Eve Todesco
email: eve.todesco@aphp.fr

Nesrine Day

Nesrine DAY b Corinne Amiel

Stéphane Elaerts

Véronique Schneider

Laurent Roudiere

Stéphane Hue

Jean-Yves Liotier

Julie Bottero

Thomas L'yavanc

Michel Ohayon

Daniel Gosset

Vincent Thibault

Laure Surgers

Julie Chas

Sepideh Akhavan

Annie Velter

Christine Katlama

Georges Kreplak

Anne-Geneviève Marcelin

Marc-Antoine Valantin N Institional

High Clustering of Acute HCV Infections and High Rate of Associated STIs Among Parisian HIV-Positive Male Patients

Keywords: Hepatitis C, Coinfections, HIV, Sexually Transmitted Infections, Phylogeny, Homosexuality, Male

in 2007 [START_REF] Van Der Helm | The hepatitis C epidemic among HIV-positive MSM: incidence estimates from 1990 to 2007[END_REF]. In the Swiss cohort of patients living with HIV, the incidence of HCV infection among MSM patients increased by a factor of 18 between 1998 and 2011 (0.23/100 PY to 4.09/100 PY) [START_REF] Wandeler | Hepatitis C virus infections in the Swiss HIV Cohort Study: a rapidly evolving epidemic[END_REF]. This phenomenon has also been observed in cities such as Amsterdam [START_REF] Urbanus | Hepatitis C virus infections among HIV-infected men who have sex with men: an expanding epidemic[END_REF],

New York, or Sydney. Phylogenetic analyses revealed extensive networks of HCV transmission. And lastly, in Amsterdam, an expansion to non-HIV-infected MSM with highrisk behaviors and under Pre-Exposure Antiretroviral Prophylaxis (PrEP) was highlighted [START_REF] Hoornenborg | Men who have sex with men starting pre-exposure prophylaxis (PrEP) are at risk of HCV infection: evidence from the Amsterdam PrEP study[END_REF].

Furthermore, a high rate of reinfections among HIV-infected MSM was recently reported in an European study [START_REF] Ingiliz | HCV reinfection incidence and spontaneous clearance rates in HIV-positive men who have sex with men in Western Europe[END_REF], the UK [START_REF] Martin | Hepatitis C virus reinfection incidence and treatment outcome among HIV-positive MSM[END_REF], and the Netherlands [START_REF] Lambers | Alarming incidence of hepatitis C virus re-infection after treatment of sexually acquired acute hepatitis C virus infection in HIV-infected MSM[END_REF]. In France, the annual incidence of HCV was estimated at 3.42% in 2015 among the subgroup of MSM presenting a high risk behavior towards contamination [START_REF] Virlogeux | 14 Modeling HIV-HCV coinfection epidemiology in the direct-acting antiviral era: the road to elimination[END_REF]. Indeed, many studies have shown that AHC are associated with recreational drug use and risky sexual behaviors (e.g. unprotected anal intercourse, bleeding during sex, "fisting") [START_REF] Urbanus | Hepatitis C virus infections among HIV-infected men who have sex with men: an expanding epidemic[END_REF][START_REF] Larsen | Gaining greater insight into HCV emergence in HIV-infected men who have sex with men: the HEPAIG Study[END_REF], and links between past history of associated sexually transmitted infections (STIs) and HCV seroconversion have been identified [START_REF] Wandeler | Hepatitis C virus infections in the Swiss HIV Cohort Study: a rapidly evolving epidemic[END_REF].

In this context, many acute HCV infections were diagnosed in a restricted geographical area of central Paris (called « le Marais ») among HIV-positive MSM in 2014-2016. We explored the phylogenetic clustering of these infections and characterized associated STIs, in order to better understand the dynamics of acute hepatitis C transmission in this area and support the need of efficient screening and management strategies in a high risk population. France). Clinical information was retrospectively extracted from hospital electronic database or medical records and anonymized prior to analysis. This information included age of the participants, reported sexual orientation, HIV status, time of last negative HCV test (when applicable), HCV and HIV VL at sampling (when applicable), previous HCV genotype (when applicable), ALT, and STIs detected ≤ 1 month before the acute HCV diagnosis (for syphilis:

new diagnosis or reinfection defined as increase of Venereal Disease Research Laboratory (VDRL) titer by a factor ≥ 4). All subjects were informed by an information note and gave their consent to the study.

A C C E P T E D M A N U S C R I P T

Polymerase sequences (NS5b, 334 nucleotides, amino acids 228 to 338) were obtained by Sanger Sequencing from the first HCV-positive sample, as described before (Genbank accession numbers: MH378080-MH378160) [START_REF] Sandres-Sauné | Determining hepatitis C genotype by analyzing the sequence of the NS5b region[END_REF]. Subtype determination was performed using the Geno2pheno[HCV] tool [START_REF] Kalaghatgi | Geno2pheno[HCV] -A Web-based Interpretation System to Support Hepatitis C Treatment Decisions in the Era of Direct-Acting Antiviral Agents[END_REF].

Maximum likelihood phylogenies were reconstructed using FastTree 2.1.7 [START_REF] Price | FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix[END_REF], under the general time reversible model of nucleotide substitution (GTR+CAT with 20 rate categories)

and branch supports were estimated by Shimodaira-Hasegawa-like test [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]. Publically available consensus sequences (n=93 confirmed HCV genotypes/subtypes) and HCV sequences from local database (n=450, including 131 genotype 1a, 52 genotype 3a and 37 genotype 4d) were added to the dataset, after removing of the duplicates [START_REF] Smith | Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource[END_REF]. Clades with a branch probability≥0.80 and intra-clade genetic distances <0.02 nucleotide substitutions per sites were considered as representing transmission chains.

A C C E P T E D M

A N U S C R I P T Acute HCV infections were found in twenty-two transmission chains, (52/81 acute Hepatitis C viruses sequenced (64.2%; 1 short sequence of genotype 3a excluded), of which 13 reinfections), divided in 11 pairs and 11 clusters from 3 to 7 patients (Figure 1).

In detail, 27/39 acute genotype 1a hepatitis C, 5/8 acute genotype 3a hepatitis C and 20/32 acute genotype 4d hepatitis C infections were detected among the 10 (5 pairs and In addition, prevalence of STIs was not higher among reinfections than in first HCV infections (p=0.615).

A C C E P T E D M

A N U S C R I P T These results raise concerns about the transmission of drug resistant viruses, as previously described [START_REF] Abravanel | Transmission of HCV NS5A Inhibitor-Resistant Variants Among HIV-Infected Men Who Have Sex With Men[END_REF].

In addition, in our work, STIs were detected in 20% of the cases, which was very high given the very strict criterion of time (≤1 month before the AHC diagnosis). These results corroborated a global rise in sexual risk behavior well described since the early 2000s.

Furthermore, it is well known that STIs could facilitate permucosal infectious agent transmission through mucosal damage, increasing infectiousness of a low inoculum.

In this study, most of the patients were co-infected with HIV, which might has a critical role in HCV transmission. Indeed, it has been shown that HIV increases HCV plasma VL compared to mono-infected patients [START_REF] Sherman | Viral kinetics in hepatitis C or hepatitis C/human immunodeficiency virus-infected patients[END_REF], and causes defects in the gastrointestinal immune system [START_REF] Lackner | The gastrointestinal tract and AIDS pathogenesis[END_REF]. Lastly, HIV-serosorting practice to avoid condom use had been supposed to also have enhanced HCV infection or other STIs within HIV-infected men. With PrEP, the spread of HCV infections might change from HIV-positive to HIV-negative patients, as already described in Amsterdam [START_REF] Hoornenborg | Men who have sex with men starting pre-exposure prophylaxis (PrEP) are at risk of HCV infection: evidence from the Amsterdam PrEP study[END_REF]. In this study, surprisingly, 11.5% of HIV-infected patients had a detectable HIV VL. The median VL was relatively low but it reached up to 13,251 copies/mL, in a context of high-risk behavior. As expected, the most represented genotypes were 1a and 4d, the latter has been emerging

for several years among MSM [START_REF] Van De Laar | Acute hepatitis C in HIV-infected men who have sex with men: an emerging sexually transmitted infection[END_REF][START_REF] Vogel | Phylogenetic analysis of acute hepatitis C virus genotype 4 infections among human immunodeficiency virus-positive men who have sex with men in Germany[END_REF]. One genotype 3a cluster was detected in this study.

