE experiments in the presence of DC and tobramycin were performed for 4 days, at 37 °C and 250 rpm, using 28 replicate populations belonging to PA14 wild-type strain and 6 different mutational backgrounds  in fresh LB medium containing a combination of DC-tobramycin (28 populations), a combination of DC-ceftazidime (28 populations), each single compound (84 control populations) or absence of compound (28 control populations). The concentration of drugs in the pairs was the same that those used individually. 10 μg/ml of DC were added for each genetic background and tobramycin or ceftazidime were added at the concentration that hinders—but allows—the growth of each P. aeruginosa  using serially diluted concentrations of DC, tobramycin and a no-drug control, in 96-U-well plates. For that, 90 μl of MH medium with DC (4 to 40 μg/ml) or tobramycin (0.0625 to 4 μg/ml) were added to each well of 96-U-well plates for the analysis of each mutational background with exception of MDR12, for which tobramycin concentrations were higher (1 to 64 μg/ml). 10 μl of cells were inoculated into each well to a final OD600nm of 0.01. Bacteria were grown at 37 °C for 48 h without shaking. The fraction inhibitory concentration (FIC) of DC and tobramycin was calculated as the MIC of the combination of DC with tobramycin divided by the MIC of each of the compounds alone. FIC index resulted from the addition of the FICs of both compounds. A FIC index value of <0.5 or >4 was considered to indicate synergy or antagonism, respectively80.Seven standard checkerboard broth microdilution assays were performed with PA14 wild-type strain and 6 different mutants (Further information on research design is available in the Supplementary MaterialPeer review fileReporting Summary


An 18-year-old male, previously diagnosed with hereditary hemorrhagic telangiectasia (HHT), presented to the outpatient department with a complaint of generalized seizures and fever for the past five days. He had a history of recurrent epistaxis, progressive shortness of breath, and cyanosis. Magnetic resonance imaging (MRI) of the brain revealed an abscess in the temporoparietal region. A computed angiogram of the pulmonary vasculature showed the presence of arteriovenous malformation (AVM). A four-weekly antibiotic regimen was initiated, which resulted in a profound improvement in symptoms. A brain abscess can arise as a complication of vascular malformation in a patient with HHT, providing a nidus for bacteria to migrate toward the brain. Early recognition of HHT is essential in these patients and their affected family members, as screening can help us prevent complications at an earlier stage. Hereditary hemorrhagic telangiectasias (HHT), also known as Osler-Weber-Rendu syndrome, is an autosomal dominant genetic disorder characterized by arteriovenous malformations (AVMs). AVMs are found predominantly in the lungs (50%), liver 30-70%), brain 10%), and a very small proportion (less than 1%) in the spinal cord 0%, and a. About 70-70%, brAn 18-year-old male presented to the outpatient department with complaints of high-grade fever, generalized tonic-clonic seizures, and dry cough for the last five days. The fever was continuous in nature with rigors and chills. The cough was non-productive and occurred in bouts. There was no associated history of rash, joint pain, oral ulcers, abdominal pain, chest pain, hemoptysis, or any specific urinary complaint. The episodes of seizures usually self-resolved in 20 to 30 seconds, and some of them were associated with frothing. Past medical history revealed a diagnosis of hereditary hemorrhagic telangiectasia (HHT) two years back. The diagnosis was made based on a prolonged history of recurrent atraumatic nosebleeds for the past 10 years that were self-limiting. These episodes of epistaxis occurred at variable intervals. They were not associated with bleeding from any other site or orifice, easy bruising, bleeding gums, hemoptysis, or hemarthrosis. Furthermore, for the last five years, he was also experiencing shortness of breath on exertion. Shortness of breath was progressive, continuous, and occurred on walking around 100 meters but subsided with rest. It was not associated with hemoptysis, orthopnea, pedal edema, or abdominal distention. However, his parents noticed cyanosis of the fingers, lips, and tongue, which became permanent for the last six months. A similar pattern of symptoms was also present in the immediate and paternal family. On general physical examination, the patient was of average height and lean built, responsive, and oriented to time, place, and person. The patient was vitally stable except for a fever of 103 Fahrenheit. Anemia, odontogenic infection, jaundice, and edema were not observed. The jugular venous pressure was raised to about 9 cm and the lymph nodes were not palpable. His oxygen saturation (SaO2) was revealed to be 88%. He had florid central cyanosis visible on the tongue, lips, and face as well as on the hands along with prominent clubbing. On cardiac examination, the apex beat heaved in character in the fifth intercostal space in the midclavicular line, 9 cm from the sternal border. No parasternal heave or murmur was appreciable, but there were epigastric pulsations, loud S1, and normal S2. The peripheral pulses were of high volume. On CNS examination, Glasgow Coma Scale (GCS) was 15/15 and higher mental functions were intact. Motor, sensory, and cerebellar examinations were normal. Signs of meningeal irritation were absent and fundoscopy revealed no abnormalities. On pulmonary examination, there were no intercostal or substernal recessions. No use of accessory muscles was observed and the chest expansion was bilaterally symmetric. Normal vesicular breathing was present on the left side and upper and middle lobes on the right side. Crepitations were audible in the right lower lobe posteriorly, which did not improve or change character during coughing. On abdominal examination, pulsations were observed in the epigastric region. No scars, striations, or dilated veins were visible. The liver was palpable one finger below the costal margin with a soft and smooth lower border and a span of 14 cm. The spleen was mildly enlarged and