Frantz's tumor of the pancreas is a rare phenomenon, and it accounts for 1–3% of all neoplasms of the pancreas. Its percentage is much higher in younger persons, especially in younger women, as compared to the rest of the population.The present study describes a 32-year-old female patient in whom a preoperative imaging diagnosis confirmed a mass in the junction of the pancreas' body and tail. Based on the anamnesis, the preoperative diagnosis, and the patient's general status, the decision was made to performed laparoscopic enucleation of the pancreatic tumor. The operation and postoperative recovery passed without complications. Definitive histopathological and immunohistochemical findings confirmed a solid pseudopapillary neoplasm of the pancreas.Depending on the localization and the size of the tumor, surgical options range from typical and atypical resections of the pancreas to minimally invasive surgical procedures, such as local excision and enucleation. Laparoscopic procedures have a comparative advantage in cases of enucleation and resection of the pancreas. The low frequency of recidivation and a favorable prognosis, even after repeated surgery, are additional reasons for favoring the laparoscopic approach over the classical surgical approach.A minimally invasive surgical approach should be applied whenever the dimensions and the localization of the tumor permit it, bearing in mind all the benefits and advantages that this surgical technique offers. •Solid pseudopapillary neoplasm of the pancreas, is known as Frantz's tumor.•Around 1,000 cases of Frantz's tumor have been described.•32-year-old patient underwent laparoscopic enucleation of an 18 mm pancreatic tumor.•Laparoscopic enucleation is a feasible option for the treatment of these masses. It mainly occurs in younger individuals, predominantly women . Frantz'Ultrasonography (USG) of the abdomen is the initial diagnostic method; nevertheless, computerized tomography (CT) and magnetic resonance imaging (MRI) of the abdomen are much more precise and superior methods. Frequently, CT and MRI can indicate the presence of a mass in the pancreas, which has the characteristics of cystic degeneration, bleeding within the cyst, and the presence of a capsule . DiffereThe only curative treatment modality is surgical treatment. Within this modality, the available options range from resection procedures on the pancreas, depending on the tumor's localization, to a minimally invasive surgical procedure on the pancreas in the form of the enucleation of these neoplasms .The present study aims to present a rare disease of the pancreas, which was treated safely and efficiently by applying laparoscopic enucleation with all the benefits this approach offers when used appropriately. A review of the literature is also performed. The work has been reported in line with the SCARE guidelines .29. No other alterations were encountered. An abdominal ultrasound was done, without any abnormal findings, so an MRI was ordered, detecting a focal area with a distortion in the parenchyma, diagnosing an 18 mm pancreatic mass in the junction of the corpus and tail of the pancreas. There were no signs of infiltration of the surrounding vascular structures and no connection to the duct of Wirsung  . A drainPostoperative recovery went uneventful. The abdominal drain was removed on the third postoperative day. On the fourth postoperative day, the patient was discharged from the hospital with normal laboratory findings. A month after surgery, ultrasound imaging of the patient's abdomen was performed, and without abnormalities. At this time, laboratory analyses were also performed, and the results were within the normal reference ranges. Six months after the operation, a follow-up MRI of the abdomen was conducted without any abnormal findings. The patient is currently on her 12 month follow-up without complications nor recurrence.Definitive histopathological and immunohistochemical findings confirmed a solid pseudopapillary neoplasm of the pancreas. The tumor did not show cytological or histological elements of malignancy .Fig. 3Hi3The solid pseudopapillary neoplasm of the pancreas was first mentioned in the literature in 1933 . HoweverThese tumors most commonly remain asymptomatic or cause non-specific complaints, regardless of their size. In addition to the compressive effect on the surrounding structures and the occurrence of a palpable mass in the abdomen once they reach a specific size, occasional acute or chronic pain, loss of appetite, vomiting, and a loss in body mass, may also occur .In the patient described in the present study, symptoms were present around two months, in non-specific pain below the left costal arch. The pain was of lower intensity, more discomfort in that part of the abdomen, and occasional intensification in the pain scale.Appropriate diagnosis plays an essential role in deciding on further treatment. The initial diagnostic methods applied are US and CT of the abdomen. A heterogeneous lesion can be seen in the latter, which often demonstrate a peripheral enhancement of contrast, characteristic of the fibrous pseudocapsule. MRI of the abdomen is a more sensitive method than CT . Some auFrantz's tumor has a low malignant potential. There are rarely metastases, and they are most commonly localized in the liver. Even when there are metastases, the overall 5-year survival is around 97% .Surgery is the only and primary curative form of treatment. Depending on the localization and the size of the tumor, surgical options range from typical and atypical resections of the pancreas to minimally invasive surgical procedures, such as local excision and enucleation . LaparosDue to the favorable localization of the tumor, its size, and bearing in mind all the advantages of a minimally invasive surgical approach, we chose a laparoscopic enucleation of the tumor, which went uneventfully.4With its mainly non-specific symptomatology, Frantz's tumor represents a diagnostic challenge and is usually detected as a coincidental finding during other examination procedures. As it mostly occurs in the younger population, especially in younger women between 18 and 35 years old, one must consider the best therapeutic approach to treat this tumor. Laparoscopic enucleation is a feasible option for the treatment of these masses.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.All procedures performed in this study were reviewed by Ethics Committee and complied with the principles laid down in the Declaration of Helsinki.This research group did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Dr. Mauricio Gonzalez-Urquijo.Conceptualization, D.E.; V.M; M.G.U; Writing-review and editing, B.T, M.V; N.G; D.J Literature review, M.B.; V.M; M.G.U; B.T Writing draft, M.V.; M.G.U, D.E.; Supervision, M.G.U.Not commissioned, externally peer-reviewed.Ethics Committee of the Clinical Center of Tecnologico de Monterrey.Laparoscopic Enucleation of Frantz's Tumor of the Pancreas: Case Report and Literature Review to be conducted.DECISION: We approve the academic clinical work No. 01134TM.Date: 26/01/2021.Name of the registry: Laparoscopic Enucleation of Frantz's Tumor of the Pancreas.Unique Identifying number or registration ID: NCT04728750.https://clinicaltrials.gov/show/NCT04728750.Hyperlink to your specific registration (must be publicly accessible and will be checked): None.


We investigate the secure degrees of freedom (s.d.o.f.) of three new channel models: broadcast channel with combating helpers, interference channel with selfish users, and multiple access wiretap channel with deviating users. The goal of introducing these channel models is to investigate various malicious interactions that arise in networks, including active adversaries. That is in contrast with the common assumption in the literature that the users follow a certain protocol altruistically and transmit both message-carrying and cooperative jamming signals in an optimum manner. In the first model, over a classical broadcast channel with confidential messages (BCCM), there are two helpers, each associated with one of the receivers. In the second model, over a classical interference channel with confidential messages (ICCM), there is a helper and users are selfish. By casting each problem as an extensive-form game and applying recursive real interference alignment, we show that, for the first model, the combating intentions of the helpers are neutralized and the full s.d.o.f. is retained; for the second model, selfishness precludes secure communication and no s.d.o.f. is achieved. In the third model, we consider the multiple access wiretap channel (MAC-WTC), where multiple legitimate users wish to have secure communication with a legitimate receiver in the presence of an eavesdropper. We consider the case when a subset of users deviate from the optimum protocol that attains the exact s.d.o.f. of this channel. We consider two kinds of deviation: when some of the users stop transmitting cooperative jamming signals, and when a user starts sending intentional jamming signals. For the first scenario, we investigate possible responses of the remaining users to counteract such deviation. For the second scenario, we use an extensive-form game formulation for the interactions of the deviating and well-behaving users. We prove that a deviating user can drive the s.d.o.f. to zero; however, the remaining users can exploit its intentional jamming signals as cooperative jamming signals against the eavesdropper and achieve an optimum s.d.o.f. P is the total average transmitted power. The s.d.o.f. have been considered in the literature in many multiuser channel models, such as helper wiretap channel [X-channel [Y channel [Physical layer security techniques allow secure transmission of information (in absolute sense) without the need for encryption keys . Consequ channel ,14, mult channel ,15,16,17 channel ,20,21,22-channel ,24, half-channel , compoun-channel , diamond-channel , MIMO wi channel , multius channel ,31,32,33 channel .In this work, we investigate extended versions of BCCM, ICCM, and MAC-WTC channel models. Information-theoretic security for discrete memoryless interference and broadcast channels with confidential messages were studied in . BCCM co1/2 . ICCM coach user . With anser [1/2 . In bothexact sum s.d.o.f. and the entire s.d.o.f. region, respectively, of the MAC-WTC. The exact sum s.d.o.f. of a K-user MAC-WTC is In MAC-WTC, which was introduced in ,35, multK(K−1)+1 . The achaltruistic and follow a prescribed transmission policy in order to maximize the sum secure rate of the entire system. In this paper, we investigate BCCM, ICCM, and MAC-WTC channel models in the case of selfish and malicious behavior, where the users/helpers do not perform the system-wide-optimal altruistic behavior but apply a selfish strategy and/or take sides by aiming to help one user and potentially hurt the other. These new models are extensions of the ones studied in [All these works assume that all nodes are udied in ,5,13 andBCCM with combating helpers, there are two helpers, where each helper takes the side of one of the receivers and at the same time aims to hurt the secure communication