Genotype 3a, which is highly prevalent among European injecting drug users, was previously very rarely described in MSM cluster, contributing to the controversy about the importance of HCV contamination by injecting drug.

Our study presents some limitations: chains of transmission are certainly non-exhaustive, as it is usually the case in phylogenetic studies. Indeed, in the present study, acute HCV infection diagnosis was realized on virological results during HIV usual monitoring or in the event of symptoms. Moreover, number of reinfections might be underestimated as reinfections with the same genotype were not detected. In addition, even if the results were in accordance to previous reports, most patients were HIV-positive, a group generally more closely monitored than HIV-negative MSM before the spread of PrEP in France, potentially representing another recruitment bias of acute HCV infections. Nevertheless, 3 major hospitals and one of the main private laboratory of the area under consideration have participated to the study and conclusions of the study are meaningful. Moreover, it has been recently showed by modeling data that HCV eradication in Europe will be based in large parts on control of infections in the subgroup of MSM engaging in high risk behavior towards contamination, for whom high treatment coverage will be needed [START_REF] Virlogeux | 14 Modeling HIV-HCV coinfection epidemiology in the direct-acting antiviral era: the road to elimination[END_REF][START_REF] Martin | Can Hepatitis C Virus (HCV) Direct-Acting Antiviral Treatment as Prevention Reverse the HCV Epidemic Among Men Who Have Sex With Men in the United Kingdom? Epidemiological and Modeling Insights[END_REF].

In conclusion, our results highlight the need for frequent screening of STIs and HCV among HIV-positive MSM. HCV-screening by PCR could be considered to reduce the diagnostic window period. Moreover, the high clustering of acute HCV infections calls for a rapid treatment of not only chronic but also acute HCV infections among this population.

Preventive behavioral interventions are also urgently required.

1 HIGHLIGHTSINTRODUCTION

 1 High clustering of acute HCV infections among parisian HIV-positive male patients  High rate of associated sexually transmitted infections  Call for a rapid treatment of not only chronic but also acute HCV infections  Need of preventive behavioral interventionsEmerging acute hepatitis C virus (HCV) infections among HIV-positive men having sex with men (MSM) have been described worldwide in recent years. For instance, rising incidence of HCV infections in HIV-infected MSM patients has been estimated by European data network CASCADE to range from 0.09-0.22 per 100 person-years (PY) in 1990 to 2.34-5.11 per 100 PY

6 METHODS

 6 This retrospective, observational study enrolled 85 male patients from the geographical area « le Marais » of Paris, for whom an acute hepatitis C infection was diagnosed between May 2014 and April 2016 on virological results carried out by Virology departments of Pitié-Salpêtrière, Saint Antoine and Tenon hospitals, and by Cerballiance Laboratory. Highly likely acute hepatitis C was defined as a positive serology test and/or a positive HCV viral load (VL) associated with a negative HCV serology within the previous 12 months, or a positive HCV VL beyond 24 weeks of a successful treatment or spontaneous clearance with modification of HCV genotype (n=72). Possible acute hepatitis C was defined as a positive HCV VL with increase of alanine aminotransferase (ALT) ≥10 upper limit of normal (ULN) without any other etiology of hepatitis, or a positive HCV VL beyond 24 weeks of a successful treatment or spontaneous clearance without modification of HCV genotype (n=13). Highly likely acute and possible acute hepatitis C were treated as acute HCV infections in the study, without distinction. No contact tracing was performed. Patients were followed by the Departments of Infectious Diseases of the Pitié-Salpêtrière, Saint Antoine and Tenon hospitals or outside the hospitals by their referring doctors (Paris,

5 clusters from 3 8 Treponema pallidum, 5

 385 to 6 patient), 1 (1 cluster of 7 patients) and 11 (6 pairs and 5 clusters from 3 to 5 patients) transmission chains identified by genotype, respectively. Nine transmission chains were composed of acute HCV infections only (4 pairs and 5 clusters from 3 to 6 patients, including 4 reinfections in 1 pair and 2 clusters) whereas 13 were mixed with acute and chronic HCV infections from the background controls (7 pairs and 6 clusters from 3 to 7 patients, including 9 reinfections in 2 pairs and 4 clusters and 18 controls). Three reinfections A C C E P T E D M A N U S C R I P T were part of the genotype 3a cluster of 7 patients. The rate of reinfection was not different between the linked cases and the unlinked ones (p= 0.868). STIs were detected in 17 patients (20%) ≤1 month before the acute HCV diagnosis (Neisseria gonorrhoeae, 7 Chlamydia trachomatis, and 1 Giardia intestinalis). It must be noted that 56.2% of the STIs were detected among patients included in a HCV chain of transmission (n= 9/16, 1 non amplified), without any statistical significant difference with patients not included in a chain of transmission (p= 0.507). Two patients exhibited the same STIs among a cluster of 5 patients (Neisseria gonorrhoeae and Chlamydia trachomatis) and 2 among a cluster of 4 patients (Neisseria gonorrhoeae).

10 10 DISCUSSIONA

 1010 large number of acute HCV infections has been diagnosed among HIV-infected MSM patients in Paris during the period 2014-2016 and, in this study, many of them were part of a transmission chain, revealing a strong infection dynamic among this local population, as also suggested by the high number of reinfections. This is in line with recent report by Ingiliz et al. who showed that among 8 European centers from Austria, France, Germany, and the UK, the highest incidence of HCV reinfections was in Paris (21.8/100PY, 95% CI 11.3-41.8) [5].

Figure 1 .

 1 Figure 1. Phylogenetic tree of HCV subtype 1a (a), subtype 3a (b) and subtype 4d (c).

8 8 RESULTS

 8 Patients were mainly MSM (at least 81.2%, n=69; 16 sexual orientations unknown), with a median age of 41.3 years (IQR:35-46 years). Median HCV VL at sampling was 5.5 log 10 IU/mL

	(IQR:4.4-6.3 log 10 IU/mL), median ALT was 320 IU/L (IQR:113-581 IU/L) and 24.7% of the 85
	HCV infections were reinfections (n=21). Patients were infected with HCV genotype 1a
	(47.6%), 4d (39.0%), 3a (11.0%) and 2k (2.4%; 3 viruses not amplified) and at least 91.8% of
	them were co-infected with HIV (n=78; 2 HIV status unknown). No acute HIV infection was
	reported. Among the co-infected patients, 9 had a detectable HIV VL, from 20 to 13251
	copies/mL (IQR:29-364), in a context of low level viremia (n=3), blips (n=2), recent treatment
	initiation (n=1), absence of ART treatment (n=1), treatment non-compliance (n=1), and

resistance to the received treatment (n=1, loss to follow-up; Tenofovir resistance mutations: M41L, D67N, T215F, Emtricitabine: M184V and Raltegravir: N155H).

Acknowledgments

We thank all the patients who agreed to participate at the Green Way-Acute Hepatitis C study, all the participant doctors who followed the patients, in particular Drs Bernard CARDON, Laurent FONQUERNIE, Jean-Philippe GRIVOIS, Gérard ISRAEL, Myriam KIRSTETTER, François LAYLAVOIX, Bénédicte LEFEBVRE, Jean-Paul PACANOWSKI and Laurent WORMSER.

Declarations


M Gastaldi

D Dragomirescu

A Takacs

V Armengaud

Compact Phase Shifting Cell Based on Micro-Strip Slow Wave Lines

This paper addresses the design, the simulations and the measurements results of an original and compact MMIC phase shifter using micro-strip slow wave lines implemented in a 0.25µm SiGe BiCMOS process for Ku-band applications. The simulations and the experimental results demonstrate interesting performances compared to classical phase shifter topologies.

I. INTRODUCTION

Phase shifters are key components in phase array systems where they achieve beamforming functions. The modern phase array systems developed for telecommunications applications need an increased flexibility and real-time electronic command and control functions. A typical core-chip used for beamforming applications requires a large number of RF phase control points. The performance of such core-chip circuits drives the performance of the final beamforming systems [1]. As example, the classical solution adopted for the beamforming system of the broadcasting satellites involves a 'hybrid' approach. The digital circuits used for command and control functions in the beamforming system are implemented in CMOS based technology while the analog RF functions (phase-shifting and attenuator cells) are mainly implemented in GaAs technology. This 'hybrid' approach increases the complexity, the size and the cost of the beamforming system.