palpable one finger below the costal margin. No fluid thrill or shifting dullness was present.Laboratory investigations and biochemical and hematological tests, including complete blood count (CBC), electrolytes, serum urea, serum creatinine, and liver function tests (LFT), were all within normal parameters except for high leukocyte count 17000/μL). Viral markers including hepatitis B surface antigen and hepatitis C antibody were non-reactive. Prothrombin time (PT) was 20.6 seconds, activated partial thromboplastin time (aPTT) was 38.8 seconds, and the international normalized ratio (INR) was 1.9. The arterial blood gas showed a pH of 7.45, partial pressure of oxygen in arterial blood (PaO2) of 53.2 mmHg, partial pressure of arterial carbon dioxide (PaCO2) of 33.1 mmHg, HCO3 of 22 mEq/L, and oxygen saturation of 88%. Blood and urine cultures were taken, which turned out to be unremarkable. Table 7000/μL. Taking into account the alarming history of progressive dyspnea in a young patient and the history of HHT, contrast-enhanced computed tomography (CT) angiogram of the chest Figure , which d It displayed communication with dilated right pulmonary artery, right inferior pulmonary vein, and multiple collaterals in the mediastinum around azygous and hemizygous veins, giving a picture of arteriovenous malformations. A decision of embolization was made to prevent any event of hemoptysis; however, the patient did not agree to undergo the procedure. During the second day of the hospital stay, the patient developed an episode of tonic-clonic seizure, which resolved within 15 seconds. Magnetic resonance imaging (MRI) of the brain was sought. A T1-weighted scan revealed a 5.2 x 2.7 x 4.7 cm brain abscess in the temporoparietal region  is an autosomal dominant vascular disorder that manifests as epistaxis, cutaneous telangiectasia, and visceral arteriovenous malformations (AVMs) as apparent in our patient. These arteriovenous malformations can arise in the pulmonary, portosystemic, cerebral, or spinal cord circulations and hence explain the neurological manifestations of HHT . These mAs our study reports, when a patient with a previous diagnosis of HHT presents with acute neurological symptoms, such as seizures, clinicians must maintain a high degree of suspicion and perform all relevant imaging modalities at an early stage to offer a better prognosis and significantly reduce the mortality rate due to patients with HHT that carry a significant risk of developing other neurological complications such as brain abscesses.


Genetic group, age at entry into confinement and at slaughter, are characteristics that have an important influence on lamb performance and carcass. The aim of this study was to evaluate the performance, carcass characteristics and non-carcass components from different genetic groups (Santa Inês and ½ Dorper x ½ Santa Inês) sheep, submitted to different feedlot entry and exit strategies. Were used 72 lambs males and castrated; 36 Santa Inês (SI) and 36 crossbred (Dorper x Santa Inês–DSI), with 6 months of average initial age. The groups were established in a completely randomized experimental design, in a 2x3x4 factorial arrangement, from the combination of genetic groups (GG), body weight at the beginning of confinement (WBC) and length of stay in confinement (LSC). The body weight classes at the beginning of confinement were: light (25 kg), intermediate (28 kg) and heavy (31 kg), for Santa Inês and crossbreeds, respectively. Slaughters were carried out every 28 days of confinement, in four LSC: 0, 28, 56 and 84 days. The GG did not influence performance, carcass and non-carcass component traits of lambs (p > 0.05). There was an effect of the WBC on the weights: final (FW), metabolic (MW), body at slaughter (BWS), empty body (EBW), hot carcass (HCY) and cold (CCW), loin, shoulder, leg musculature; loin eye area (LEA) and loin fat (p < 0.05). There was also an effect on LSC, for FW, average daily weight gain (ADG), MW, weight and yield of body components, weight of cuts and tissue ratio components of cuts (p < 0.05). In non-carcass components, effect on full and empty weight of: omasum, rumen-reticulum, small intestine; empty large intestine, liver and kidneys, paws and skin, and perirenal, pelvic and inguinal fat (p < 0.05). Interaction double effect on the tissue muscle/fat:bone ratio (MF:B) and for the full omasal component (p < 0.05). And triple interaction effect for ADG, full omasum and perirenal fat (p < 0.05). Weight at the beginning of confinement and confinement time are the characteristics that most influence performance, quantitative characteristics of carcass and non-carcass components. Regardless of the genetic group and age class, the animals reach the same weight after 84 days of confinement. Thus, the confinement of heavier lambs (31 kg) can be a profitable alternative, as they presented the highest weights for the most commercially valued cuts (shank and loin). The confinement strategy must adapt to market situations. The increase in consumption and demand for lamb meat has stimulated research in search of strategies that can help producers in decision-making and increase herds productivity , 2. In tHowever, even in confinement, the intended responses depend on the individual genetic material, still linked to race or genetic group . Every sThe Santa Inês breed, for example, is known for its maternal ability, hardiness and production of low-fat meat, essential characteristics that have led to crossbreeding with breeds specialized in meat production, such as the Dorper breed, with the possibility of increasing the productive efficiency of the herd , 10. HowIn addition, the age and weight of lambs at slaughter are among the main factors that affect meat quality , 14, as Our hypothesis is that genetics, age at slaughter and confinement time can influence performance and carcass yield in lambs. Thus, the objective was to evaluate the performance, carcass characteristics and non-carcass components of Santa Inês and ½ Dorper x ½ Santa Inês sheep, submitted to different feedlot entry and exit strategies.The procedures with the animals were approved by the Ethics Committee for Research with Animals and Experimentation (CEPAE