to the other receiver. The two helpers have contradicting objectives and hence are combating. Helpers in this model do not coordinate with the transmitter as in [In the first model, which is the er as in . We use er as in , which ier as in  in a recper user , as if bICCM with selfish users, there is an external system helper. In this model, the users do not coordinate as in the optimal strategy in [In the second model, which is the ategy in  instructategy in . There iMAC-WTC with deviating users, we first consider the case where M out of K users deviate by not transmitting cooperative jamming signals. We start by evaluating the achievable sum s.d.o.f. when the remaining users do not change their original optimum strategies. We show that the sum s.d.o.f. of the system decreases, and deviating users do not benefit from their actions. Then, we consider two possible counterstrategies by the remaining users: In the first strategy, all users decrease their rates to ensure that all message-carrying signals are protected by the remaining cooperative jamming signals, and leakage s.d.o.f. is zero. We show that, in this case, the individual s.d.o.f. of the deviating users increase. Hence, deviating users gain at the expense of well-behaving users. In the second strategy, we allow the leakage s.d.o.f. to be nonzero but constrain leakage in a single dimension. We show that, although the sum s.d.o.f. of the system is lower than in the case of the first counterstrategy, this strategy decreases the individual s.d.o.f. of the deviating users and increases the s.d.o.f. of well-behaving users. Next, we consider a more severe form of deviation by considering one user turning malicious and sending intentional jamming signals. As this deviating user has infinite power, it can wipe out all communication, secure or otherwise, if it sends Gaussian signals. For the sake of a meaningful formulation, we restrict the strategy set of this deviating user to be of structured signaling and alignment type. Under this restriction, we formulate the problem as an extensive-form game [In the third model, which is the orm game . We showOrganization: In n is the length of the codeword. Each message ith receiver, while being kept secure from the jth receiver, X and Y, and ith receiver. The s.d.o.f. P is the transmitter power constraint In BCCM, the transmitter has two private messages one of the receivers. The input/output relations for the BCCM with combating helpers Ycombating as they maximize the s.d.o.f. of one user only, while hurting the other user by sending jamming signals. The transmitter acts in even transmission frames, and helpers respond in odd frames. Each node has perfect channel state information (CSI) and knows the actions of others at the end of every frame. We require that the action of a helper does not hurt its own receiver (in terms of s.d.o.f.) if no new jamming signals are produced by the other helper. Consequently, we formalize the role of the ith helper as:jth user in the kth transmission frame, where k is odd. On the other hand, the transmitter does not take the side of any of the users and maximizes the sum s.d.o.f. of the system, i.e., transmitter’s role in even encoding frames is:The helpers are k, all secure and jamming signals are picked from PAM constellation set We use recursive real interference alignment as the achievable strategy for our model. At encoding frame ith helper sends a structured jamming signal Frames 0 and 1 are considered transient frames. For frame 0, the transmitter performs the optimal strategy in the presence of helpers  and sendThen, the received signals are:Although The transmitter knows that a new irrational dimension is generated within frame , β2 see . The recConsequently, the system retains full s.d.o.f. .If To calculate the s.d.o.f., we need the following lemma.Lemma 1.If every message-carrying signal is protected by a cooperative jamming signal, then the s.d.o.f. is given by:where Proof. From , the folkth frame at receiver 1, and P. Hence, the probability of error converges to zero as Let ry as in  as:(20)Since we designed the coding scheme at each frame so that Now, we are ready to formally calculate the resulting s.d.o.f. from the recursive real interference alignment in the following theorem.Theorem 1.For BCCM with combating helpers under the constraint of not decreasing the s.d.o.f. of their own receivers due to helper actions, the s.d.o.f. of each user evolves as:i.e., the combating behavior is asymptotically neutralized.Proof. k. For the base step Using Lemma 1, we have rms with . For k=2k is odd and For the induction step, assume that Solving these two equations gives ith receiver, while being kept secure from the jth receiver, Z. Inputs satisfy power constraints ith receiver in the kth transmission frame, and In ICCM, each transmitter has a message selfish and malicious. User i maximizes the individual s.d.o.f. at receiver ith user’s role is:less stringent than in the BCCM model, since in the ICCM model, we allow the users to hurt their own receivers if they hurt the other receiver more. On the other hand, the system helper does not take the side of any of the users and maximizes the sum s.d.o.f. of the system:The users are Similar to the BCCM, we propose using recursive interference alignment using the PAM constellation selfless strategy as in [All nodes perform the optimal gy as in . The traThe received signals at both receivers are as in :(36)Y1[0i maximizes j keeps its strategy as in frame 0. Each user prefers to jam the other user directly, even if it results in partial decrease of its own s.d.o.f. (by creating an extra dimension at its receiver), since in this case, it can drive the s.d.o.f. of the other user to zero and maximize the s.d.o.f. difference. Thus:User Hence, the received signals in this case are:Both users know that their communication links are jammed during frame Consequently, k is odd/even. If k is odd, each user chooses to jam all dimensions of the other user’s secure signals. This choice leads to k is even, each user takes advantage of the generated jamming by the other user plus the extra jamming signal from the system helper to protect more signals.The s.d.o.f. differs based on whether Theorem 2.For the ICCM with selfish users in the presence of a system helper, assuming that users maximize the s.d.o.f. difference for every transmission frame, the s.d.o.f. evolves as:i.e., selfishness eventually precludes secure communication.Proof. k is even. This follows by induction: For i jams all secure dimensions of user j in frame i in frame k to protect From , the ratk is even and expression Y1=∑Z, which is also subject to the power constraint Kth user without loss of generality, see k are: In the second part of the section, we consider a severe form of deviation where one user transmits intentional jamming signals. To distinguish that user and its jamming signal, we denote its channel input as M users have deviated from the optimum strategy in [Consider that ategy in  Xi=t C . The conhosen as :(55)Q=P1Consequently, the received signals are see :(56)Y1=∑Let j}. From ,15, the For the first term M dimensions lack cooperative jamming signals, i.e., we have For the second term Substituting (62) and (67) into , and takThat is, the sum s.d.o.f. decreases by timal in . This afIn this section, we consider two achievable schemes resulting from two different responses of the remaining users.In this achievable scheme, all users decrease their secure rates, i.e., decrease the number of message-carrying signal components to ensure that all of them are aligned with cooperative jamming signals. Specifically, the first Consequently, the received signals are see :(70)Y1=∑Let es using , after cThe components of Since all message-carrying signals are jammed by cooperative jamming signals, we have Substituting (75) and (79) into , and takThe resultant sum s.d.o.f. is less than the optimal in . Howeverategy in .In this achievable scheme, we allow one rational dimension to be leaked. This dimension is not secured by a cooperative jamming signal. This results in the ability of injecting an extra message-carrying signal component for each user. All these extra signals are aligned in the same rational dimension at the eavesdropper. The transmitted signals are see :(81)Xi=∑Although the sum s.d.o.f. in this case is smaller than in , the indKth user) sends intentional jamming signals. The deviating  user is restricted to using structured signals. In this section, we show that, when the malicious user acts, it can drive the sum s.d.o.f. to zero. However, when the remaining users respond, the sum s.d.o.f. is raised to In this section, we consider a more severe form of deviation, where a user (say the N rationally independent dimensions In any encoding frame, each user sends its message-carrying signals Then, the jammer designs structured jamming signals Consequently, the received signal at the legitimate receiver is:Hence, each message-carrying signal is aligned with a jamming signal. Let Hence, N dimensions:Since structured jamming signaling suffices to jam the system, the jammer sends structured signals in Users make use of the generated jamming signals to hide extra secure signals from the eavesdropper. Users send:We note that the received signals at receiver K-user MAC-WTC with M external altruistic helpers, as a result on its own. We note that this Further, since every message-carrying signal is protected by a cooperative jamming signal, We introduced three new channel models, namely, BCCM with combating helpers, ICCM with selfish users, and MAC-WTC with deviating users. These new models aimed at studying the effects of selfishness and malicious behavior on the secure rate in networks. We investigated the achievable s.d.o.f. in these models. The presented schemes are only achievable; new role-based converse arguments are needed.For the BCCM with combating helpers, we formulated the problem as an extensive-form game. We assumed that each helper wants to minimize the s.d.o.f. of the other receiver without sacrificing the s.d.o.f. of its receiver and analyzed schemes that employ recursive real interference alignment. In this case, we showed that the malicious behaviors of the combating helpers are neutralized and the s.d.o.f. of both users converge to For the ICCM with selfish users, we changed the objective function of the users to maximizing the difference of the s.d.o.f. between the two users. By similar analysis to BCCM, we showed that the selfishness precludes any secure communication, and the s.d.o.f. of two users converge to zero.Finally, for the MAC-WTC with deviating users, we considered two types of deviation: First, in the case when some of the users stop transmitting cooperative jamming signals as required by the optimal scheme, we evaluated the corresponding s.d.o.f. and proposed counterstrategies to respond to the deviation. Second, we investigated an extreme form of deviation, where a user sends intentional jamming signals. We showed that although a deviating user can drive the sum s.d.o.f. to zero, the jamming signals can be exploited as cooperative jamming signals against the eavesdropper to achieve an optimum s.d.o.f.