The integration capability on a silicon chip is a key issue allowing a significant footprint reduction beside the low power consumption [START_REF] Hangai | S-and C-Band Ultra-Compact Phase Shifters Based on All-Pass Networks[END_REF]- [START_REF] Noh | Ku-band Upconverter Multi-function MMIC using 0.25 µm SiGe BiCMOS Technology[END_REF]. Consequently, due to the characteristics of Si technologies, a large number of low cost phase shifters have been investigated [START_REF] Min | Single-Ended and Differential Ka-Band BiCMOS Phased Array Front-Ends[END_REF]- [START_REF] Kalyoncu | A 4-bit SiGe passive phase shifter for X-band phased arrays[END_REF].

In this paper, we introduce a novel and original BiCMOS phase shifting cell/topology based on the use of micro-strip Slow Wave Lines (SWL). This topology leads to a compact footprint (compared with the footprint of 'classical' topologies [START_REF] Noh | Ku-band Upconverter Multi-function MMIC using 0.25 µm SiGe BiCMOS Technology[END_REF]- [START_REF] Kalyoncu | A 4-bit SiGe passive phase shifter for X-band phased arrays[END_REF]) while a phase shift below 22.5° is targeted. Also, this topology constitutes a new alternative to other SWL phase shifters [START_REF] Bakri-Kassem; Mansour | Novel millimeter-wave slow-wave phase shifter using MEMS technology[END_REF].

The section II focuses on the proposed topology of the SWL Phase Shifter (SWL PS). The simulated and experimental results obtained for the manufactured 11.25° SWL PS cell are presented in Section III. The performances of the SWL PS cell can be further improved by using body floating or substrate connection techniques as discussed in Section IV.

II. PHASE SHIFTER DESIGN

This phase shifter uses SiGe cold FETs (no bias current) as switches. The phase shift is obtained as a delay between the two signal paths (Fig. 1 The SWL used to obtain the targeted phase shift is a two layer S-MSL [START_REF] Gastaldi | Micro-strip slow wave line for phase shifting cells[END_REF] that allows a more compact structure compared to the classical Shielded Coplanar Waveguide (S-CPW). The stack layer of the process is presented in Fig. 2. It offers two thick layers (TM2 and TM1) fitted for carrying the signal and three thin layers (M3, M2 and M1).

As depicted in Fig. 3, the conducting strip is located on the top metal layer (TM2) while the metal strips composing the ground plane are located on the two layers (TM1 and M3). This disposition of the ground strips on two metal layers prevents the E-field from penetrating the silicon and improves the Q factor [START_REF] Dinc | An X-Band Slow Wave T/R Switch in 0.25um SiGe BiCMOS[END_REF].

The slow wave line path has been implemented in 0.25µm SiGe BiCMOS process and measured by using a VNA (Anritsu ME7808A). The S-MSL was obtained after the optimization of the Strip Length (SL), the Strip Spacing (SS) and the Gap parameters. The experimental results show an improvement of the phase constant by a factor 2.4 compared to a classical MSL (at 15GHz) while the attenuation constant remains almost equivalent [START_REF] Gastaldi | Micro-strip slow wave line for phase shifting cells[END_REF].

III. RESULTS

The phase shifting cell was fabricated using IHP 0.25µm BiCMOS process. The S-Parameters measurements were performed with a VNA using a standard SOLT calibration procedure. A photo of the dye can be seen on Fig. 4. The size of the phase shifter excluding the pads is 300µm x400µm. In order to simulate the behaviour of the cell, SWLs were electromagnetically simulated on HFSS and then imported on ADS to be combined with the simulation of the transistors. The targeted phase shift was 11.25° (with an authorized deviation of +/-3°) in the Ku-band: 10.7 GHz -14.5 GHz. The experimental results for ON (V c =2.5 V) and OFF (V c =0 V) states are shows in Fig. 5 (insertion loss) and Fig. 6 (return loss). The measurements show important insertion loss for the phase shifting cell. This drawback can be explained by the fact that the bulk of every transistor is directly connected to the ground. Solutions to correct this issue are proposed in section IV. The return losses are under -8.5dB for the targeted band (10GHz -15GHz). The difference between the simulations and the measurements can be explained by (i) the impact of the pads (aluminium) that is not taken into account in simulations and (ii) the insertion losses of the transmission lines (especially S-MSL) that are underestimated in HFSS. Insertion loss of the SWL PS for ON/OFF states Fig. 6. Return loss of the SWL for the ON/OFF states Fig. 7 shows the simulated and measured phase shift of the SWL PS as function of frequency. The differences between the simulations and the measurement are due to the overestimated (in the simulation) phase advance of the SWL line. The FETs being used as switches do not consume DC current (the measured DC current is less than 0.1 µA). Moreover the SWL PS exhibits a quite small linear variation of the phase shift in the targeted frequency band that is very useful from an application point of view. This cell is even more promising when compared to a 'classical' MMIC phase shifter [START_REF] Hangai | S-and C-Band Ultra-Compact Phase Shifters Based on All-Pass Networks[END_REF]- [START_REF] Min | Single-Ended and Differential Ka-Band BiCMOS Phased Array Front-Ends[END_REF]. A 11.25° hybrid/classical phase shifter was fabricated on the same run for comparison purposes. Unfortunately this circuit was impacted by a (not detected during design rule check) layout error and the experimental results are not exploitable. However a 45° classical phase shifter cell was fabricated on the same run. The inductors and capacitors values were calculated by the equations given in [START_REF] Min | Single-Ended and Differential Ka-Band BiCMOS Phased Array Front-Ends[END_REF] (L s =450pH, L p =1050pH, C=105fF). This cell allows us to check the correlation between the adopted simulation technique under Cadence/ADS and the experimental results. Fig. 8 shows the simulated and the experimental results for the 45° classical PS cell.

Fig. 9 demonstrates that the SWL-PS topology is more compact as compared with the classical PS topology. The footprint of the SWL-PS is reduced by a factor 3.5 as compared with the footprint of the 'classical' phase shifter when a phase shift of 11.25° is targeted. Size comparison between hybrid topology and SWL based solution

IV. DISCUSSION

As presented in section III, the insertion losses of the phase shifting cell are quite important. Retro-simulations were performed in order to isolate the source of the losses and two solutions are proposed to correct this drawback. It was been found that the main part of the losses was generated by the transistors. The losses added by the switching FETs can be reduced by [START_REF] Dinc | An X-Band Slow Wave T/R Switch in 0.25um SiGe BiCMOS[END_REF]: (i) connecting the bulk of the FETs to their source (Bulk Connected to Source: BCS) and (ii) using the Body Floating (BF) technique. BF technique consists of the connecting a 5kΩ resistor to the bulk of the transistors [START_REF] Dinc | An X-Band Slow Wave T/R Switch in 0.25um SiGe BiCMOS[END_REF]. BF technique improves also the linearity and the return loss of the cell. The impact of these techniques (BF and BCS) was simulated by using a co-simulation approach. HFSS software was used for the electromagnetic simulation of the S-MSL. The obtained S-parameters (by HFSS simulation) were injected in ADS software that was used for circuit simulation. Return loss (simulation) of the SWL PS by using BF (diamond marker) and BRC (square marker) techniques compared with the measured results of the fabricated SWL PS (no marker). A new run including an optimized version of the 11.25° SWL-PS cell with pads allowing the access to the bulk is currently in fabrication and more experimental results will be available for the Conference.

P1dB (dBm)

VI. CONCLUSION

This paper presents an original and compact phase shifter topology using micro-strip slow wave lines (SWL-PS). The proposed phase shifter exhibits a very low DC power consumption and is a more compact solution compared to the classical hybrid topologies: the footprint is reduced by a factor 3 for a phase shift of 11.25°. This solution remains more compact that the classical topology for a phase shift below 22.5° and constitutes a new alternative to the classical phase shifters. The experimental results for the first manufactured sample based on this innovative SWL-PS show good results in terms of phase shift and return loss. The insertion loss is quite important but as demonstrated by the simulation results these insertion losses can be drastically reduced by using BF or BCS techniques. It was also proven that the BF technique improve the input 1dB compression point with at least 8 dB for the SWL PS. Moreover the experimental results demonstrate that the SWL PS topology exhibits a small linear variation of the phase shift over a large frequency bandwidth. This characteristic can be very attractive from critical application (e.g. broadcasting satellite telecommunications) where large uplink and downlink bands should be covered by the same beamforming system.

  a). The first transmission line is a classical Micro-Strip Line (MSL) and the other transmission is a Shielded Micro-Strip Line (S-MSL). For comparison purposes, the 'classical' topology is represented in Fig 1 b. This topology is valid for phase shifter between 90° and 11.25° [4][5].