protocol n° 97.2015) of the Federal University of Pará.The research was conducted in Castanhal, Pará, Brazil (1°17’S and 47°55’W), climate type Am, according to Köppen, average annual temperature of 26.8°C and relative humidity around 85%.Seventy-two castrated male lambs were used; 36 Santa Inês (SI) and 36 mestizos (½ Dorper x ½ Santa Inês–DSI), with an average initial age of 6 months. The experimental groups were formed based on the combination of genetic groups (GG), body weight at the beginning of confinement (WBC) and length of stay in confinement (LSC). Three body weight classes were formed at the beginning of confinement: Light , Intermediate  and Heavy , for Santa Inês and mestizos, respectively. Slaughters were performed every 28 days, setting up four LSC (days): 0 ; 28  56  and 84 .The animals were weighed, dewormed and identified; and confined in individual wooden stalls (1.2 m x 1.0 m), with a concrete floor lined with thick sawdust litter, provided with a feeder and drinker, located in a masonry shed with an opening for natural ventilation.Urochloa humidicula (Quicuio-da-Amazônia), with supplementation of 0.3% of live weight  and mineral salt ad libitum. All animals underwent an adaptation period (15 days) to the experimental conditions , during which they were weighed, identified, dewormed and vaccinated.Before confinement, the animals were raised in a continuous grazing system on the grass Pennisetum purpureum) silage (SIL), 16.30% soybean meal (Glycine max L.) (SM), 49.32% ground corn grain (Zea mays L.) (CG), 1.03% calcitic limestone (CL) and 1.35% mineral and vitamin supplement (SUP). With 2.31 of Metabolizable Energy (ME) , 84.4% of Dry Matter (DM), 12.53% of Crude Protein (CP), 1.92% of Ether Extract (EE), 62% of Neutral Detergent Fiber (NDF), 42.63% of Fiber in Acid Detergent (ADF) (The diet was offered twice a day (8 am and 5 pm) with a ratio of 40:60 (Forage:Concentrate). Was composed of 32% elephant grass (nt (ADF) .The DM, CP, EE, NDF and ADF were determined according to the Association of Official Analysis Chemists . For estThe productive performance of the lambs was evaluated by weighing the animals individually on the first experimental day and on the last day (which varied according to the treatments), always in the morning, before providing the first meal.Thus, total weight gain (TWG) was determined by the difference between final and initial body weight; and the average daily gain (ADG) dividing the TWG by the number of confinement days (TWG/70). With total daily dry matter intake  and average daily gain (ADG), metabolic weight (MW:BW0.75), feed conversion (FC: DMI/ADG ratio) and feed efficiency (FE) was calculated by the following formula: FE = total ADG/DMI. Where: total DMI = total daily dry matter intake; ADG = daily weight gain, in kg/day; and FE = feed efficiency.The lambs were slaughtered in an experimental slaughterhouse, following the Regulation of the Industrial and Sanitary Inspection of Products of Animal Origin—RIISPOA . Body weAfter 24 hours in a cold room (4°C), the carcasses were weighed, obtaining the cold carcass weight (CCW), used to calculate the cold carcass yield (CCY = 100 * CCW/BWS). Rib eye area (REA) and subcutaneous fat thickness (SFT) were also evaluated . The cooThe percentage of muscle, bone and fat (subcutaneous and intermuscular) was calculated by dividing the total of each tissue by the corrected cold carcass weight, then multiplied by 100. The relationships between the various tissues were calculated by dividing the absolute values totals of each component by the other: muscle/bone ratio (M:B) = Total weight of muscles in the carcass/total weight of bones in the carcass; muscle/fat ratio (M:F) = total weight of muscles in the carcass/total weight of fat in the carcass; fat/bone ratio (F:B) = total carcass fat weight/total bone weight in the carcass. Percentages and ratios were found following the methods of , 23.The ratio of the total edible portion to total bones was obtained by adding the total weight of carcass fat and muscle and dividing by the total bone weight: muscle + fat/bone ratio (MF:B) =  / .Statistical analyzes were performed in the caret package of the R version 3.5.1 software . The experimental design was completely randomized in a 2 x 3 x 4 factorial arrangement, considering two genetic groups (SI x DSI), three body weights at the beginning of confinement (25 x 28 x 31 kg) and four times of confinement permanence (0 x 28 x 56 x 84 days).The normality of data for each variable was verified using the Shapiro-Wilk test  and the The GG did not influence (p > 0.05) performance, carcass traits and non-carcass components of lambs. The WIC was significant (p ≤ 0.05) on the FW and MW variables. The highest averages for both FW (39.74 ± 4.35 kg) and MW (15.81 ± 1.30 kg) were obtained by the heaviest class. For these two variables, there was an effect (p ≤ 0.05) of TCP, with greater weights obtained at 84 days (FW = 40.91 ± 3.21 kg and MW = 16.17 ± 0.95 kg) .There was a triple interaction effect (p ≤ 0.05) between the GG, WIC and TCP factors for the TWG , with siThe REA and SFT values were affected by TCP, increasing as the confinement time increased, except for REA, where there was no difference between days 28, 56 and 84 . There wHigher loin and shoulder weights were obtained (p < 0.05) by the heaviest confined animals, with mean weights of 1.13 ± 0.21 kg and 3.0 ± 0.53 kg, respectively . RegardiOnly LSC was significant (p ≤ 0.05) on carcass tissue components and their relationships , with hiIn the tissue composition of the cuts and ratios, there was an effect of WBC on the leg and loin musculature, with higher averages for the heavy class, with an average weight of 3.63 ± 0.71 and 0.65 ± 0.15 kg, respectively (p ≤ 0.05) ; and loiIn the components of the gastrointestinal tract, LSC influenced the empty rumen-reticulum, with lower weight at the beginning of confinement (0.81 ± 0.10 kg) and increase in the other LSC, but there was no difference between them (p ≤ 0.05) . The samThe LSC was the factor that most influenced the non-carcass components . In periThere was an interaction effect (p ≤ 0.05) between GG, WBC and LSC for the lung-trachea component and perirenal fat . In