Streptococcus sanguinis, S. gordonii, S. oralis, and S. mutans were used as streptococcal bacterial strains. These bacterial cultures were grown for 24 h on CpTi and TZP. The number of bacterial adhesions was estimated using an ATP-bioluminescent assay, and scanning electron microscope (SEM) observation of the adhered bacterial specimens was performed. No significant differences in surface roughness or wettability were found between CpTi and TZP. In XPS analyses, outermost layer of CpTi included Ti0 and Ti4+, and outermost layer of TZP included Zr4+. In the cell adhesion assay, the adherences of S. sanguinis, S. gordonii, and S. oralis to TZP were significantly lower than those to CpTi (p < 0.05); however, significant difference was not observed for S. mutans among the specimens. The adherence to CpTi and TZP of S. mutans was significantly lower than that of S. sanguinis, S. gordonii, and S. oralis. These results were confirmed by SEM. S. sanguinis, S. gordonii, and S. oralis adhered less to TZP than to CpTi, but the adherence of S. mutans was similar to both surfaces. S. mutans was less adherent compare with the other streptococci tested in those specimens.The purpose of this study was to evaluate the adherence of streptococci to disks of titanium  and zirconia . CpTi and yttria-stabilized TZP disks with a mirror-polished surface were used as specimens. The arithmetic mean surface roughness (Ra and Sa) and the surface wettability of the experimental specimens were measured. For analyzing the outermost layer of the experimental specimens, X-ray photoelectron spectroscopy (XPS) analysis was performed.  Periodontal diseases are the result of an infection process in microbial colonization referred to as dental plaque. The dental plaque is primarily formed by early-colonizing bacteria (Streptococci), following which it is formed by late-colonizing pathogenic bacteria . Some lain vitro papers which focused on attachment of late-colonizing anaerobic bacteria in peri-implantitis [Peri-implantitis is defined as a pathological condition occurring in the tissues surrounding dental implants, characterized by inflammation of the peri-implant connective tissue and progressive crestal bone loss . In termlantitis ,9. Howevlantitis . CurrentStreptococcus sanguinis, S. gordonii, S. oralis, and S. mutans, were detected in subgingival and submucosal plaque samples in peri-implantitis [Recently, streptococcal species, including lantitis ,12. It iSince the 1990s, Zirconia (zirconium dioxide) has been gradually used in dentistry. Compared with other ceramics, zirconia, particularly tetragonal zirconia polycrystals (TZP), exhibit superior biomechanical properties, including a high toughness of fracture and bending strength . These pin vivo and in vitro studies have been performed; however, the results reported have been controversial concerning the numbers of adhered bacteria on CpTi vs. TZP [in vivo study, compared to CpTi abutments, TZP abutments revealed lower plaque accumulation and less BoP (bleeding on probing) [S. gordonii and few reports of S. oralis. S. sanguinis is also under discussion. Therefore, it is important to assess whether the adherence of Streptococci is dependent on the material properties of CpTi and TZP.In terms of microbial colonization and biofilm formation on TZP compared with CpTi surfaces, both  vs. TZP , 15–17. probing) . Howeverprobing) ,15–17. AThe purpose of this study was to evaluate the adherence of streptococci to disks of CpTi and TZP, and compare the adherence to CpTi with that to TZP.CpTi  and yttria-stabilized TZP  were used in this experiment. CpTi specimens measuring 13 mm in diameter and 0.5 mm in thickness were used. They ground gradually with silicon carbide paper down to 1,200 grit, following which they were polished with 3-μm diamond pastes and 0.06-μm colloidal silica. TZP specimens measuring 13 mm in diameter and 0.5 mm in thickness were also used. They were ground with 70- and 45-μm diamond disks, following which they were finely polished with 9- and 3-μm diamond pastes and 0.06-μm colloidal silica. A polishing machine  was used. They were cleaned by ethyl acetate, acetone, and distilled water, ultrasonically. Then, the washed disks were sterilized in an autoclave . The sterilized disks were stored for at least 24 h until use in dry conditions.The surface roughness was characterized using a 3D measuring laser microscope . The arithmetic mean of the departures of the roughness profile from the mean line and defined for a profile  was measured with measured length 4 mm and cut-off value 0.8 mm. In addition, the arithmetic mean of the departures of the roughness area from the mean plane  was measured with measured area 645×645 μm and Gaussian filter size 80×80 μm.Five specimens from each group were tested.The surface wettability of the specimens was measured by a contact angle meter , using double distilled water. Contact angles were measured at three different locations on each of the five samples, 15 s after application of the droplets. The volume of the distilled water drop was kept at 4 μL. Five specimens from each group were tested.XPS analysis was carried out to determine the composition of the outermost surface and chemical shift using an X-ray photoelectron spectrometer  with an X-ray source of Al Kα (monochromator), 15 kV and 15 mA to determine the intensity of Ti, O, and C for the CpTi specimen, and Zr, Y, O, and C for the TZP specimen. The binding energy of each spectrum was calibrated with a C1s of 285.0 eV.S. sanguinis ATCC 10556 , S. gordonii ATCC 10558, S. oralis ATCC 35037, and S. mutans ATCC 25175 were used. The bacteria were grown on plates containing Brain heart infusion  and 1.5% agar (Wako) for plate cultures. Pre-culture was performed in an anaerobic chamber  at 37°C for 1 day. A single colony on the plate was cultured in BHI broth for further 24 h. A fresh culture, following liquid culture for another 4 hours, was used for the adherence experiment. The optical density of every bacterial suspension was regulated with BHI broth to 0.2 at 660 nm by a spectrophotometer . The bacteria for subsequent experiments were grown on disks in 24-well plates.In terms of streptococcal species, The number of adherent bacteria to the disk surface was measured using an ATP-bioluminescent assay with a commercial kit . Following incubation with bacteria for 24 h, all bacterial fluids on the disks were removed and washed with PBS. BacTiter-Glo reagent was applied to the disk surface at 200 μL. Following this, the activity of ATP in the solution was measured using an automatic luminometer , and the relative luminescence was determined. Seven specimens from each material were used and at least three experiments were performed.The streptococci were cultured as described in the cell adhesion assay above. Following culture on the disks, the specimens were fixed with 1.25% glutaraldehyde in PBS for 2 h at room temperature. Then, the specimens were washed three times with PBS, and dehydrated using a graded ethanol series . The specimens were freeze-dried, sputter-coated with Au–Pd, and observed using SEM .The results are expressed as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism . The data were tested by Shapiro-Wilk test and they were parametric. The data were analyzed using the student’s t-test, and probability (p) values of <0.05 were considered statistically significant. In the cell viability assay between the materials, one-way analysis of variance, followed by Tukey’s multiple comparison test, was used.The Ra and Sa values of the experimental specimens are shown in The contact angles on the experimental specimens against double distilled water and a cross-sectional view of the water droplet are shown in The results of the XPS analyses of the outermost surface of as-polished CpTi and TZP are shown in S. sanguinis, S. gordonii, and S. oralis on TZP were significantly lower than those on CpTi (p < 0.05). However, no significant difference was observed in S. mutans between the specimens. The differences in bacteria are shown in S. sanguinis, S. gordonii, and S. oralis were significantly higher than that of S. mutans (p < 0.05).The cell viability of the four streptococcal strains examined in this study that adhered after 24 h was measured by the relative light unit (RLU), as shown in S. sanguinis, S. gordonii, and S. oralis, the same findings as in the cell viability assay, that bacterial adherence on TZP was significantly lower than on CpTi, were confirmed by SEM  were applied. The results indicated that S. sanguinis, S. gordonii, and S. oralis were less adherent to TZP than to CpTi, although the adherence of S. mutans to the former was similar to that to the latter. S. mutans was less adherent than the other streptococci tested in those specimens.The present In material assessment, Ra were 0.021–0.022 μm for CpTi and TZP disks. It was reported that Ra of a representative commercial Ti abutment, standard Brånemark abutment, was 0.21 μm. It indicated that a reduction in surface roughness (less than a roughness of 0.2 μm) had no major effect on the bacterial attachment ,20. It wS. sanguinis, S. gordonii, and S. oralis to TZP were lower than to CpTi; however, the difference was not observed in the adherence of S. mutans between the specimens. In a previous in vitro study, it was reported that S. sanguinis adhered more to CpTi than to TZP specimens [S. gordonii and S. oralis adhered more to CpTi than to TZP specimens. Song et al [S. epidermidis adhesion was positively correlated with the stiffness of this material, with Young’s modulus of 0.8–80 MPa. Besides adhesion, Saha et al [Escherichia coli and Lactococcus lactis grow better on softer  than harder  thin polyelectrolyte multilayer films. In the present study, S. sanguinis, S. gordonii, and S. oralis may have adhered more and grown faster on CpTi  than TZP . As the Young’s moduli of the materials in the present study were far from those of previous reports, the direct influence of Young’s modulus may be limited. The underlying mechanism still remains unknown, however, it becomes clear that the material stiffness affects bacterial adhesion and the physiology of the adhered cells [In the cell adhesion and SEM assays, the adherences of pecimens . In adding et al  investigng et al . Thus, bng et al . The isong et al . According et al . Reportsng et al . It was ng et al . Therefong et al  suggesteha et al  found thed cells .S. mutans to CpTi was similar to that to TZP. It has been reported that S. mutans exhibits a similar behavior in sandblasted acid-etched CpTi and machined CpTi surfaces [The present study found that the adherence of surfaces .S. mutans was less adherent than S. sanguinis, S. gordonii, and S. oralis to the CpTi and TZP disks. There may be two reasons for this. First, in general, it is reported that there are sucrose-independent and sucrose-dependent attachments in S. mutans. A higher concentration of sucrose exposure increases adherence compared with that in the absence of sucrose for S. mutans [S. mutans is sucrose-independent. This may have caused the reduced adherence of S. mutans. Second, compared with culture without disks in a 24-well plate, the adherence of S. mutans was ~20 times higher without disks than with the CpTi and TZP disks (Sup 1). Therefore, the reduced adherence may be due to the presence of the CpTi and TZP disks.In the comparison to each species, . mutans . In thisS. mutans, culture medium that does not contain sucrose may influence the result of the adherence assay. However, S. mutans was detected in subgingival and submucosal plaque in the peri-implantitis site,8 and the culture in this environment can be sucrose-independent.As a limitation to this study, in the culture of S. sanguinis, S. gordonii, and S. oralis were less adherent to TZP than to CpTi, although the adherence of S. mutans was similar to both CpTi and TZP. S. mutans was less adherent to CpTi and TZP compared with the other streptococcal species. It is possible that streptococci are relevant to peri-implantitis as early-colonizing bacteria on the machined TZP and CpTi abutment surfaces, to which attachment is difficult. In the future, effective prevention of peri-implantitis may be possible if it is possible to remove streptococci, the early-colonizing bacteria, as target. Further investigation is required to investigate the properties of late-colonizing pathogenic bacteria adherence following early-colonizing bacteria adherence.In the present study, S. sanguinis, S. gordonii, and S. oralis were less adherent to TZP than to CpTi, although the adherence of S. mutans to TZP was similar to that to CpTi. S. mutans was less adherent compared with S. sanguinis, S. gordonii, and S. oralis.S1 FigS. mutans was ~20 times higher without disks than with the CpTi and TZP disks.Compared with culture without disks in a 24-well plate, the adherence of (TIFF)Click here for additional data file.