Fig. 1 .

 1 Fig. 1. Proposed phase shifting cell based on SWL (a) proposed topology (b) example of the classical topology

Fig. 2 .

 2 Fig. 2. S-MSL topology and cross section of the BiCMOS process

Fig. 4 .

 4 Fig. 4.Photo of the phase shifting cell (250µm x 400µm excluding pads)

Fig. 7 .

 7 Fig. 7.

Fig 8 .

 8 Fig 8.Phase shift of the 'classical' 45° PS cell

  Fig 9.Size comparison between hybrid topology and SWL based solution

Fig. 10 (

 10 insertion losses) and Fig. 11 (return losses) show the experimental and simulated results for the manufactured SWL-PS compared with the simulated results obtained by using the proposed BCS and BF techniques. As depicted in Fig. 10 and Fig. 11 the use of BF or BCR techniques allows the reduction of the insertion loss and the improvement of the return loss. The BF technique improves also the linearity of the PS cells as represented in Table I (simulation results). The 1dB input compression point is improved with at least 6 dB as function of the topology and of the state (ON/OFF) of the PS.

Fig 10 .

 10 Fig 10. Insertion loss (simulation) of the SWL PS by using BF (diamond marker) and BRC (square marker) techniques compared with the measured results of the fabricated SWL PS (no marker).

Fig 11 .

 11 Fig 11.Return loss (simulation) of the SWL PS by using BF (diamond marker) and BRC (square marker) techniques compared with the measured results of the fabricated SWL PS (no marker).

P1dB (dBm):OFF state ON state

	PS 45°	3.65	17.9
	PS 45° + BF	11.55	24.55
	PS 11.25°	3	12
	PS 11.25° + BF	11	22
	SWL PS	15	19
	SWL PS +BF	23	29.2
	TABLE I.
	1dB COMPRESSION POINT OF THE PHASE SHIFTER CELLS

ACKNOWLEDGEMENT

The authors would like to acknowledge CNES (French National Spatial Center) for supporting this work by a research grant. They will also like to thank both Thales Alenia Space and CNES for the Ph.D. grant of Mathieu Gastaldi.


Zhening Yang

Alexandru Takacs

Samuel Charlot

Daniela Dragomirescu

Kapton Flexible Technology for V-band Applications

come

I. INTRODUCTION

Nowadays, there are increasing demands for flexible and wearable electronics. Also the targeted frequency dedicated for emerging applications increases. The millimeter spectrum and especially Vband are now addressed for short-range and high bit-rate communications. For such high frequencies, Kapton flexible substrate has become a trusted candidate. The Kapton exhibits: good RF and thermal properties, very good flexibility over a wide temperature range and good tolerance for many chemical solvents. This paper focus on the recent advances in the development of Kapton flexible technology for V-band applications. Exhaustive investigations of the Kapton behavior in V-band including the quantified impact of the ambient humidity are presented. An assembling process of electronic chips by heterogeneous integration on flexible Kapton is also proposed.

II. V-BAND HUMIDITY MEASUREMENT OF KAPTON

Three passive devices were selected as benchmark for this technology: a ring resonator, a microstrip patch antenna and a crossed slot dipole antenna. All above mentioned devices, represented in Fig. 1, are appropriate for humidity tests due to their intrinsic sensitivity to dielectric property variation. The humidity test method is based on IPC-TM-650 2.6.2.1 standard technique [1]. The test samples were firstly dry out in an oven for over 30 minutes at 150 °C, each of them was individually weighed immediately. Then the test samples were immersed in a crystallizing dish filled with deionized water for 12 hours (hr). Each sample was removed independently from the water and sprays dried with nitrogen and weighed again. The same process was repeated for 24 hr and 48 hr respectively. The weight was increased with 1.64% after 12 hr, 1.78% after 24 hr and 1.81% after 48 hr.

The S-parameters measurements were carried out right after the weighing of the samples. Fig. 2 shows the results obtained for the ring resonator, the patch antenna and the crossed slot dipole antenna. The extracted values for the relative dielectric permittivity are reported in Table I. taking into account the associated tolerances.

III. FLIP CHIP ASSEMBLING TECHNIQUE

For the purpose of integration of different components on Kapton polyimide, different dummy circuits were mounted by using a flip chip technique [START_REF] Jatlaoui | Wireless communicating nodes at 60 GHz integrated on flexible substrate for shortdistance instrumentation in aeronautics and space[END_REF]. probe method was used to the bumps resistivity as shown in Fig. 3.a). A resistance about 10 mΩ was measured for the gold bump, which proves a very good DC contact.

VI. CONCLUSION

In this paper, several resonant passive devices were fabricated on Kapton flexible substrate. A standard humidity test was performed in V band. The experimental results demonstrates that the dielectric constant of the Kapton increases with maximum 15.6 % (up to 3.7 from 3.2 the initial value) and the dielectric losses increases with maximum 181% (up to 0.045 from the initial value of 0.016) when the Kapton structure were immersed in water after 48 hours. A limitation of the water absorption was also observed after 24 hours of the water immersion. The DC and RF measurements of dummy circuit mounted with flip chip technique show very promising results for a future 3d heterogeneous integration.

2 .

 2 . 1. a) Ring resonator; b) Microstrip antenna; c) Crossed slot dipole antenna aMeasured S parameter of a) ring resonator; b) microstrip antenna and c) crossed slot dipole antenna

Fig. 3

 3 Fig. 3. a) Four probe measurement; b) 50 Ω CPW transmission line mounted on Kapton; c) Measured Sparameters of CPW transmission line with flip chip mounted transition For the RF performance evaluation, a 50 Ω CPW transmission line on was mounted on Kapton as shown in Fig. 3.b). The measurement results of S-parameters show us a good RF performance in Fig. 3.c).


Prasanta Sarkar
email: prasanta.sarkar@univ-grenoble-alpes.fr

Giovanni Ghigliotti

Marc Fivel

Jean-Pierre Franc

Numerical investigation of the dynamics of pressure loading on a solid boundary from a collapsing cavitation bubble

Keywords: cavitation, cavitation erosion, bubble dynamics, fluid-structure interaction

The motivation behind this research lies in understanding the physical mechanism of cavitation erosion in compressible liquid flows, with applications in the field of aerospace, hydrodynamics, diesel injectors etc. As a consequence of collapsing vapor cavities in cavitating flow near solid boundaries, high pressure impact loads are generated. These pressure loads are believed to be responsible for the erosive damages on solid surface observed in most applications. For our investigation, the initial geometry is a single vapor bubble near a solid boundary collapsing due to the pressure difference between the bubble and surrounding liquid. The numerical approach employs a simplified homogenous mixture or 'single fluid' model with barotropic assumption in a fully compressible finite-volume fluid solver. The numerical method is validated against the well-known Rayleigh collapse of a pure 3D vapour bubble. It is then used for the simulation of a 2D vapour bubble collapsing in the proximity of a solid boundary placed at a specified distance from the centre of the bubble. The pressure loads are computed from the evolving dynamics of collapsing bubble near a solid boundary which can be used to determine the resulting surface deformation. The developed compressible cavitation solver in the CFD code 𝑌𝐴𝐿𝐸𝑆2 can efficiently model small and large scale cavitating structures in a fully resolved three dimensional flow.