periThe crossbred DSI lambs obtained characteristics similar to those of the Santa Inês breed, data consistent with previous reports –28, indiThe DM intakes for the two genetic groups were similar to those found by  for the The fact that FC and EF, indices commonly used to measure nutritional performance, did not show significant effects, indicates that the lambs, regardless of GG and WBC, were similar in the transformation of dry matter into body weight. On the other hand, the justification for the feed conversion not being different for the genetic groups was probably due to the fact that CMS and ADG did not present significant differences for the means of these traits. These results are similar to those found by , 33, 34,It was expected that confined animals (heaviest group) would obtain higher carcass, REA and STF yields. On the other hand, there was an increase in non-carcass components, mainly viscera, with the exception of the omasum, a factor that may be directly linked to the proportion of energy supplied in the diets. Highly energetic diets produce a greater deposition of fat in the carcass and viscera, causing the involution of the omasum . FurtherIt should be noted that the weights of the body components are related to the stage of development of the animal, that is, the greater its degree of physiological maturity and body weight, the greater the weight of the cuts, and are also influenced by the supply of a diet concentrated , 36, 37.That said, the heavy class obtained higher values for FW, MW, BWS, EBW, HCW, CCW and commercial cuts loin and shoulder. In addition, they showed an increase in all characteristics of body components, yields and weight of the cuts over the confinement time. Confining with 31 kg also resulted in higher final weight at 84 days, when compared to the weight ranges of 25 and 28 kg. Furthermore, greater weights of the tissue components of the ham muscle and loin fat were observed in this class. It is important to emphasize that the loin and shank are the most commercially valued cuts , and theThe increase in the weight of the cuts in all periods with the longest confinement time was due to the longer time for the animals to adapt and body development, which favored the weight gain of the meat cuts. The results are in agreement with those obtained by , who alsIn terms of physiological priority, early development of bone tissue, intermediate development of muscle fibers, and late development of adipose tissue are observed , 40. TheCrossbred and SI lambs in this study had an average age of 6 months at the beginning of confinement, being considered animals closer to the adult stage, which occurs at 1 year of age in lambs. In this phase, the animals are already finishing muscle deposition and starting the process of adipose tissue deposition . As the Thus, the increase in fat deposition, with the time spent in confinement, influenced the M:F ratio, which suffered a decreasing effect, as the lambs increased their weight, demonstrating that the greater the age at slaughter, the greater the weight, adiposity and carcass conformation , 46, witThe edible portion increased with the confinement time. Thus, interpreting the existing relationship between the tissue composition  present in the carcass makes it possible to determine the best period for slaughter, providing the market with carcasses with greater quantity and better distribution of muscularity and adiposity, and meat with better quality sensory, thus obtaining attractive quality carcasses for consumers –51.The variables abomasum (full and empty), rumen-full reticulum and the organs: lung/trachea, heart, spleen and tongue, were not influenced by the factors. Non-carcass components such as: omasum, rumen and reticulum, present late development, unlike the abomasum (early), and the percentages of some non-carcass components decrease with increasing live weight of animals . HoweverLiver and kidneys, and confinement time were the most relevant factors. This fact may have occurred in response to a higher metabolic rate, due to the level of concentrate present in the diet, and it can be inferred that the development of the organs is also linked to the size of the animals, consumption and composition of diet , 54, whiThe non-carcass component (omasum full) showed an increase in weight at the beginning of confinement, but it was not effective over time. Organs and viscera develop early and the phenomenon occurs with greater intensity in the early stages of the animal’s life, shortly after weaning and with the beginning of feeding, mainly forage , 57. WitThus, perirenal fat deposits increased with time spent in feedlot for heavy classes (SI) and light class for lambs (DSI), in confirmation of age in relation to the growth of adipose tissue. But, it is necessary to consider the extent to which fat is interesting in the animal’s carcass, as in large amounts it will harm the producer . In someEvaluating different slaughter weights , Siqueira  observedIn general, confining lambs with 31 kg implies a higher final weight at 84 days, when compared to the other weight ranges (25 and 28 kg), with higher values for BWS, EBW, HCW, CCW of the cuts and non-carcass components. In the relationship between GG, WBC and LSC, feedlot SI lambs with higher initial weight had higher weight of non-carcass components, indicating that part of the BWS is due to the viscera. In the case of DSI lambs, a lower viscera weight was observed at the end of 84 days, when confined with 31 kg. However, the tissue ratio of the edible portion for the two genetic groups increased over time in confinement, indicating that a longer stay in confinement brings good results.The weight at the beginning of confinement and the time spent in confinement are characteristics that most influence performance, quantitative characteristics of the carcass and non-carcass components. Regardless of the genetic group and age class, the animals reach the same weight after 84 days of confinement. Thus, the confinement of heavier lambs (31 kg) can be a profitable alternative, as they presented the highest weights for the most commercially valued cuts (shank and loin). The confinement strategy must adapt to market situations.