Periploca sepium, as well as the relationship between these factors.Sand burial plays an irreplaceable and unique role in the growth and distribution of vegetation on the Shell Dike Island in the Yellow River Delta. There are still some unknown on the effects of sand burial on the morphology, biomass, and especially the stoichiometry of P. sepium. Shallow sand burial shortened the emergence time and improved the emergence rate, morphological and biomass of P. sepium compared to deep burial and the control. Burial depth significantly affected the nitrogen (N) and phosphorus (P) contents of the leaves. With deep burial, the carbon/nitrogen (C/N) and carbon/phosphorus (C/P) ratios decreased firstly and then increased with depth, while the nitrogen/phosphorus ratio (N/P) presented the contrary trend. Correlation analysis showed that the stoichiometry of N/P was positively correlated to morphology and biomass of P. sepium at different burial depths. Structural equation model analysis revealed that N was the largest contributor to P. sepium biomass.Shell sand burial depth had a significant influence on seedling emergence, growth, and biomass of P. sepium. Stoichiometry has an important influence on the morphological formation and biomass accumulation.Optimal burial depth is beneficial to the seedling emergence, growth and nutritional accumulation of  The Shell Dike Island, which locates in the southwest coast of the Bohai Sea in China, is formed by the confluence of the Yellow River and the Bohai Sea Estuary. It is the only shell dike in the world produced by the coexistence of old and new shells. The dike formed from the shells and debris of shellfish living in the intertidal zone, which has been carried by waves and deposited near the high-tide line. The wildlife resource in the dike and surrounding intertidal wetlands provides rich biological diversity and, is vital to studies of biodiversity and marine ecology . AffecteSowing and afforestation are important methods to restore the vegetation of the sandy land . CompareCarbon (C), nitrogen (N), and phosphorus (P) are the three main biological elements in plants , and theP. sepium, a deciduous shrub, is a dominant species with excellent medicinal value and plays an important role in the ecological protection of the Shell Dike Island. Due to human and natural factors, the distribution of P. sepium on the Shell Dike Island decreased. Therefore, it is important to study the physiological and ecological factors affecting P. sepium to protect and restore the ecology of the Shell Dike Island. Previous studies of the Shell Dike Island mainly focused on plant physiology, soil physical and chemical properties, and the biological diversity [P. sepium seedling could be influenced by elemental stoichiometry. Therefore, the aim of this study was to evaluate the effects of shell sand burial depth on the seedling emergence, morphology, biomass allocation, and nutrient content of C, N and P in leaves. Our specific objectives were to: (i) quantify the response of seedling emergence, morphology, and biomass allocation to shell sand burial depth; (ii) determine how leaf C, N, P concentrations and the leaf C: N: P ratio respond to different burial depths; (iii) identify the relationships between C, N, P concentration and total biomass. Our results would provide scientific support for the coastal environmental restoration. Further, it will provide a reference for judging how the stoichiometry affect plant biomass.iversity , 16. HowP. sepium seedlings first increased and then decreased . Burial depth of 4–5 cm inhibited seedlings emergence, emergence rate was only 26.7% of the depth of 5 cm, which was significantly lower than the untreated control .With increasing shell sand depth, the emergence rate of sed Fig. . There wP. sepium was suitable for shallow burial depth. Burial depth significantly affected the initial emergence time of P. sepium seedlings . These results showed that shallow sand burial was beneficial to P. sepium for shortening the time of first emergence.Burial depth had a significant influence on the seedling emergence process. Increased sand burial depth generally led to slower emergence rates Fig. . The emengs Fig. . The shoSeedling height, base diameter, leaf number, the third leaf area, root length and primary root number were measured for plants buried at different depths. Increasing shell sand burial depth initially led to increasing seedling height, but deeper burial depth resulted in decreasing height Fig. a. The plP. sepium varied with shell sand depth, the number of leaves initially increased with depth but decreased at larger depths  was observed at 3 cm burial depth, but this did not differ from the 2 cm treatment group. The leaf area of the control group was 1.52 cm2, and did not differ from the 5 cm treatment group.The leaf characteristics of ths Fig. c. Leaf nths Fig. d. The laP. sepium .Burial depth had a significant impact on the growth of the underground part of ium Fig. e; the roium Fig. f. The 0 P. sepium dry weight were shown in Table The effects of different shell sand burial depths on P. sepium first increased with burial depth and then decreased .The overall and above-ground biomass of sed Fig. a, and diium Fig. b. In terP. sepium leaves did not vary with burial depth, but the N and P contents first increased and then decreased with burial depth , with a standardized path coefficient of 0.96. This result showed that the N element in the leaf was the major contributor to the biomass of P. sepium, while C and P did not play a significant role.The relationships between the C, N, and P contents, total biomass and burial depth were further analyzed by structural equation model Fig. . It is rP. sepium, both of which are susceptible to external factors. Therefore, the experiment on the response of the seeds and seedlings to the depth of shell sand can reflect the survival, formation and distribution of P. sepium under shell sand burial.Sand burial influences the growth and distribution of vegetation on the Shell Dike Island in the Yellow River Delta, and plants have a certain tolerance limit to sand burial. If this factor does not exceed the tolerable limit of the plant, it will promote plant growth and development. However, if the burial depth exceeds tolerable limit, it will inhibit the normal growth and development of the plant . Seed geP. sepium have small material energy. When buried too deep, it is impossible for the seeds to unearth properly due to energy material depletion. Previous study has shown that excessive burial depth reduced the permeability of shell sand, led to seed dormancy, and affected the formation of seedlings [P. sepium leaves under sand burial, indicating that the stoichiometry of the leaves also played an important role in seedling morphology. This role may be very significant due to the fact that variation in the burial depths changed the microenvironment at the time of seedling emergence, and that seeds are sensitive and critical to changes in environmental conditions, and thus, affecting the morphogenesis of seedlings.The relationship between the burial depth and the rate of seed germination and seedling emergence is important for quantifying the effect of shell silt accumulation . Our reseedlings . Deep bueedlings . Light ceedlings , 26. WatP. sepium is distributed more to the stem when buried at 2 cm, resulting in an increase in above-ground biomass and a decrease in the proportion of below-ground biomass. This is consistent with studies which showed that sand burial changed the biomass distribution of seedlings and induced seedlings to allocate more resources to the above-ground or below-ground parts. For example, sand burial can reduce the biomass ratio of Nitraria sphaerocarpa on the ground and increase the biomass ratio of underground roots [Scaevola plumieri, sand burial improves stem growth and the replacement of leaf area [Sand thickness has an important influence on the biomass distribution of plant seedlings . The resnd roots . In Scaeeaf area . Howevereaf area . These seaf area .P. sepium in the present study. These results showed that burial depth had a significant effect on the nutrient utilization of P. sepium seedlings. Although the C content in P. sepium leaves was the largest at 2 cm depth, the difference between the treatment groups was not significant. The contents of N and P, however, first increased and then decreased with depth. At 2 cm, the contents were the largest and the differences were significant. These results indicated that P. sepium regulated the N and P inputs of the leaf tissue, but not the content of C to ensure its normal life activities under the sand burial.During plant growth and development, the accumulation of biomass is inseparable from the contents of C, N, and P . In thisP. sepium leaves. The changes of biomass and N and P content were similar with depth. The above indexes reached their maximum values at 2 cm depth, indicating that this is the optimal depth for biomass accumulation and nutrient utilize. Other studies have shown that stoichiometry affected the distribution of plant biomass, high N content can increase leaf photosynthesis, increase the accumulation of assimilation products, and significantly reduce the root stem ratio . Conversely, low N content increases the plant R/S [P. sepium leaves was unrelated to R/S. Thus, the stoichiometry of P. sepium leaves plays an important role in the accumulation of biomass during the seedling stage, but does not play a decisive role in the distribution of biomass during growth. This may be due to the fact that sand burial changes the soil microenvironment around plant roots, such as nutrient, which has an important impact on the distribution of plant biomass [Studies have shown that N and P in plants promote photosynthesis and growth and that the contents of N and P in the leaves are closely related to the chlorophyll content . N or P lant R/S . In this biomass . However biomass , 38.P values [P. sepium leaves were higher than the global average levels except the 2 cm burial group in the present study [P. sepium leaves was less than 14. According to the theory of nutritional restrictions [P. sepium seedlings are mainly limited by N. Judging from the N/P of P. sepium at different burial depths, the N/P was the highest at the 1-2 cm depths. These results demonstrate that shell sand burial at1–2 cm is the most favorable for P. sepium nutrient absorption.The C/N and C/P of plant represent the ability of the plant to assimilate and accumulate C, which reflects the nutrient use efficiency of the plant body. The use of nutrient elements by plants can be reflected by its C/N and C/P values . The C/Nnt study , 41. Thirictions , the groP. sepium life cycle. Future work should investigate how depth affects other stages of growth, development, and adaptation of P. sepium.The influences of different shell sand depths were investigated for seedling emergence, growth and nutrient utilization in the present study. However, we did not examine the dynamics of burial depth in different periods of the P. sepium showed similar patterns of variation with increasing shell sand burial depth. The leaf ratio of P. sepium first decreased and then increased with depth, while the root ratio showed the opposite pattern, and the stem ratio did not vary except at 2 cm depth. (3) The C content of the leaves did not change with increasing depth, but the N and P contents initially increased and then decreased. The contents of N and P in