Introduction

The collapse of cavitation structures formed due to the breaking down of the continuous liquid medium under very low pressures, near a solid surface leads to surface damage known as cavitation erosion. The driving mechanism for the cavitating structures is the local decrease in liquid pressure below typically vapor pressure at a given temperature [START_REF] Franc | Fundamentals of Cavitation[END_REF]. The cavitating regions consist number of cavitation bubbles due to the rapid growth of initially present air nuclei in the liquid flow. These cavitation bubbles move along the liquid flow until they collapse in the areas of high pressure and disappear. The presence of air nuclei, amount of dissolved gas in the flow are determining factors for the amount and size of cavitation bubbles. The motivation of this research is to numerically investigate the unsteady compressible cavitation bubble behaviour in a continuum liquid flow, with associated compressibility effects inside the flow domain. It will be used to predict accurately the material surface response to these small time-scale, highly unsteady flow characteristics. The vapor bubble collapse is usually characterized by the shrinking of bubble surface, acceleration of liquid flow towards the centre of bubble and shock propagation during the collapse. During the final stages of the collapse, due to the collision of bubble surface and liquid jet at the center, a high impulse shock wave is produced and propagated into the surrounding liquid. The collapse of a cavitation bubble close to a solid boundary is significantly affected by the non-dimensional stand-off parameter (), defined as the ratio of distance of the bubble center from the solid boundary to the maximum bubble radius. The dynamics of the bubble collapse and its interaction with the solid boundary such as the liquid micro-jet formation, splashing and surface erosion is strongly dependent on the stand-off parameter. Comprehensive description of the effect of the stand-off parameter may be found in the paper of Philipp and Lauterborn [START_REF] Philipp | Cavitation erosion by single laser-produced bubbles[END_REF]. For an isolated bubble, the collapse is basically symmetric and a shock wave forms near the center of the bubble which propagates into the surrounding liquid. For an asymmetric bubble collapse near a solid boundary, a liquid re-entrant micro-jet pierces through the bubble surface opposite to the solid boundary accelerating one side of the bubble towards the opposite surface. The liquid jet is directed towards the solid boundary and the estimated speed of the micro-jet is usually very high, several hundred meters per second. Multiple shock waves are emitted due to the impact of the liquid jet with the opposite bubble surface and eventually, the solid boundary. The high amplitude, localized pressure impulse at the solid boundary due to the emitted shock and micro-jet results in material surface damage or erosion. The collapse of a pure vapor bubble surrounded by a liquid can be represented as as figure 1 where 𝑃 𝑏𝑢𝑏𝑏𝑙𝑒 is the pressure inside the bubble and 𝑃 𝑙𝑖𝑞𝑢𝑖𝑑 is the surrounding liquid pressure such that 𝑃 𝑙𝑖𝑞𝑢𝑖𝑑 ≫ 𝑃 𝑏𝑢𝑏𝑏𝑙𝑒 . Since one of the primary consequence of cavitation is its effect on nearby solid boundaries, a detailed investigation is needed on the final stage of bubble collapse with high temporal and spatial resolution. A feedback of solid material response can then be introduced into the fluid domain using a two-way coupled fluid-structure interaction model. The numerical investigation of collapsing cavitation bubble is challenging due to the need of high spatial and temporal resolution to capture the instantaneous high amplitude pressure loads.

CFD Solver 𝒀𝑨𝑳𝑬𝑺𝟐

A pressure-based semi-implicit algorithm is used in the multi-physics solver YALES2 [START_REF] Moureau | An efficient semi-implicit compressible solver for large-eddy simulations[END_REF] for compressible flows with a fractional-step method [START_REF] Kim | Application of a fractional step method to incompressible Navier Stokes equations[END_REF] which is based on a characteristic splitting of the Navier-Stokes equation. The method consists of an advection or predictor step and a pressure-correction step separating the acoustics from the advection. In the pressure-correction step, a Helmholtz equation for pressure is solved implicitly to remove the acoustic CFL limitation. The compressible formulation of the governing flow equations allows its hyperbolic treatment to include the time dependent flow characteristics in the solution. Liquid flows with cavitation undergo strong negative pressure gradients for liquid break up to initiate cavitation. The speed of sound may be quite small in the two-phase region of a cavitating flow. For cavitation phase transition in a compressible liquid flow numerical methodology should resolve a wide range of Mach numbers (𝑀), defined as the ratio of flow velocity to sound speed in the flow field. In the twophase liquid-vapor mixture, local Mach number can be extremely high 𝑀 ≫ 1 whereas pure liquid phase stays close to low Mach number regime 𝑀 → 0. In the low Mach limit, the compressible 𝑌𝐴𝐿𝐸𝑆2 solver tends towards an incompressible solver inheriting the same stability and efficiency. In the solver, the wall boundary condition is treated implicitly by imposing a no-slip velocity in the advection or predictor step. Outlet boundary conditions are treated explicitly with classical Navier-Stokes Characteristic Boundary Conditions (𝑁𝑆𝐶𝐵𝐶) [START_REF] Poinsot | Boundary Conditions for Direct Simulations of Compressible Viscous Flows[END_REF] to enforce acoustically nonreflecting boundaries. A consistent equation of state is used for the pure liquid phase using the Tait's equation of state and for the two phase liquid-vapor mixture following an isentropic phase transition model proposed in Egerer et al. [START_REF] Egerer | LES of turbulent cavitating shear layers[END_REF]. The two phase modelling using a homogenous mixture or 'single fluid' is widely accepted and implemented with the barotropic fluid assumption which specifies that the pressure is only a function of the fluid density. The model uses a single set of governing equations for all phases with consistent equations of state and fluid viscosities. Since the cavitation model is assumed to be barotropic, the energy equation is decoupled from the system of governing equations in the solver. The speed of sound in the pure liquid phase is computed as 𝑐 = √𝜕𝑃 𝜕 ⁄ whereas in the liquid-vapor phase it is constant equal to the speed of sound at  𝐿 𝑠𝑎𝑡 . This is implemented to eliminate numerical instability of the pressure correction equation due to steep change in the speed of sound at the interface of pure liquid and liquid vapor mixture phase.

Results

The two phase model for compressible liquid is validated for an isolated 3D single bubble collapse case with the analytical model of Rayleigh-Plesset described in [START_REF] Franc | Fundamentals of Cavitation[END_REF]. The analytical model considered assumes liquid incompressibility and no liquid viscosity, gravity and surface tension. The analytical model considers a bubble saturated with pure vapor whose pressure is equal to vapor pressure 𝑃 𝑣𝑎𝑝 = 2194 𝑃𝑎 and surrounding liquid pressure 𝑃  = 10 𝑀𝑃𝑎. The numerical simulation is carried out with a bubble filled with two phase mixture of 99% saturated vapor content inside or simply, a vapor void fraction  = 0.99 . The isentropic cavitation model implemented assumes that phase change from pure liquid to liquid-vapor mixture starts at 𝑃 𝑣𝑎𝑝 = 2340 𝑃𝑎. The pressure decreases along an isentropic path as the density of liquid-vapor phase decreases, in other words, with increasing vapor void fraction in the bubble. At initialisation, the bubble is filled liquid-vapor mixture of phase density 10 𝑘𝑔/𝑚 3 and phase pressure of 2194 𝑃𝑎. The 3D spherical bubble is initialised inside the flow domain at time 𝑡 = 0 𝑠 with initial bubble radius 𝑅 0 = 500 µ𝑚. The surrounding liquid is initialized with a 𝑃  = 10 𝑀𝑃𝑎 and the bubble starts to collapse at 𝑡 > 0 𝑠 under the influence of the surrounding liquid pressure. The computational domain for the 3D simulation is cubical in shape with numerical boundaries located at 25 𝑅 0 away from the bubble center in all dimensions. The bubble is spatially resolved with 100 cells in the initial bubble radius 𝑅 0 . The bubble interface is defined by the variation of vapor void fraction  = 0 in the pure liquid to  = 0.99 inside the bubble. Further, the collapse of an isolated vapor bubble attached to a solid boundary is investigated in a 2D setup. The initial radius of the bubble is 𝑅 0 = 500 µ𝑚 and the bubble is placed near the boundary as such to obtain a stand-off parameter of 0.8. The bubble is initialised with a vapor void fraction  = 0.99 saturated inside at vapor pressure 𝑃 𝑣𝑎𝑝 = 2194 𝑃𝑎 and surrounded by liquid pressure of 𝑃  = 100 𝑀𝑃𝑎. The bubble is placed on the bottom surface of a rectangular domain which is numerically treated as a rigid boundary. An acoustically non-reflecting outlet boundary condition is imposed at other faraway boundaries of the rectangular domain situated 25𝑅 0 distance away from bubble center to avoid the interaction of reflected shock waves with the final stages of the collapse. The bubble is spatially well resolved with 100 cells along the initial bubble radius 𝑅 0 . The problem was simulated with a temporal resolution of 𝑡 𝐶𝐹𝐷 = 5. 10 -10 𝑠 , chosen from the minimum time step computed from CFL and acoustic CFL condition during the moment of collapse and kept constant throughout the computation. Figure 3a shows the initial set up of the 2D bubble geometry at 𝑡 = 0 𝑠. The bubble interface remains well defined during the complex collapse phase and a liquid micro-jet is formed during the final stages of the collapse from the opposite side of the rigid boundary due to asymmetry of the problem. The pressure evolution at a point K on the bottom boundary surface is presented in figure 3b, extracted at every 0. The remaining bubble ring after the liquid micro-jet impact contracts due to the surrounding liquid pressure towards the center of the bubble. The impacting liquid jet rebounds from the solid boundary and contributes to the shrinking of remaining vapor bubble ring. The remaining bubble ring is thus, impacted by an inward moving flow surrounding the bubble and an outward moving flow from the liquid jet rebound. As the bubble ring collapses, two shock waves are emitted that travel through the domain with liquid speed of sound. The second pressure peak of 3920 𝑀𝑃𝑎 is created at 𝑡 = 3.2079 𝑠 due to the interaction of the travelling shock waves at the bubble center as seen in figure 5.