Popillia japonica (Coleoptera: Scarabaeidae), is an emerging invasive pest in Europe and America. In the Azores, this pest was first found on Terceira Island during the sixties and soon spread to other islands. The rate of infestation differs between islands, and we hypothesized that microbiome composition could play a role. Therefore, we sampled 3rd instar larvae and soil from sites with high and low infestation rates to analyze the microbiome using next-generation sequencing. We analyzed twenty-four 16S DNA libraries, which resulted in 3278 operational taxonomic units. The alpha and beta diversity of the soil microbiome was similar between sites. In contrast, the larvae from high-density sites presented a higher bacterial gut diversity than larvae from low-density sites, with biomarkers linked to plant digestion, nutrient acquisition, and detoxification. Consequently, larvae from high-density sites displayed several enriched molecular functions associated with the families Ruminococcaceae, Clostridiaceae and Rikenellaceae. These bacteria revealed a supportive function by producing several CAZyme families and other proteins. These findings suggest that the microbiome must be one drive for the increase in P. japonica populations, thus providing a checkpoint in the establishment and spread of this pest. Popillia japonica Newman  is a highly voracious herbivorous insect that consumes a wide variety of plants, many of which are of high economic value. P. japonica has been designated as one of 2019’s priority pests by the European Commission. Adults are attracted to foliage, flowers, and fruits and can cause significant damage to ornamental plants and fruit and vegetable crops. Larvae develop in the grass and can cause serious damage to turfgrasses and pastures. P. japonica is native to Japan; thus, it is commonly known as the Japanese beetle (JB). It became an invasive pest in the 20th century; it had arrived in the United States by 1916 [P. japonica was first noticed in the mid-1960s on Terceira Island near a military airport used by the US Air Force [Bacillus popilliae strains [P. japonica. Moreover, traps were placed in all ports and airports of the archipelago islands to serve as warning stations. Despite this, in 2013, the presence of the pest in another seven islands was reported by the Agricultural Services of Azores , appareP. japonica whose immature stages spend more than 75% of its life cycle in the soil, feeding not only on roots but also on organic matter, as recently revealed [This unequal distribution can be influenced by many factors such as vegetation, predators, pathogens, climatic variables, anthropogenic actions, etc. Microbial communities in the soil and associated with the insect could be factors driving the insect population. Aiming to understand the role of the microbiome in population rates we chose two islands with quite similar ecosystems but with different population rates to analyze and compare the microbiome of soils and gut larvae. The soil microbiome has an important impact on insects, particularly on soil-dwelling insects, that is the case of revealed , thus exrevealed ,14. A ferevealed .P. japonica grubs and soil from populations with low density (São Miguel Island) and from populations with high density (São Jorge Island) and analyzed the bacterial composition of these samples using next-generation sequencing (NGS) methods.Moreover, the gut microbiome of insects is known to have a great impact on their survival and expansion. Previous studies have demonstrated the fundamental importance of microbes in the adaptation of insect pests to new environments ,17,18. TP. japonica which corresponded to São Jorge Island (HD) with an annual mean of 38,323 captured adults/km2 and a site with low infestation levels which corresponded to São Miguel Island (LD) with 1106 captured adults/km2, based on 10 years record of JB adults collected in the Azores supported by the Regional Secretariat for Agriculture and Rural Development of the Government of the Azores [2 of study sites in which three stations with identical altitudes and vegetation among those lands used for pasture and with the highest known infestation rates per island (Trifolium repens L.), perennial ryegrass (Lolium perenne L.), and annual ryegrass (Lolium multiflorum Lam.) [Rubus spp. . Subsequently, we randomly chose two sampling spots from each station and collected a total of six soil and larvae samples per density between April and May 2021  . The pasMay 2021 .We collected the soil samples (1 kg each) by mixing ten subsamples, kept them cold during transport to the laboratory, and finally stored them at −80 °C. Also, we collected six biological replicates of larvae, each consisting of a pool of five larvae samples from the same random spots where we collected the soil samples. The larvae samples were collected in refrigerated absolute ethanol and transported in a thermo-container at 4 °C. Approximately 16 h after collection, the larvae were surface-sterilized using the protocol described by Montagna et al.  and thenTM 2.0, Singapure) and dsDNA BR Assay kit  with quantity thresholds of 73 and 270 ng/µL per sample, respectively.From each soil sample, we performed five DNA extractions from 10 mg of soil which were gathered together to be sequenced as one representative sample. DNA was extracted from a gut homogenate and soil using a DNeasy Power Soil Kit , following the manufacturer’s instructions, and preserved at −80 °C. The DNA quality and purity were assessed with a fluorometer  were obtained from official stations located in Velas (São Jorge) and Ponta Delgada (São Miguel). The mean climatic values were calculated based on the annual period of larvae from January to May and from October to December, from 2020 to 2021.The collected soil samples were analyzed at the Laboratory of Soil and Plant Analysis at the University of the Azores, with the following order of tasks: the soil was dried in an oven with forced air circulation at 40 °C for two to three days and sifted using a 2 