the leaves increased first and then decreased. The C/N and C/P first decreased and then increased with depth. The opposite pattern was observed for N/P. (4) There was a strong correlation between the stoichiometry of the P. sepium leaves and the seedling morphogenesis and biomass, indicating that the stoichiometry was an important factor in P. sepium seedlings morphogenesis and biomass accumulation. The study of stoichiometry and growth adaptation of the dominant plants on the Shell Dike Island under sand burial is of great importance to coastal environmental restoration efforts.There is a direct relationship between the depth of burial and the germination of seeds and seedling emergence. The proper burial depth can provide suitable environmental conditions for the seedling emergence. The seeds may fail to emerge as lack of oxygen or excessive mechanical resistance when the burial depth is too great. The depth of burial not only affects seedling emergence, growth and morphology, but also the seedling’s absorption of nutrient elements. The results of this study demonstrated that (1) shallow burial (1–2 cm) was conducive to seedling emergence, as emergence rate decreased with increasing depth. (2) The morphological characters and biomass of P. sepium seeds and shell sand were collected from May to October 2016 at the Shell Island and the National Nature Reserve in Binzhou . The seeds were collected in the experimental area of the Shell Dike Island. P. sepium is a deciduous shrub of the Asclepiadaceae family according to the Flora of China, which was formally identified by Professor Jingkuan Sun in our laboratory. The experimental techniques and plant materials used were strictly adhere to regulations of the People’s Republic of China on nature reserves. Ripe P. sepium seeds were naturally dried and stored at 4 °C. A voucher specimen has been deposited at the Shandong Provincial Key Laboratory of Eco-Environmental Science for Yellow River Delta, Binzhou University . The shell sand was sifted to remove impurities.P. sepium seeds were dibbled in a plastic basin filled with shell sand for each pot. Each treatment was repeated 3 times. The depths of seed burial were 0, 1, 2, 3, 4, and 5 cm, according to the methods described by Mou et al. [Thirty u et al. . The seeu et al. , 45).P. sepium were fully ground to assess the C and N contents using a Vario EL III Element Analyzer . The P content of the leaves was determined by molybdenum-antimony anti-spectrophotometry (perchloric acid - concentrated sulfuric acid) [After characterization of the physical parameters, the roots, stems and leaves were packed into self-sealing bags. Then, these materials were washed with distilled water and dried to constant weight. The dry weight of each part was measured and the leaves of ic acid) .Statistical analyses were conducted using SPSS 19.0 software . One-way ANOVA and Duncan multiple contrasts were conducted for significance analysis. Pearson correlation analysis was performed on the leaf stoichiometry and the morphological and biomass measurements. Structure equation model was used to analyze the relationship between depth, total biomass, and the leaf contents of C, N, and P by AMOS 17.0.2 software . The structure equation model was tested using the maximum likelihood (ML) method and the root-mean—square-error of approximation (RMSEA) indicator was used to evaluate the model fit. Origin 9.0 software  was used to draw the figures.Additional file 1: Fig. S1. Effects of different burial depths on the first emergence time of P. sepium. Different letters denote significant differences at P < 0.05.


T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda.Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples.We collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense.In total, 39/2,088  cattle, 25/400  pigs and 40/2,184  tsetse, were positive for T. b. gambiense positives in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l.The lack of  Trypanosoma brucei gambiense, the parasite responsible for the disease. Our study found T. brucei s.l. in local cattle, pigs and tsetse flies, with their respective prevalences as follows, 1.9%, 6.3% and 1.8%. Further analysis to establish identity of these positives to the sub-species level found that no cattle, pigs or tsetse were carrying the pathogen responsible for Gambian sleeping sickness. Our work highlights the difficulty of establishing the absence of a disease, especially in an extremely low endemic setting, and the limitations of some of the most commonly used methods.The decline of annual cases of West-African sleeping sickness in Uganda raises the prospect that elimination of the disease is achievable for the country. However, with the decrease in incidence and the likely subsequent change in priorities there is a need to confirm that the disease is truly eliminated. One unanswered question is the role that domestic animals play in maintaining transmission of the disease. The potential of cryptic-animal reservoirs is a serious threat to successful and sustained elimination of the disease. It is with the intent of resolving this question that we have carried out this study whereby we examined 2088 cattle, 400 pigs and 2184 tsetse for  Trypanosoma brucei gambiense. Similarly, East African sleeping sickness or Rhodesian HAT (r-HAT) results from an infection caused by T. b. rhodesiense. While T. b. rhodesiense has long been known to have a primarily zoonotic lifecycle, T. b. gambiense is considered to be largely anthropophilic with the parasites largely circulating between tsetse and humans only. T. b. gambiense has been identified in domestic animals such as pigs, sheep and goats [T. b. gambiense some of which were shown to be infective to tsetse. These observations suggest that it may be possible for animals to act as reservoirs hosts for T. b. gambiense [T. b. gambiense caught in areas without cases of g-HAT [T. b. gambiense has significant consequences if elimination by 2030 is to be achieved [th century there have been three major outbreaks of sleeping sickness resulting in hundreds of thousands of deaths. Crucially the third outbreak occurred after intense control efforts had reduced the number of HAT cases to near-elimination levels [The term "human African trypanosomiasis" (HAT) is used to describe two diseases that are clinically, geographically and parasitological distinct. The majority of HAT cases 98%) occur in West and Central Africa and are referred to as West African sleeping sickness or Gambian HAT (g-HAT) indicating the geographical range of the disease and the protozoan parasites responsible, nd goats , 2 as wend goats , 4. Simiambiense –7 and pl% occur in levels . The thrT. b. gambiense, and the evidence that tsetse can become infected through animal hosts, it is not known if zoonotic cases of T. b. gambiense act as cryptic reservoirs that play a role in sustaining transmission of gHAT. Modelling studies [Despite the well documented reports of animals infected with  studies –13 have  studies .T. b. gambiense from animal-infective T. b. brucei [T. brucei sub-species, although possible, has limited sensitivity, as single copy regions of the genome must be targeted [T. brucei s.l. will need to be assessed to verify there is enough DNA present to undergo the less sensitive sub-species PCR assay [T. brucei s.l. lack sufficient DNA to undergo the sub-species-specific detection assay.A limiting factor to analysing the role and importance of non-human hosts is the type of diagnostic method used to detect the presence of trypanosomes. Classically, microscopic detection of parasites in blood of a human is regarded as evidence of infection. However, for animal hosts this method is unable to distinguish human-infective . brucei . Molecul. brucei . Howevertargeted . IdeallyCR assay . To dateCR assay . It is lAn alternative to molecular methods are serological techniques including the card agglutination test for trypanosomiasis (CATT) and the trypanolysis (TL) test, however the CATT can produce false positives due to malaria and transient trypanosome infections , 20.The T. b. gambiense in N.W. Uganda. These assays were used to screen the local vector population and two potential, animal-reservoir populations; cattle and pigs. This work was carried out in a large-scale, using molecular methods to first identify cases of T. brucei s.l. and subsequently the sub-species of T. brucei with PCR assays.Here we use currently available molecular assays to determine the presence or absence of T. b. gambiense infection, although three participants did test positive for having had T. b. gambiense in the past [The North West of Uganda has nine districts, Nebbi, Arua, Koboko, Yumbe, Moyo, Adjumani, Maracha, Amuru and Gulu, of which Arua, Koboko, Yumbe, Moyo, Maracha, Amuru and Adjumani have historic sleeping sickness foci . Recordsthe past .In this study tsetse were caught in the district of Koboko and screened for trypanosomes and in both Koboko and Arua cattle were sampled and screened for trypanosomes. No sampling for tsetse was carried out in Arua due to a vector control programme being carried out in this district . Pigs weA total of 12,152 tsetse flies were caught along the Kochi River in the district of Koboko over a period of 16 months from April 2013 to July 2014. To catch tsetse, pyramidal traps  were depOf the tsetse caught, 6,664 were dissected at the field laboratory (333842N-269418E) to screen for trypanosomes in their mouthparts, salivary glands and midguts . Followietal. [After transportation to the Liverpool School of Tropical Medicine, at room temperature, samples were stored at 4ᵒC until DNA extraction, after which the samples were stored, in a separate fridge, at 4ᵒC. Each individually-preserved tsetse tissue underwent DNA extraction, previously described in Cunningham etal. . BrieflyT. brucei s.l. using the FIND-TBR primers (27). Alongside the cattle sampling, in August 2013, 400 pigs were sampled from seven sites across in Arua  vein. Blood was collected with three 50mm heparinised capillary tube which collected 35μl of blood. Two tubes were centrifuged at 8,000 rpm for three minutes and the buffy coat layer examined as a wet preparation at x400 magnification using a compound-microscope with a dark-field filter. The contents of the third capillary tube was transferred to a Whatman FTA card  and left to air dry before it was heat sealed in a foil pouch with a packet of silica gel to ensure the sample remained desiccated.To extract the DNA from the FTA card, a modified version of the method described by Ahmed was carried out as follows: 10x 2mm hole-punches were taken from each bloodspot, using a Harris micro-punch. The punches were then washed three times in 1ml of distilled water and then 135μl of a 1% Proteinase K/10% Chelex TE suspension was added to each batch of 10-hole punches. These were then incubated at 56ᵒC for an hour followed by 93ᵒC for 30 minutes. In total 14 sampling sites  were useT. brucei s.l.. Tsetse were processed with a nested multiplex primer set that targeted T. brucei s.l., T. congolense and T. vivax, whereas cattle and pig samples were screened with the FIND-TBR primers [etal. [T. congolense and T. vivax as well as T. brucei. The outer nest utilised the previously published forward and reverse primers that target the internal transcribed spacer region (ITS) of the trypanosome genome [T. congolense Kilifi (accession number U22317), T. congolense Forest (accession number U22319), T. congolense Savannah (accession number