Conclusion

This work has been devoted to the investigation of the dynamics of a single vapor bubble collapse. The fluid is assumed to be compressible and viscous for the problem. Using the described numerical method, a 3D bubble collapse in an unbounded fluid has been realised and compared with the analytical Rayleigh-Plesset result. There is good agreement between the numerical simulation and analytical result for the 3D bubble collapse case. The compressible cavitation solver developed is used to investigate the collapse dynamics of a vapor bubble near a solid boundary in a 2D set up, for an initial bubble radius 𝑅 0 = 500𝑚 and stand-off parameter  = 0.8. The bubble collapses under the influence of surrounding liquid pressure 𝑃  = 100 𝑀𝑃𝑎 and two distinct pressure peaks are observed. The first peak of 1986 𝑀𝑃𝑎 is due to impacting liquid micro-jet and a much stronger second pressure peak of 3920 𝑀𝑃𝑎 from the interacting shock waves after the collapse of bubble ring. This predicted instantaneous pressure will be used in the FSI methodology to predict the solid boundary response.

Figure 1 :

 1 Figure 1: Vapor bubble collapse due to imposed pressure difference Pliquid >> Pbubble; collapse of an isolated bubble -symmetric collapse (top); bubble collapse near a solid boundary -asymmetric collapse (bottom)

  The time step for the resolved problem is 𝑡 𝐶𝐹𝐷 = 510 -10 𝑠 based on CFL and acoustic CFL condition. The total simulated collapse is completed within the computed Rayleigh time 𝑡 𝑟𝑎𝑦𝑙𝑒𝑖𝑔ℎ of 4.610 -6 s. The comparison of the simulated 3D bubble collapse by the developed 𝑌𝐴𝐿𝐸𝑆2 compressible cavitation solver and theoretical Rayleigh-Plesset collapse is shown below in figure2. The plot shows the evolution of bubble radius 𝑅 with respect to time 𝑡, both normalized by initial bubble radius 𝑅 0 and Rayleigh time 𝑡 𝑟𝑎𝑦𝑙𝑒𝑖𝑔ℎ respectively. The solid line is the solution obtained from Rayleigh-Plesset analytical equation and the dots represent the equivalent radius from the bubble vapor volume obtained from the simulation. A decent agreement between the analytical and numerical result is obtained during the collapse, demonstrating the ability of the developed compressible cavitation solver to predict the dynamics of bubble collapse effectively.

Figure 2 :

 2 Figure 2 :Simulation of a single 3D spherical bubble collapse with 𝑌𝐴𝐿𝐸𝑆2 and comparison with Rayleigh Plesset collapse model for Rmax= 500 m, P=10 MPa. Numerical bubble collapse setup (left); evolution of non-dimensional radius with respect to non-dimensional time (right)

  1 𝑛𝑠 during the simulated collapse. The considered point K is located right under the bubble to capture the pressure evolution due to impacting liquid jet and subsequent shock waves. The initial pressure on the point is the saturation vapor pressure 𝑃 𝑣𝑎𝑝 = 2194 𝑃𝑎 due to the attached bubble on the boundary. It is assumed that there is no liquid film layer between the attached bubble and solid boundary. Until 𝑡 = 3.0326 𝑠, the pressure at the point remains at 𝑃 𝑣𝑎𝑝 as the bubble shrinks and formation of micro-jet starts. Two distinct pressure peaks can be seen in the pressure plot at 𝑡 = 3.0715 𝑠 and 𝑡 = 3.2079 𝑠 of 1986 𝑀𝑃𝑎 and 3920 𝑀𝑃𝑎 respectively. The first observed pressure peak is due to liquid jet impact and second peak due to the collapse of the bubble ring or torus, similar to the observations made in Chahine et al.[START_REF] Chahine | Modelling cavitation erosion using fluid-material interaction simulations[END_REF].

Figure 3a (

 3a Figure 3a (left) : : Initial bubble setup with represented point K in the bottom solid boundary where pressure evolution is plotted; Figure 3b(right): Pressure evolution at point K in the bottom solid boundary, Rmax = 500 m,  = 0.8, P = 100 MPaThe corresponding pressure and density contours for the observed pressure peaks are shown next in figure4and figure5. The first pressure peak of 1986 𝑀𝑃𝑎 is the liquid jet with an estimated velocity of 880 𝑚/𝑠 impacting the solid boundary and producing an instantaneous water hammer like pressure load at 𝑡 = 3.0715 𝑠.

Figure 4 :

 4 Figure 4: Pressure (top) and density (bottom) contour for a 2D bubble collapse showing the liquid micro-jet impacting the solid boundary, corresponding to the first pressure peak of 1986 MPa seen at time t = 3.0715 s

Figure 5 :

 5 Figure 5: Pressure (top) and density (bottom) contour for a 2D bubble collapse showing the interaction of shock waves after the collapse of the bubble torus, corresponding to the second pressure peak of 3920 MPa at t = 3.2079 s


Lucy Alford

Richard Marley

Anthony Dornan

Jean-Sébastien Pierre

Julian A T Dow

Ronald J Nachman

Shireen A Davies

Assessment of neuropeptide binding sites and the impact of biostable kinin and CAP2b analogue treatment on aphid (Myzus persicae and Macrosiphum rosae) stress tolerance

Keywords: Aphicide, Cold Tolerance, Desiccation, Ligand-Binding, G-Protein Coupled Receptors Receptor-Mapping, Pest Control

BACKGROUND: Neuropeptides are regulators of critical life processes in insects and, due to their high specificity, represent potential targets in the development of greener insecticidal agents. Fundamental to this drive is understanding neuroendocrine pathways that control key physiological processes in pest insects and the screening of potential analogues. The current study investigated neuropeptide binding sites of kinin and CAPA (CAPA-1) in the aphids

Accepted Article

Myzus persicae and Macrosiphum rosae and the effect of biostable analogues on aphid fitness under conditions of desiccation, starvation and thermal (cold) stress. RESULTS: M. persicae and M. rosae displayed identical patterns of neuropeptide receptor mapping along the gut, with the gut musculature representing the main target for kinin and CAPA-1 action. Whilst kinin receptor binding was observed in the brain and VNC of M. persicae, this was not observed in M. rosae. Furthermore, no CAPA-1 receptor binding was observed in the brain and VNC of either species. CAP2b/PK analogues (with CAPA receptor cross-activity) were most effective in reducing aphid fitness under conditions of desiccation and starvation stress, particularly analogues 1895 (2Abf-Suc-FGPRLa) and 2129 (2Abf-Suc-ATPRIa), which expedited aphid mortality. All analogues, with the exception of 2139-Ac, were efficient at reducing aphid survival under cold stress, although were equivalent in the strength of their effect. CONCLUSION: In demonstrating the effects of analogues belonging to the CAP2b neuropeptide family and key analogue structures that reduce aphid fitness under stress conditions, this research will feed into the development of second generation analogues and ultimately the development of neuropeptidomimetic-based insecticidal agents.

Introduction

With a global dependence on broad-spectrum insecticides, the damaging effects of which are well documented, [START_REF] Pimentel | Environmental and economic costs of pesticide use[END_REF][START_REF] Wilson | Why farmers continue to use pesticides despite environmental, health and sustainability costs[END_REF] there is increasing need for the development of greener, target-specific insecticides. The development and employment of neuropeptide synthetic analogues offers a promising avenue in the drive for greener and target-specific insecticidal agents. Within the insects, neuropeptides are regulatory peptides with functional roles in growth and development, behaviour and reproduction, metabolism and homeostasis, and muscle movement. [START_REF] Altstein | Neuropeptide signaling in insects[END_REF] Due to their high specificity, neuropeptides and their cognate receptors (Gprotein coupled receptors, GPCRs) may be developed as an insecticidal target system [START_REF] Nachman | Interaction of mimetic analogs of insect kinin neuropeptides with arthropod receptors[END_REF][START_REF] Audsley | G protein coupled receptors as targets for next generation pesticides[END_REF] to selectively reduce the fitness of target pest insects, whilst minimising detrimental environmental impacts.