mm mesh sieve to obtain fine soil.Subsequently, chemical analysis of the following parameters was carried out: texture, using the densimeter method, according to Bouyoucos ; total o4C2H3O2) at pH 7 by atomic absorption (1:10) , whereas phosphorus (P) was determined using the Olsen method modified by extraction of sodium hydrogen carbonate at pH 8.5 using an AutoAnalyzer (1:20) .The macronutrients potassium (K), magnesium (Mg), and calcium (Ca) were determined by extraction in ammonium acetate  on an Illumina MiSeq platform  for the V3–V4 variable regions of 16S rRNA, using 300 bp paired-end sequencing reads with the forward (5′ CCTACGGGNGGCWGCAG 3′) and reverse (5′ GACTACHVGGGTATCTAATCC 3′) primers. The library preparation protocol was based on the Illumina 16S rRNA Metagenomic Sequencing Library. The quality of the reads was assessed using FastQC software version 0.11.8  before fRaw sequencing reads were pre-processed using Cutadapt software version 3.4  to removFor whole metagenome sequencing, the DNA was first analyzed to ensure that the samples had sufficient integrity and concentration for optimal library preparation. As a standard procedure, 1 μL of gDNA from each sample was used to test integrity and purity by 1.5% agarose gel electrophoresis. Subsequently, a Qubit fluorometer was used to quantify DNA and determine whether it was sufficient to proceed; the integrity of this DNA was also verified. Thereafter, the samples were used for library preparation and sequencing. The generated DNA fragments (DNA libraries) were sequenced using an Illumina Novaseq platform , using 150 bp paired-end sequencing reads.Phyloseq package [ampvis2 [From the taxonomic classification of the OTUs, data were imported into R software version 4.1.3  for stat package . The rel[ampvis2  which isvegan—Adonis package) [Diversity was assessed using the Simpson and Shannon indices for alpha diversity and the Bray Curtis index (with UPGMA cluster and NMDS representation) for beta diversity (package) , and thep < 0.05.The differentially abundant taxa between groups were determined using linear discriminant analysis effect size  . For allcorrplot and VIF test from usdm R package. The filtered variables were assessed to the microbiome abundance using canonical correlation analysis (CCA) constrained to the density variable and the data was transformed initially by applying the Hellinger transformation [The bacterial abundance was correlated with the soil and climatic parameters to estimate the impact of these variables on the microbial community of the soil and gut. Environmental variables were analyzed and filtered for correlation and multicollinearity with ormation .tidyverse package in R [The raw reads generated from whole metagenome sequencing were initially filtered to remove low-quality and adapter sequences. This was achieved using Trimmomatic version 0.39 . Any remage in R  to generTo perform functional enrichment analysis, we used the KEGG Mapper Reconstruct tool to group and summarize the gene count tables into pathways and gene/protein families. Differential abundances between the groups were elucidated using LEfSe analysis. Circos plots  were genNext-generation sequencing (NGS) generated 2,445,131 raw bacterial reads from all larval and soil samples (Accession Code: PRJNA933566). A total of 1,276,377 raw reads were generated from 12 larval samples. Of these, 642,033 larval sequences were obtained from the HD site (6 samples), while 634,344 larval sequences were obtained from the LD site (6 samples). A total of 1,168,754 raw reads were generated from the 12 soil samples. Of these, 594,334 sequences were obtained from the HD site (6 samples) and 574,420 sequences were obtained from the LD sites (6 samples). After quality control and denoising of the raw sequences, a total of 598,595 high-quality sequences were obtained: 380,328 from larvae and 218,267 from soil . The rarA total of 3278 bacterial OTUs were identified in the samples. Interestingly, the number of OTUs was similar between soil groups: 1240 and 1165 OTUs at HD and LD sites, respectively. In contrast, the number of OTUs obtained from larvae from the HD site was higher than that from larvae from the LD site, with 1472 and 1222 OTUs, respectively. For diversity analysis, counts were rarefied to a sample depth of 9659, resulting in the removal of 40 OTUs. In total, 94% and 84.6% OTUs were classified at the taxonomic level of family and genus, respectively, and only 1134 (34.6%) were classified at the species taxonomic level, which is average for metabarcoding studies with the 16S rDNA gene.p < 0.05), compared with that of larvae from the LD site . The larval microbiome differed between the HD and LD sites . In the PCA, larval samples from the HD and LD sites formed separate cluster groups, indicating that there was a significant difference in the larval gut microbiome composition between the two sites. Regarding soil microbiome, the samples from the HD and LD sites were mixed in the dendrogram and overlapped in the PCA, indicating non-compositional differences .The structural composition assessed using the Bray-Curtis beta diversity index was analyzed using a principal component analysis (PCA)  and a clTo determine the taxonomic composition of the bacterial communities, OTUs were used to calculate the relative abundance of taxa. To avoid misclassification, we evaluated the bacterial community up to the family level, since the V1–V3 region of the 16S rRNA gene has high-level similarity between closely related taxa.Firmicutes being dominant (24.1%), followed by Proteobacteria (23.2%), Actinobacteriota (12.3%), Verrucomicrobiota (9.8%), Acidobacteriota (9.6%), Planctomycetota (9.1%), Chloroflexi (4.6%), and Bacteroidota (3.3%) (Bacillaceae (18.1%) from Firmicutes and Xanthobacteraceae (11.4%) from Proteobacteria  A. The mobacteria B.Coriobacteriia and WS2 were distinctive of HD soils, whereas Thermoleophilia, Gemmatimonadota, Pla4_lineage, and Subgroup_25 were distinctive of LD soils   between HD and LD soils in the less abundant