U22315), T. brucei s.l. (accession number JX910373), T. vivax (accession number U22316), T. godfreyi (accession number JN673383) and T. simiae (accession number AB742533). A new universal forward primer and three species-specific reverse primers were designed and used in a multiplex. The location of the new primers are shown in a diagrammatic form in relation to the universal primers designed by Adams etal.  to 433bp (T. congolense Savannah and T. congolense Forest). The products for T. brucei s.l. and T. vivax measure 342bp and 139bp, respectively  from tsetse.The PCR reactions were carried out at a final volume of 25μL each containing the following reagents: 2.5μl of 10X PCR Buffer , 200μM of each of the deoxynucleotide triphosphates (dNTPs) , 1.2mM of MgCl2 , 0.4μM of both the forward and reverse primers and 10μL of BIOTAQ Red DNA Polymerase (Bioline). The first reaction of the two nested PCRs and standard PCR used 5μL of DNA template. For the nested PCRs second reaction 1μL of the PCR product from the first reaction was used as the template. The primers used to detect T. brucei s.l. positive samples using either the multiplex ITS primers or the FIND TBR-PCR primers [T. b. gambiense species specific primers (TgsGP) [T. brucei s.l. positive samples were not identified down to the sub-species due to the lower sensitivity of the TgsGP primers [Following the detection of  primers , the pos primers . The FIN primers  whereas  (TgsGP) , target  primers , 36.T. brucei s.l. there was a need to determine what sub-species of T. brucei the sample belonged, be it T. b. brucei, T. b. gambiense or T. b. rhodesiense due to the significance of the presence of a human infective sub-species. The PCR assays for positive identification of T. b. rhodesiense and T. b. gambiense rely on the amplification of a single copy gene unique to either the West or East African parasite. If neither Gambian or Rhodesian HAT is detected, then it can be assumed the organism present is T. b. brucei. However, due to the difficulty of reliably amplifying a single copy gene, and therefore the low sensitivity of the two sub-species specific assays, there was a chance that a negative result occurs due to insufficient target DNA. To help increase the confidence of a negative result it was possible to determine if there was sufficient quantity of DNA by running an assay that amplifies a single copy gene ubiquitous to all three sub-species. The multiplex designed by Picozzi et al [T. b. rhodesiense. The multiplex consisted of universal Trypanozoon primers that target the T. brucei s.l. single copy gene, phospholipase C (PLC), as well as primers that target the serum resistance associated gene (SRA) for T. b. rhodesiense. Among the variable surface glycoprotein (VSG) genes there are regions with some sequence identity to the SRA gene. Between the VSG and SRA genes there is an internal deletion within the SRA genes that allows it to be distinguished between any VSG amplification and SRA amplification. Two pairs of primers were designed that amplify across this deleted region to allow for clear size distinction between a SRA PCR product and a VSG PCR product. The combination of primers results in the amplification of a 324 bp product for all Trypanozoon species, a 669 bp product for T. b. rhodesiense and a >1 Kb for any VSG products amplified  tsetse were positive for trypanosomes comprising 73 single midgut infections, 69 single mouthpart infections, nine mixed mouthpart-midgut infections, a single salivary gland-midgut infection and six cases where all three tissues were infected with trypanosomes. Of the 2,877 blood films examined from cattle, trypanosomes were identified in 568  samples, however, it was not possible to identify down to the species level using the microscopy methods.T. brucei s.l.. The number of tsetse positive for the three Salivarian species of trypanosomes were as follows: T. vivax 46/2184 , T. brucei s.l. 40/2184  and T. congolense 58/2184 . Of the T. brucei positive tsetse, seven had a single positive mouthpart, nine were single salivary gland positives, 20 were single midgut positives, two were mixed salivary gland and midgut positive and the remaining two had all three tissues positive. Only the presence of T. brucei s.l. in the mouthparts deviates from the accepted life cycle of this trypanosome and is either due to a transient presence of trypanosomes that have passed along the proboscis during a feed or the result of cross contamination between tissues during dissection  cattle and 25/400  pigs examined using the FIND-TBR primers were positive for ssection .Of the 2,184 samples screened with multiplex ITS primers, trypanosomes were observed by microscopy in 62 samples (49 flies), comprising 30 midguts, four salivary glands and 28 mouthparts. Of the microscopy positives the molecular assay identified 48 of 62 positive tissues, .T. brucei s.l. positive cattle, pig and tsetse samples were processed using the T. brucei s.l. multiplex assay to screen for T. b. gambiense, T. b. rhodesiense and the number of samples with enough DNA to detect down to a single-copy gene. While not expecting to identify any cases of T. b. rhodesiense the inclusion of a positive control for the East African form of the disease acts as another quality control , 15 (60%) and 25 (56%) were positive for the PLC gene, respectively, suggesting that sufficient DNA was present to detect DNA from T. b. rhodesiense or T. b. gambiense if it were present.The 103  control . None ofT. b. gambiense primer, TgsGP. Among the cattle and pig samples there was no amplification of a 308 bp product however 17 tsetse samples produced a band approximately 308 bp in size.The samples that proved to have enough genetic material for the amplification of the single copy PLC gene were then screened using the sub-species-specific T. b. gambiense using the NCBI database resulted in only a 90% identity and a query cover of 16% Of the 17 bands, a subsample of three were sent for sequencing to determine the specific product size and sequence. The samples were sent to SourceBioscience using both forward and reverse primers. The results of the sequencing showed that the three bands sent were identical and that the product was 281 bp (inclusive of primers) in length. The sequence when aligned against reference sequences for T. b. gambiense in local tsetse, cattle and pig populations from N.W. Uganda. The successful identification of T. b. gambiense would suggest that transmission of sleeping sickness in the area was continuing and the identification of the disease in either cattle or pigs would help resolve the role of animal reservoirs in the transmission of the disease.The aim of this paper was to determine the prevalence of T. brucei s.l. positive by PCR 56% were found to have enough DNA for the amplification of a single-copy gene region. Sixteen produced faint bands of approximately 300bp, comparable in size to the expected band size for T. b. gambiense. However sequencing results showed that the size of the product generated by the samples was smaller than the expected size at only 281bp compared to the expected 308bp sized product. There was also significant variation in the 281bp sized sequences compared to T. b. gambiense sequence. Based on the sequencing results these positive samples cannot be unequivocally identified as T. b. gambiense.Of the 40 tsetse samples identified as T. brucei gambiense, this could be due to T. b. gambiense no longer being transmitted in the area or that our sample size was too small to detect T. b. gambiense. Despite being understood as the sole vector of gHAT [T. b. gambiense under laboratory conditions have often proven unsuccessful [T. b. gambiense may be as low as 1 in 4,000 flies [T. b. gambiense being found, the tsetse population studied were actively transmitting T. b. brucei. Third, the TgsGP primers cross-reacted with DNA from an unidentified source and produced a band, similar in size, to T. b. gambiense, this raises concerns about the specificity of the TgsGP primers are and the potential for erroneously reporting the presence of T. b. gambiense.Three conclusions arise from the tsetse survey, the first is that despite screening 2,184 tsetse, no tsetse were found to be positive for  of gHAT  the prev of gHAT , 41 and ccessful . Studies00 flies . HoweverT. brucei or T. congolense similarly of the positive mouthparts all were infected with either T. congolense or T. vivax with no mono infections of T. brucei s.l. identified, although in cases of mixed infections T. brucei s.l. was detected in the mouthparts.T. brucei s.l. however there was one instance of a single T. congolense infection. The presence of a mITS positive does not indicate an infection of a specific tissue by the trypanosome detected but merely the DNA, which could be a transient trypanosome, free floating DNA or DNA introduced during the dissection step; previous studies have found similar results [The infected salivary glands were predominantly positive for  results . Althoug results , howeverT. b. gambiense infections nor where there any cases similar to those found in the fly samples, where non-target DNA was amplified. However, cases of T. b. brucei were found in both animal populations with pigs proving to have the higher prevalence of T. b. brucei infection. This is typical of trypanosome epidemiology which has been shown to be highly localised in other studies [T. b. gambiense in the two animal populations sampled correlates with a previous study carried out in the same area [No animal samples (pig or cattle) produced a band of either 308bp or 281bp when screened with the TgsGP primers, indicating that there were no zoonotic  studies . The lacame area , indicatame area , 46, andT. b. gambiense [T. brucei sub-species [T. b. gambiense are limited due to the practical aspect of conducting these assays in the field and the limited diagnostic markers available. As mentioned previously the sensitivity of T. b. gambiense specific PCR is limited to detecting a single copy gene, although it is possible that the sensitivity TgsGP PCR could be improved if the PCR was nested. However, how much more sensitive this assay would be is not known and it is likely that the sensitivity would remain lower than that of both the ITS and TBR PCR assays. Some molecular assays attempt to overcome this problem by relying on the human serums ability to lyse all salivarian trypanosomes (except for T. b. gambiense and T. b. rhodesiense) therefore any T. brucei s.l. identified in a human sample would be one of the two HAT species [T. brucei species group. Using this method any positives would have to be one of the two HAT species, however as the treatment of the two diseases differs and the only option to try and identify if it was an East or West African sleeping sickness infection would be to try and determine the geographical location of where the individual was infected. This approach would also only be limited to humans and could not be used in either xenodiagnoses or screening animals, as all three T. brucei sub-species could be present in the vector or animal populations. To put these differences of sensitivity in perspective, we can look at the limit of detection (LoD) of the number of trypanosome per mL, between multiple diagnostic methods (The diagnostic methods used in this paper involved both microscopy and PCR, of which only PCR has the potential to discriminate sub-species of ambiense , 34. The-species , 17, 34. species . This al methods ..T. b. gambiense in both the human, vector and local animal populations is sorely needed if the hope of eliminating sleeping sickness by 2020 is to be achieved. The importance of such a new diagnostic is also relevant to answering the question of potential human reservoirs of disease. Typically, HAT is still described as being a fatal disease if left untreated, leading to a general view that there are little to no human-reservoirs. However, there is evidence that the disease can remain un-detected for prolonged periods of times in asymptomatic humans, raising the very clear possibility that humans may themselves be cryptic reservoirs of disease [The lack of a highly specific, sensitive and field-friendly assay that is capable of screening for  disease . WithoutT. brucei s.l.. Despite not finding T. b. gambiense in the tsetse population of Koboko vector control has been calculated to being essential to reach the elimination goal of 2030 [This lack of positive samples reflects the overall low prevalence of the disease and the continued decrease in the number of cases in Uganda . This st of 2030