The two neuropeptide families selected for study include the insect kinins and CAP2/CAPA peptides. Insect kinins are multifunctional neuropeptides which share a conserved C-terminal pentapeptide motif Phe-X 1 -X 2 -Trp-Gly-NH2, where X 1 can be His, Asn, Ser or Tyr, and X 2 can be Ser, Pro or Ala. [START_REF] Holman | Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly, Musca domestica[END_REF] The insect kinins have been identified in most insects, with the exception of Coleoptera, [START_REF] Halberg | Tracing the evolutionary origins of insect renal function[END_REF] and have diverse roles in the stimulation of muscle, [START_REF] Schoofs | The myotropic peptides of Locusta migratoria: structures, distribution, functions and receptors[END_REF] fluid secretion in renal tubules, [START_REF] Coast | The diuretic activity of a series of cephalomyotropic neuropeptides, the achetakinins, on isolated Malpighian tubules of the house cricket Acheta domesticus[END_REF][START_REF] Dow | Insights into the Malpighian tubule from functional genomics[END_REF] digestive enzyme release, [START_REF] Harshini | Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae)[END_REF] inhibition of larval weight gain [START_REF] Nachman | A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae[END_REF] and the desiccation and starvation stress response. [START_REF] Cannell | The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin Accepted Article neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster[END_REF][START_REF] Zandawala | Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila[END_REF] The second family, the CAPA peptides, were first identified from the moth Manduca sexta (CAP2b) [START_REF] Huesmann | Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta[END_REF] and have since been identified in many insect families. [START_REF] Davies | Signaling by drosophila capa neuropeptides[END_REF] Although function varies depending on insect species, life stage, and lifestyle, CAPA peptides play a key role in myomodulation and osmoregulation [START_REF] Davies | Signaling by drosophila capa neuropeptides[END_REF] and have more recently been linked to desiccation and cold tolerance in Drosophila species. [START_REF] Terhzaz | Insect capa neuropeptides impact desiccation and cold tolerance[END_REF][START_REF] Terhzaz | Renal neuroendocrine control of desiccation and cold tolerance by Drosophila suzukii[END_REF]

Accepted Article

The CAPA peptides belong to the PRXamide superfamily which can be further subdivided into three major classes: CAPA peptides, pyrokinins (PK) and ecdysis triggering hormone (ETH). [START_REF] Predel | Biology of the CAPA peptides in insects[END_REF] The pyrokinins are further subdivided into diapause hormone (DH) and pheromone biosynthesis activating neuropeptides (PBAN) and by their C-terminal motifs WFGPRLamide and FXPRLamide respectively. [START_REF] Predel | The FXPRLamide (Pyrokinin/PBAN) Peptide Family, in Handbook of biologically active peptides[END_REF] The GPCRs of this ligand group form a homologous cluster, suggesting co-evolution of ancestrally related ligand-receptor partners.

As a result, some cross activity by analogues of the ligand sub-groups with respective, recombinant receptors has been observed. [START_REF] Jiang | Functional phylogenetics reveals contributions of pleiotropic peptide action to ligand-receptor coevolution[END_REF][START_REF] Jiang | Functional characterization of five different PRXamide receptors of the red flour beetle Tribolium castaneum with peptidomimetics and identification of agonists and antagonists[END_REF] For this reason, certain PK analogues that have previously demonstrated cross activity on recombinant CAPA receptors of Tribolium castaneum have been included in this study. In particular, analogue 1895 (Table 1) has exhibited agonist activity, and analogues 1896 and 1902 (Table 1) antagonistic activity on T. castaneum TcCAPAr. [START_REF] Jiang | Functional characterization of five different PRXamide receptors of the red flour beetle Tribolium castaneum with peptidomimetics and identification of agonists and antagonists[END_REF] Furthermore, PRXamide analogues with the addition of hydrophobic moieties at the N-terminus have been shown to display greater biostability in-vivo, [START_REF] Zhang | Disruption of insect diapause using agonists and an antagonist of diapause hormone[END_REF] as featured in 1895, 1896 and 1902. Subsequently, 2089, 2123, 2125 and 2129 (Table 1) were designed and synthesized as second generation analogues of 1895, 1896 and 1902 to be evaluated in the current study.

Insect kinins are prone to rapid degradation by peptidases within the insect haemolymph and bound to tissues. The angiotensin converting enzyme (ACE) from the housefly is capable of cleaving insect kinin at the primary hydrolysis site, and neprilysin (NEP), at both the primary and secondary hydrolysis sites, [START_REF] Cornell | Cloning and expression of an evolutionary conserved singledomain angiotensin converting enzyme from Drosophila melanogaster[END_REF][START_REF] Lamango | Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family[END_REF][26][START_REF] Nachman | Active conformation and mimetic analog development for the Pyrokinin/PBAN and Myosuppressin insect neuropeptide families[END_REF][START_REF] Nachman | Aib-containing analogues of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensinconverting enzyme and potent diuretic activity[END_REF] thus representing biostability issues. In order to overcome this issue, incorporation of a single α-amino isobutyric acid (Aib) into the third position of the insect kinin active core protects the primary hydrolysis site from tissue-bound peptidase. [26][START_REF] Nachman | Active conformation and mimetic analog development for the Pyrokinin/PBAN and Myosuppressin insect neuropeptide families[END_REF][START_REF] Nachman | Aib-containing analogues of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensinconverting enzyme and potent diuretic activity[END_REF][START_REF] Taneja-Bageshwar | Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae)[END_REF][START_REF] Taneja-Bageshwar | Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs[END_REF] Incorporation of a second Aib residue adjacent to the secondary peptidase hydrolysis site further enhances biostability. 26 Insect kinin analogues that incorporate

Accepted Article

sterically-bulky Aib residues, adjacent to both primary and secondary peptidase hydrolysis sites were previously evaluated on two recombinant receptor assays from the southern cattle tick, Rhipicephalus (Boophilus) microplus, [START_REF] Holmes | Cloning and transcriptional expression of a leucokinin-like peptide receptor from the southern cattle tick Boophilus microplus (Acari: Ixodidae)[END_REF][START_REF] Holmes | Functional analysis of a G protein-coupled receptor from the southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor[END_REF] and the dengue vector, the mosquito Aedes aegypti. [START_REF] Pietrantonio | The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins[END_REF] These results demonstrated that biostable Aib analogues of the insect kinins can retain potent activity on these two receptors. [START_REF] Taneja-Bageshwar | Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs[END_REF] Recent studies have documented the potent effects of biostable neuropeptide analogues on pest insects. [START_REF] Taneja-Bageshwar | Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs[END_REF][START_REF] Smagghe | Antifeedant activity and high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) induced by biostable insect kinin analog[END_REF][START_REF] Zhang | Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs[END_REF] By employing two aphid pest species, kinin [START_REF] Radford | Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles[END_REF] and CAPA [START_REF] Terhzaz | Mechanism and function of Drosophila capa GPCR: a desiccation stressresponsive receptor with functional homology to human neuromedinU receptor[END_REF] receptor sites were first mapped in aphid tissue. The potential effects of kinin and CAP2b/PK analogues on aphid stress tolerance and fitness (desiccation, starvation, cold) were subsequently screened, representing the first study into the role of these neuropeptide families in aphid stress tolerance. Aphids (Hemiptera: Aphididae) are one of the most significant groups of agricultural pests [START_REF] Christie | In silico analyses of peptide paracrines/hormones in Aphidoidea[END_REF] and are vectors in the transmission of approximately 50% of all insect transmitted plant viruses. [START_REF] Nault | Arthropod transmission of plant viruses: A new synthesis[END_REF] The primary study species for the current study, the peach potato aphid Myzus persicae, is the most economically important aphid crop pest worldwide, 40 with a global distribution and host range encompassing more than 400 species in 40 different plant families. [START_REF] Blackman | Aphids on the World's Crops: An Identification Guide[END_REF] The secondary study species, the rose aphid Macrosiphum rosae, was selected to represent a major pest of horticulture. M. rosae is an important pest of cultivated species of Rosa and is a vector in the transmission of 12 plant viruses including the strawberry mild yellow edge virus. [START_REF] Blackman | Aphids on the World's Crops: An Identification Guide[END_REF] The results of this study will inform design and development of novel, specific insecticidal agents.