phyla. LD soils C. Soils  (<0.1%) D.Firmicutes (66.9%), Bacteroidota (21.4%), and Desulfobacterota (7.4%), which together accounted for over 95% of the relative abundance in larvae from both the HD and LD sites , Lachnospiraceae (13.4%), Christensenellaceae (9.7%), Clostridiaceae (8.1%), Oscillospiraceae (4.1%), Sporomusaceae (3.4%), and Bacillaceae (2.9%), whereas Rikenellaceae (18.3%) from Bacteroidota, and Desulfovibrionaceae (7.8%) from Desulfobacterota were also highly represented. Although the most abundant phyla were similar at both infestation sites, the relative abundance of families was different (The gut microbiome was predominantly composed of LD sites A. Withinifferent B.Firmicutes and Desulfobacterota (>7%) and the phyla Chloroflexi, Deferribacterota, Planctomycetota, Myxococcota, and Alphaproteobacteriota (<1%) were distinctive of larvae from the HD site, whereas the phyla Bacteroidota (>20%) and BRH_c20a (<1%) were distinctive of larvae from the LD site  between larvae from both sites. The phyla  LD site C.Rikenellaceae (22.5%) and Dysgonomonadaceae (2.8%) were more abundant in larvae from the LD site, whereas 15 families, led by Desulfovibrionaceae (9.4%) and Clostridia vadinBB60 group (5.1%), were more abundant in larvae from the HD site. Additionally, four families  were found exclusively in the larvae from the HD site.At the family level, we found 19 significantly discriminative taxa (LEfSe: abs LDA score > 2.0) in the gut microbiome of larvae from the two sites D. The faP. japonica larvae. Among these, 33 pathways and 18 gene/protein families were significantly discriminative between the microbiome of larvae from the LD and HD sites (LEfSe: abs LDA score > 2.0). Moreover, we identified 13 biomarker pathways in the microbiome of larvae from the LD site and 20 from the HD site . From these, 355,460 to 517,742 genes were annotated, resulting in 13,926 distinct KEGG orthology molecular functions. In total, 267 pathways and 54 gene/protein families were identified in the gut microbiome of  HD site A. Additi HD site B.A few relevant pathways and proteins were found among the discriminative functional biomarkers, such as the lysine, valine, leucine, and isoleucine degradation pathways and the folate biosynthesis, fatty acid degradation, glutathione metabolism, bisphenol degradation, and biosynthesis of cofactors pathways, which were all enriched in larvae from the HD site. Additionally, relevant genes and proteins were enriched in larvae from the HD site, such as genes coding for cytochrome P450 and for mitochondrial biogenesis, cytokine receptor, and membrane trafficking proteins. Although larvae from the LD site had fewer enriched functional biomarkers, relevant pathways were also observed, such as those for fructose, galactose, and mannose metabolism; starch and sucrose metabolism; and two-component systems. Furthermore, genes linked to antimicrobial resistance were enriched in larvae from the LD site.Producers of CAZymes in the microbiome have been identified to play a key role in herbivorous nutrition; thus, we checked for genes encoding these enzymes in the metagenome and for their respective producers. We identified 138 CAZyme families in the gut microbiome of the larvae. LEfSe analysis performed between larval groups from the LD and HD sites revealed that 33 CAZymes were significantly different (abs LDA score > 2.0). Only 6 CAZymes  were more abundant in larvae from the LD site, whereas 27 CAZymes (mostly glycosyltransferases and some glycosylhydrolases) were enriched in larvae from the HD site .Ruminococcaceae, Clostridiaceae, and Paenibacillaceae were the most relevant producers of these enzymes in larvae from LD sites, whereas, in the microbiome of larvae from the HD site, these enzymes were produced by Ruminococcaceae, Rikenellaceae, and Clostridiaceae , which was the most abundant taxon in soil microbiomes. Other bacterial families shared between larvae and soils of high and low densities were Planococcaceae , Xanthobacteraceae , Pirellulaceae , and Rhizobiaceae .The microbiomes of the soil and larvae from the HD site shared 120 OTUs (25.5%), twice the number and relative abundance of those from the LD site  A,B. ThesRuminococcaceae and one belonging to Rikenellaceae were enriched in the HD site, whereas one OTU belonging to UCG_010 and one belonging to Dysgonomonadaceae were enriched in the LD site  relative to the soil microbiome (Alistipes (11% in HD and 21% in LD), Candidatus Soleaferrea (13% in HD and 15% in LD), Tyzzerella (10% in HD and 11% in LD), Desulfovibrio (9% in HD and 6% in LD), Christensenellaceae R-7 group (7% in HD and 6% in LD), Clostridia vadinBB60 group (5% in HD and 2% in LD), Clostridia UCG-014 (2% in both sites), Clostridia UCG-10 (2% in HD and 3% in LD), and Dysgonomonas (1% in HD and 3% in LD).The gut microbiome of crobiome A. Thus, Bacillus (16% in HD and 15% in LD), Candidatus Udaeobacter (5% in HD and 7% in LD), Bradyrhizobium (3% in HD and 4% in LD), WD2101_soil_group (2% in both sites), Lysinibacillus (2% in HD and 1% in LD), and Candidatus Xiphinematobacter (2% in HD and 1% in LD).The soil microbiome contained 180 enriched bacterial genera relative to the larval gut microbiome (LEfSe: abs LDA score > 2.0) B. Thus, p < 0.05) with variations in gut microbiome composition  of this harmful compound can have on insects [Despite this, larvae from the HD site have a higher diversity of enriched bacterial families related to plant digestion and nutrient acquisition. mius sp. ,55, termmius sp. ,57, and mius sp. . These bmius sp. . Apparen insects . We hypoClostridia class. This taxonomic group has a cellulosome-like structure, which is a large protein complex responsible for the degradation of plant cell walls. This enzymatic complex allows bacteria to efficiently degrade cellulose and other plant polysaccharides [Clostridia is considered a symbiont of higher termites and was found to be localized in the gut-mixed segment of the termite Nasutitermes takasagoensis [Clostridia have been found to degrade polysaccharides