Phobocampe confusa, a Nymphalini specialist, responsible for high mortality rates in charismatic butterfly species in Europe (genus Aglais). We studied its ecology and genetic structure in connection with those of its host butterflies in Sweden. To this aim, we gathered data from 428 P. confusa individuals reared from 6094 butterfly larvae  collected over two years (2017 and 2018) and across 19 sites distributed along a 500 km latitudinal gradient. We found that P. confusa is widely distributed along the latitudinal gradient. Its distribution seems constrained over time by the phenology of its hosts. The large variation in climatic conditions between sampling years explains the decrease in phenological overlap between P. confusa and its hosts in 2018 and the 33.5% decrease in the number of butterfly larvae infected. At least in this study, P. confusa seems to favour A. urticae as host. While it parasitized nests of A. urticae and A. io equally, the proportion of larvae parasitized is significantly higher for A. urticae. At the landscape scale, P. confusa is almost exclusively found in vegetated open land and near deciduous forests, whereas artificial habitats are negatively correlated with the likelihood of a nest to be parasitized. The genetic analyses on 89 adult P. confusa and 87 adult A. urticae using CO1 and AFLP markers reveal a low genetic diversity in P. confusa and a lack of genetic structure in both species, at the scale of our sampling. Further genetic studies using high-resolution genomics tools will be required to better understand the population genetic structure of P. confusa, its biotic interactions with its hosts, and ultimately the stability and the functioning of natural ecosystems.The biology of parasitoids in natural ecosystems remains very poorly studied, though they are key species for their functioning. Here we focused on  Most biological studies of parasitoids have been done in the context of biocontrol in agricultural ecosystems. Such focus on parasitoids specialized on pest species, however, has limited our knowledge on the biology and function of parasitoids in natural ecosystems. For example, only a few of the over 100,000 ichneumonid species estimated are identified to date ,3,4. ThuIt is generally accepted that parasitoid species are sensitive to the interactions and population dynamics of their hosts and have their own habitat requirements . HoweverPhobocampe confusa is an important parasitoid of charismatic butterfly species in Europe (genus Aglais). In Sweden, P. confusa represents the second cause of larval mortality due to parasitism in A. urticae and A. io, after the tachinid Pelatachina tibialis [P. confusa is an ichneumonid of the Campopleginae subfamily. It is a solitary endoparasitic koinobiont, that is, the female lays an egg in the body of its host, which continues to function and feed until the parasitoid larva emerges, in this case before the pupation of its host. The parasitoid overwinters as a pharate adult in the cocoon [Aglais io, Aglais urticae, Araschnia levana, Nymphalis polychloros, and Polygonia c-album [P. confusa has not yet been systematically studied.tibialis . P. confe cocoon . As in H c-album , most ofP. confusa with one of its main host, A. urticae, to explore the potential biotic constraint induced by the host on the parasitoid and its dispersal. Here, we studied the ecology of P. confusa and document how it interacts with its host butterfly species. We aimed to (i) identify the temporal constraints imposed by the phenology of its main host species in Sweden, A. urticae and A. io, (ii) investigate preference of hosts, and (iii) better understand the distribution of this parasitoid species in the landscape. In addition, as the population dynamic of parasitoids are likely to be closely linked to that of their hosts, (iv) we characterized and contrasted the genetic structure of Phobocampe confusa has been recorded to parasitize several Nymphalini species but in the vast majority of the recorded cases, P. confusa emerged from two nettle-feeding butterfly species, Aglais io and A. urticae. Aglais urticae and A. io are widely distributed over most of Sweden. These species are closely related butterflies [A. urticae are partly bivoltine, depending on the weather conditions, with larvae observed in the field from May to the end of August. Populations of A. io are univoltine in Sweden and their phenology is slightly more restricted than for A. urticae, with larvae observed from late May to early August. Both Aglais species overwinter as adults.terflies  and showA. levana, has occasionally been reported to be parasitized by P. confusa. Its spatial and temporal distribution overlaps greatly with that of A. urticae and A. io. As with A. urticae and A. io, it almost exclusively feeds on nettle (Urticae dioica). The species is also batch-laying, but with comparatively reduced batch size of 10 to 40 eggs. It is an obligate bivoltine species, with larvae observed in the field from June to early September. A. levana overwinters in the pupal stage.Another Nymphalidae species which has recently established in the southern half of Sweden , A. levaA. io, A. urticae, A. levana, and V. atalanta, sampled over two years (2017–2018) and across 19 sites distributed along a 500 km latitudinal gradient in Sweden , regions (south versus north), and years. The measure of the phenological overlap is a continuous variable, varying from zero, if the time windows during which the parasitoid and the host do not overlap, to one, if these two periods are identical. The phenology of P. confusa and its host butterflies were constructed from the distribution of the number of individuals of P. confusa and the number of butterfly nests collected over all the weeks of sampling, at a site and for a given year. The phenological overlap was modelled using a linear model. The initial model included all the two-way interactions and model selection followed a backward elimination procedure. The phenological overlap between j and for year i between P. confusa and a host butterfly, or overlap parasitoid-host index (OPH), is the sum over the sampling weeks k of the minimum between the standardized abundance values of P. confusa  and the host  (Equation (1)). For P. confusa, standardized abundance data  refers to the number of individuals (NP) collected for a given sampling week k, at site j and for year i, and expressed in proportion of the total number of individuals of that species collected on all the samplings at the site j for year i (Equation (2)). For the host butterfly species (A. urticae or A. io), standardized abundance data refers to the number of nests collected for a given sampling week k, at site j, and for year i and expressed in proportion of the total number of nests of that species (NH) collected on all the samplings on the site j for year i (Equation (3)). The overlap index (OPH) is a parsimonious measure of the phenological overlap under the hypothesis that the parasitoid does not benefit from a surplus of resources [P. confusa and each of its hosts, were sampled at a site within a given year.Specifically, the phenological overlap at a site esources . The pheP. confusa attack rates on its two main host butterflies, A. urticae and A. io, in two ways. First, we studied the proportion of butterfly nests parasitized by P. confusa. This analysis was restricted to butterfly nests sampled within the time window of occurrence of P. confusa (see P. confusa was observed within a given year (n = 359 nests). Second, for the butterfly nests parasitized by P. confusa, we examined the proportion of larvae parasitized per nest by P. confusa (n = 145 nests). The proportion of butterfly nests parasitized, and the proportion of larvae parasitized by P. confusa per nest, were modelled with a binomial error distribution and a logit link function to model the number of nests and larvae parasitized out of the number of nests sampled and the number of larvae sampled in each nest. We analysed variations in parasitism rates according to butterfly host, region, larval instar at collection, the phenological overlap, the year and week of collection, and the total number of butterfly nests of both host butterflies (A. io and A. urticae) occurring in the week of sampling. Based on the nature of the distribution of such data over time, and preliminary exploration of the data, we included a quadratic term for the sampling week and phenological overlap. We also included the two-way interactions between the butterfly host and the region, the year, the larval instar at collection, the total number of butterfly nests at sampling, and the two-way interaction between region and year. Because few nests were collected at 1st instar, we pooled them with nests collected at 2nd instar. Model selection followed a backward elimination procedure. Model diagnostics were assessed using the R package DHARMa [We investigated differences in fusa see  and at se DHARMa .P. confusa. Using the models selected in the analyses of the proportion of butterfly nest parasitized and the proportion of larvae parasitized by P. confusa per nest (see above), we estimated the additional variance explained when including land cover variables. In the analyses, absences were informed by including data on butterfly nests collected at sites where P. confusa was not observed (n = 31), but that were sampled during its period of activity (https://www.naturvardsverket.se/). To assess the effect of land cover on the propensity and intensity of parasitism, we computed each metric within buffers of increasing radius  around each sampled nest. All metrics were calculated with the R packages sf [P. confusa per nest, were modelled with a binomial error distribution. Model selection followed a backward elimination procedure and model fits were assessed using the R package DHARMa [We examined how habitat heterogeneity and fragmentation influenced the distribution of activity . Land cokages sf  and rastkages sf . The lane DHARMa .P. confusa and A. urticae were studied using two types of molecular markers, a fragment of the cytochrome c oxidase subunit (CO1) mitochondrial gene, and amplified fragment length polymorphism (AFLP). AFLPs have been commonly used to study the population genetic structure of species since the publication of the method by Vos et al. [The genetic structure of Swedish s et al. . Althougs et al. ). P. confusa collected across 15 sites, and from abdomenal material of 87 adult A. urticae  collected across 8 sites spread across the latitudinal gradient using the NucleoSpin® 96 Tissue kit (Macherey–Nagel)  and we measured concentrations of about 30 ng/µL.DNA was extracted from whole body tissue of 89 adult y–Nagel) . After ehttps://www.fluxus-engineering.com/sharenet.htm). We used a maximum parsimony algorithm to infer the most parsimonious branch connections between the haplotypes.We sequenced the fragment of the CO1 gene proposed as a standard DNA barcode for animals  using LCP. confusa, only diploid females were used. Male Hymenoptera are haploids and carry only half of the genetic information that diploid females do. For this reason, using a mixture of both males and females could lead to ambiguous results. In addition, we genotyped only one individual per butterfly nest sampled in order to avoid genotyping related individuals which would, potentially, reduce the genetic variability of our sample. We kept only non-ambiguous AFLP results, which led to a total of 39 P. confusa AFLP genotypes and 86 A. urticae AFLP genotypes.To study the nuclear genetic variation of © software (Applied Biosystems). Eight individuals of P. confusa, and 12 individuals of A. urticae were genotyped twice to estimate the genotyping error rate. AFLP genotyping followed the protocol described elsewhere [We obtained the AFLP fragments from 600 ng of genomic DNA, digested successively with the TaqI and EcoRI restriction enzymes . The digested DNA was incubated at 37 °C for 3 h in the presence of adapter pairs corresponding to both types of restriction sites and T4 DNA ligase enzyme  before amplifying them by two successive PCRs using the EcoRI-A and TaqI-A primers, during the pre-selective PCR, and TaqI-AAC and EcoRI-AAC (FAM) primers, during selective PCR. The separation of the labelled AFLP fragments and the acquisition of the raw fluorescence data was performed by the “Genomics” platform of the Henri Mondor Institute by capillary electrophoresis (Applied Biosystem) in the presence of the LIZ 500 size marker. The obtained AFLP profiles were calibrated and analysed using the GeneMapperlsewhere ,32,33.a priori information about individual origin. We ran 5 replicate runs, with the number of clusters, K, ranging from 1 to 15, of a model of correlated frequencies, i.e., taking into account the similarity of the frequency of alleles between populations. We ran 100,000 iterations and sampled every 100 iterations.The genetic diversity statistics, i.e., the proportion of variable markers and gene diversity based on Nei’s formula , were caP. confusa individuals emerged from larvae collected from 146 different butterfly nests  and from 1 to 82 in 2018 . The species was absent from two sites in both years, site 31 and Odensjö. Additionally, P. confusa was not present in Ljungby and site 915 in 2017, and in 2018 it was absent from the sites 284, 569, 63, and Åsvägen. In our laboratory conditions, P. confusa adult emergence rate was of 29.0% with a total of 124 individuals that emerged, 48 males, 72 females, and 4 that we failed to sex. All the emergence of adults of P. confusa occurred within the year of its cocoon formation. The absence of emergence after winter diapause is probably the result of suboptimal husbandry of wintering cocoons.nd years . Across Phobocampe confusa is a solitary parasitoid, laying one egg per larval host in most cases. Nevertheless, we observed one case where a larva of A. io was parasitized by both P. confusa and the tachinid Blondelia nigripes. Aglais urticae and A. io were the two main hosts of P. confusa among the four butterfly species we sampled. 231 P. confusa larvae egressed from the 2254 A. urticae larvae collected, 196 out of the 2259 A. io, and 2 out of the 1583 A. levana.P. confusa and that of its host butterflies differs significantly among hosts and between regions and years . In the south, the phenology of P. confusa coincides better with that of A. urticae than with that of A. io . This difference between regions is consistent between the two years of sampling. In addition, we observe that although P. confusa rarely parasitizes A. levana (only two cases have been recorded), in the south where this butterfly is distributed, its phenology overlaps that of P. confusa in a manner comparable to the overlap observed with the native species A. io . The proportion of larvae parasitized by P. confusa per nest also varies with the larval instar at collection , deciduous forests, and artificial surfaces (building and road) . AlthougP. confusa, we obtained 88 sequences of a 613 bp fragment of the CO1 gene (GenBank Accession Numbers MT524354-MT524441). We detect 5 haplotypes . We detect 11 haplotypes . In turn, the variation in climatic conditions explains a large part of the observed decrease in phenological overlap between P. confusa and its native hosts. This decrease was most pronounced in the northern region, where we also observed in 2018 a shift toward the spring of the phenologies of the butterfly species  to 8.05% in 2018 (7 out of 84 native butterfly nests sampled). In addition to the importance of the overlap between the phenology of the host butterflies and P. confusa, the probability of detecting a case of a nest parasitized by P. confusa is strongly influenced by the larval stage at the time of collection and was highest for the nests for which the larvae were collected in the fourth larval instar. From larvae monitored in our laboratory rearing conditions (23 °C and 22L:2D light regime), we evaluate the time window of attack of a larval host by P. confusa to be of about a week (see SM 2). This time window of attack is most certainly longer in the wild, the mean temperature being lower, and probably longer for A. io than A. urticae due to its longer development time. We did not find any difference between native species in the probability of a nest to be parasitized; however, the intensity of parasitism, taken as the proportion of larvae parasitized per nest, differs between species and is significantly higher for A. urticae than for A. io. This result suggests that, at least in this study, P. confusa seems to favour A. urticae as host.The total number of larvae parasitized by cies see  and resuP. confusa. This aligns with previous studies showing that specialist species, as is the case for P. confusa, are particularly sensitive to climatic unpredictability [A. levana has recently established in Sweden, probably as a result of climate warming [A. levana larvae parasitized by P. confusa. This low level of parasitism might be explained by the enemy release hypothesis [A. levana is a potential host for P. confusa and the phenologies of these two species greatly overlap in Sweden, suggesting that A. levana could provide a refuge for P. confusa at a time when the native hosts are rare. Future monitoring of parasitism in A. levana and comparative data on the attack rate by P. confusa on A. levana in other parts of the butterfly’s range, and where the species are known to co-occur, would be insightful in that respect.The large between-year variation in climatic profile highlights the potential impact of warming on our study system. Climate change is a challenge for ectothermic species such as parasitoids and their butterfly hosts. As they do not produce heat, their development and survival rely on the temperature of their habitat . In Swedtability ,41. Howetability  reportedtability . Most imtability ,43,44, ctability , as show warming . Here, wpothesis ,47, whicP. confusa, preferentially occur in habitats characterized by vegetated open land and where deciduous forests are found in the close vicinity. At a scale of 10 m radius around the butterfly nests sampled, the surrounding habitat of 87.4% of the nests included open vegetated land and for 58.5% deciduous forests. Association with these habitats might partly be explained by the pattern of distribution of nettles, Urtica dioica, the  host plants of these butterflies. Nettles, common in northern Europe, are found in a diverse range of habitats but preferentially in nutrient-rich soils and in sites with moderate shading [P. confusa. The probability of a butterfly nest to be parasitized by P. confusa decreased significantly with increased proportion of artificial surface, but the proportion of larvae parasitized per nest significantly increases. Other studies have shown that parasitoids suffer from environmental changes such as habitat fragmentation and habitat loss , which has a significant negative impact on the propensity of a nest to be parasitized. The mechanisms by which artificial surfaces influence the distribution of P. confusa are difficult to assess and would require further investigation. Among potential explanations, the alteration (and unevenly so) of the nutritional content of nettles at close proximity to human habitation, or habitat fragmentation, may alter the parasitoid’s searching behaviour and ability to find a nettle patch and/or this proximity might be associated with higher mortality during the overwintering period, weakening the local populations . ,49. P. ciewed in ). The hiiewed in ,5.P. confusa. Here, we show that the CO1 genetic diversity is extremely low in this species, at least within the geographical scale of our study. We found only five different haplotypes which diverged by no more than 3 mutational steps  are more likely to have a history of inbreeding [P. confusa at the geographical scale of our study. In comparison, the CO1 genetic diversity observed in our samples of A. urticae was higher, with a total of 11 haplotypes  and the non-coding control region of the nuclear genome showed that at the scale of the distribution of the species high gene flow is the primary factor shaping its population genetic structure [To date, no genetic data have been made available for al steps . The lacbreeding . This laplotypes . Althouge et al.  have sugtructure .P. confusa and that of its host, since the dispersal ability of the host A. urticae is larger than the geographical scale investigated in this study. Although AFLPs were successfully used in this study , high-resolution genomics tools, such as restriction-site DNA sequencing , could Phobocampe confusa, a parasitic hymenopteran which represents one of the most species-rich insect groups [P. confusa was misidentified as Hyposoter horticola), although the species causes high mortality rates in very common and charismatic butterfly species probably throughout Europe. In particular, we showed that the occurrence of P. confusa relies on its phenological match with its host butterflies. It attacks similarly nests of A. urticae and A. io; however, the proportion of larvae parasitized per nest in our study was higher for A. urticae. Within our sample, the species occurred preferentially in vegetated open land and showed a high dependence on the occurrence of deciduous forests in the near surroundings. Artificial surfaces, however, seem to have a negative impact on the distribution of P. confusa. The genetic analyses did not reveal a genetic structure in our study population, and further work is required to understand what is structuring the population genetics of P. confusa, and to understand its dispersal abilities and its biotic interactions with its hosts. Such knowledge is crucial to further our understanding of the factors and mechanisms shaping the stability and the functioning of natural ecosystems, including for conservation efforts.In this study, we focused on t groups , to provt groups  (in whic