into acetone, alcohol, lactate, CO2, and hydrogen [Another interesting biomarker found in larvae from the HD site was the vadinBB60 group of the agoensis . Furtherhydrogen ,53,61,62hydrogen ,64, suggNitrosomonadaceae are known to participate in the nitrogen cycle and the oxidation of ammonia to nitrite [Desulfarculales participate in the conversion of sulfate to hydrogen sulfide [P. japonica in the populations from the high-density site.Equally important are the exclusive taxa detected in the gut of HD site larvae:  nitrite  and Desu sulfide . These tP. japonica gut microbiome showed enhanced functional enrichment in the HD site larvae compared to that of the LD site. The larvae from the HD site had a higher abundance of biomarker pathways, genes/protein families, and CAZymes than did the larvae from the LD site. These findings suggest that the gut microbiome of the larvae from the HD site is more enriched and has a greater potential for nutrient utilization, improved plant material degradation, detoxification, and enhanced immune response than the gut microbiome of the larvae from the LD site.The metagenomic analysis of the The different pathways found to be enriched in larvae from the HD site indicated a better capability to break down several amino acids  as a source of energy , produceRuminococcaceae and Clostridiaceae families in both JB populations, are important for the digestion of carbohydrates in insect guts. The complex carbohydrate xylan, which constitutes a significant portion of plant cell walls, is broken down by members of the enzymatic family GH5, known as xylanases [The presence of a higher number of CAZymes in the microbiome of larvae from the HD site indicated a potential advantage in comparison with those from the LD site, in terms of herbivorous nutrition. The GH5 and GH9 CAZyme families, mostly produced by ylanases . Other mylanases . In addiylanases . SimilarRikenellaceae family. These CAZymes included GH65, GT2, GT3, and GT51 family members. Enzymes of the GH65 family are known to breakdown several disaccharides, such as trehalose, maltose, cellobiose, isomaltose, sucrose, and lactose [Interestingly, the comparison between the microbiomes of larvae from the HD and LD sites showed that, in the former, more CAZymes were being produced by members of the  lactose , and tho lactose .Holotrichia parallela larvae varied significantly among ten populations from different locations [Costelytra zealandica was reported to have community variations between samples that were influenced by external factors, such as the geographic location of the population and the insects’ diet [Another relevant finding resulting from the comparison of microbiomes in the gut from HD and LD is that larvae from the HD site shared a higher number of OTUs with their soil than the populations from the LD site. This data is in accordance with Huang and Zhang that showed the composition and diversity of the dominant bacterial groups in ocations . Likewists’ diet .P. japonica larvae. Chouaia et al. compared the microbiome between soil and larvae, pupae, and adults of P. japonica and found that larvae share more microbiome components with soil than pupae and adults. In addition, the authors reported a small core bacterial community (89 OTUs) that was common throughout all life stages, proving the existence of vertical transmission [The most enriched taxa in larvae relative to the soil are all members of bacterial families known as fiber-digesting bacteria ,23,81. Csmission .Dysgonomonadaceae, Clostridia UCG-010, and Rikenellaceae. On the other hand, abundant OTUs with a negative correlation to soil parameters belonged to the biomarker families of HD larvae, such as Clostridia vadinBB60 group and the Enterococcaceae RF39. These results indicated that soil parameters may have an influence on the gut microbiota of the larvae and may help the insects adapt to different ecological circumstances.In this study, we also found the potential hint of the correlation between three soil abiotic factors  and the variations observed between the gut microbiomes of larvae from the HD and LD sites. For example, a positive correlation of these factors was observed with abundant OTUs belonging to the biomarker families of LD larvae, such as Avila-Arias et al. investigated the effects of abiotic factors on the gut microbiome of JB larvae. They found that factors such as cation exchange capacity, OM content, water-holding capacity, and texture of the soil were moderately correlated with the composition of prokaryotic microbes in the gut, particularly in the midgut. Similar to our data, these results also suggest that the microbial communities present in JB guts are partly shaped by their adaptation to local soil conditions .P. japonica larvae differs between high- and low-density populations. We could establish clear biomarkers in these microbiomes, particularly in the nondominant phyla of bacteria. In addition, the microbiome in insects from high population rates has enriched functional pathways linked to plant digestion, detoxication, and immune defenses. Furthermore, we observed that soil parameters  correlate negatively with bacterial biomarkers of P. japonica larvae from high densities. Altogether, these findings suggest that gut microbiome could be a target in a strategy to prevent pest dissemination and growth, as indicated by others [P. japonica could serve as a valuable tool to assess the risk of population spread and expansion in various regions. The findings of this study indicate a strong association between insect microbiomes and soil parameters. As a result, a new hypothesis emerges, proposing that the manipulation of soil abiotic factors could potentially influence the microbiome of JB larvae, resulting in reduced insect fitness and limited establishment in new areas. Therefore, this important research area could provide further knowledge on the intricate interactions between insects, microbes, and the environment.This study showed that the gut microbiome of y others ,82. We a