Material and methods

Accepted Article

Aphid rearing

Stock cultures of anholocyclic M. persicae were established using aphids supplied by the Smagghe laboratory, Ghent University, Belgium. Cultures were reared under a 12:12 h LD photocycle at 22°C on Chinese cabbage (Brassica rapa var. Wong Bok) contained within a BugDorm fine mesh cage (44545F) (45cm x 45cm x 45 cm). A fresh supply of Chinese cabbage of approximately 4 weeks from sowing was supplied to the cages on a once-weekly basis to maintain the aphid cultures.

M. rosae was selected as a secondary aphid species and a sub-set of experiments was performed on the species to determine the overlap in response between aphid species of different genera. Stock cultures of anholocyclic M. rosae were set up from individual aphids originally collected on Rosa species within the grounds of the University of Glasgow, Scotland, UK. A stock culture was set up within the laboratory and maintained on supermarket-bought miniature rose plants and under identical conditions to M. persicae. Prior to use, the leaves of the rose plants were thoroughly washed using 70% ethanol followed by distilled water to ensure the removal of any potential chemical residues present on the plant.

Neuropeptide synthesis

Native and fluorescently labelled neuropeptides CAPA-1 (CAPA-1-F) and kinin (Kinin-F) were synthesized by Cambridge Peptides (Birmingham, UK) as previously detailed [START_REF] Halberg | Tracing the evolutionary origins of insect renal function[END_REF] and based on the CAPA and kinin structures of Drosophila melanogaster. In brief, native kinin The synthesis of PK analogues (with CAPA receptor cross-activity) 1895 and 1902, [START_REF] Jiang | Functional characterization of five different PRXamide receptors of the red flour beetle Tribolium castaneum with peptidomimetics and identification of agonists and antagonists[END_REF][START_REF] Zhang | Disruption of insect diapause using agonists and an antagonist of diapause hormone[END_REF] CAP2b analogue 1896, [START_REF] Jiang | Functional characterization of five different PRXamide receptors of the red flour beetle Tribolium castaneum with peptidomimetics and identification of agonists and antagonists[END_REF] and insect kinin analogues 1728 and 2139 [START_REF] Taneja-Bageshwar | Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae)[END_REF][START_REF] Taneja-Bageshwar | Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs[END_REF] have been previously

described. CAP2b analogues 2089, 2123, 2125, and 2129; [START_REF] Zhang | Disruption of insect diapause using agonists and an antagonist of diapause hormone[END_REF] as well as insect kinin analogue 2139-Ac [START_REF] Taneja-Bageshwar | Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae)[END_REF] were synthesized and cleaved according to procedures that have been previously

described. The analogues were purified on a Waters Delta-Pak C18 reverse-phase column ( 8x 100 mm, 15 µm particle size, 100 Å pore size) with a Waters 510 HPLC system with detection at 214 nm at ambient temperature. Solvent A = 0.1% aqueous trifluoroacetic acid (TFA); Solvent B = 80% aqueous acetonitrile containing 0.1% TFA. Initial conditions were 10% B followed by a linear increase to 90 % B over 40min.; flow rate, 2 ml/min. Delta-Pak C18 retention times: 2089, 12.0 min.; 2123, 9.0 min; 2139-Ac, 5.9 min; 2125, 12.5 min; 2129, 7.5 min. The analogues were further purified on a Waters Protein Pak I 125 column (7.8 x 300 mm). Conditions: isocratic using 80% acetonitrile containing 0.1% TFA; flow rate, 2 ml/min. Waters Protein Pak retention times: 2089, 6.0 min; 2123, 5.5 min; 2139-Ac, 5.9 min; 2125, 5.5 min; 2129, 6.0 min. Amino acid analysis was carried out under previously reported conditions (Nachman et al., 2004) to quantify the analogues and to confirm identity: The structures of the biostable analogues are displayed in Table 1.

2089: F[1.0], P[1.0], R[1.0], T[1.0], V[1.0]; 2123: F[1.0], R[0.9], T[0.9], V[0.9]; 2139-Ac: F[2.0], G[0.9]; 2125: F[1.0], R[0.8], T[0.7], V[0.8]; 2129: A[1.0], I[0.9], P[0.9], R[0.9], T[0.9].

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Receptor mapping assay using fluorescently labelled neuropeptides

Aphids were cold anesthetised and the tissue of interest dissected out in a 1:1 solution of Schneider's insect medium and optimised saline. [START_REF] Halberg | Tracing the evolutionary origins of insect renal function[END_REF][START_REF] Dow | The Malpighian tubules of Drosophila melanogaster -a novel phenotype for studies of fluid secretion and its control[END_REF] The dissected tissue was mounted on a poly-L-lysine-covered 35mm glass bottom dish containing 1:1 saline. Nuclei were stained via incubation in DAPI (1 µg ml -1 ) for 1 minute and then washed with the optimised saline solution. A baseline image was taken to determine the level of autofluorescence and adjust exposure settings accordingly. All images were recorded on an inverted confocal microscope (Zeiss LSM 510 Meta). A labelled neuropeptide (10 -7 M) was subsequently added to the tissue and the tissue incubated for 1 minute before washing with the optimised saline solution. The sample tissue was immediately imaged. The concentration of 10 -7 M was chosen for labelled neuropeptides because it represents the minimal concentration required to produce a saturated receptor response, thereby optimising the conditions for optical detection of ligand-receptor complexes. [START_REF] Halberg | Tracing the evolutionary origins of insect renal function[END_REF] Following imagining, unlabelled neuropeptide (10 -5 M) was added to the sample and a time-lapse experiment set up to determine if the unlabelled neuropeptide outcompeted the labelled neuropeptide, thus affirming detection of the ligandreceptor complexes. Images were collected every 30 s for a duration of 20-30 m. All imaging was repeated on a minimum of three specimens to ensure consistency and further re-affirm conclusions. All images were exported as JPEG files and subsequently viewed in FIJI and Microsoft Illustrator. When specific binding was observed in muscle tissue, this was supported by the addition of rhodamine phalloidin; a high-affinity F-actin probe conjugated to tetramethylrhodamine (TRITC) that specifically binds to muscle.

Neuropeptide treatment via microinjection

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Neuropeptides were administered to test aphids via microinjection to allow for rapid mass screening of neuropeptide analogue efficacy. For this, native neuropeptides were diluted in Neuropeptide analogues which significantly impacted aphid survival under desiccation / starvation stress were further tested under non-stress conditions. For this, aphids were treated with the neuropeptide analogue as detailed above and maintained on excised leaves of the host plant for a duration of 7 days. Survival data at day 7 were arcsine square root transformed and analysed using analysis of variance (ANOVA) and Tukey's multiple range test in MINITAB version 17.

Cold tolerance bioassay

Calculation of discriminating temperatures

M. persicae and M. rosae displayed identical results in desiccation / starvation stress assays.

For this reason, and given its global pest status, only M. persicae was taken forward in cold stress assays. Survival curves were first established to determine a species-specific discriminating temperature for subsequent neuropeptide testing. Aphids were selected at the pre-reproductive adult stage for cold tolerance bioassays since aphid cold tolerance is known to significantly vary throughout an aphid's life cycle. [START_REF] Clough | Differential cold hardiness in adults and nymphs of the peach-potato aphid Myzus-Persicae[END_REF][START_REF] Powell | Intergenerational acclimation in aphid overwintering[END_REF] Temperature ranges were selected to encompass 0-100% mortality. Anholocyclic pre-reproductive adults (approximately 9 d old at 22°C) of M. persicae were exposed to a range of low temperatures (-14°C to -7°C at 1°C intervals) using a direct plunge method. [START_REF] Sinclair | Rapid cold-hardening in a Karoo beetle, Afrinus sp[END_REF][START_REF] Terblanche | Thermal tolerance in a south-east African population of the tsetse fly Glossina pallidipes (Diptera, Glossinidae): Implications for forecasting climate change impacts[END_REF] For each temperature treatment, 30 adults were placed within plastic 0.5mL Eppendorf tubes at densities of ten adults per tube, which, in turn, were placed within a glass boiling tube held within an alcohol bath (Haake G50 and PC200; Thermo Scientific, Germany) pre-set to the desired temperature. Pieces of cotton

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wool were used to stopper the boiling tubes to limit air circulation and to ensure a more stable internal temperature within the tubes. Adults were held at the desired exposure temperature for 1 h. Following exposure, aphids were allowed to recover at the culture temperature in microcages containing excised leaves of the host plant and survival was assessed after 48 h.

The procedure was repeated for each exposure temperature.

Survival data were analysed using Probit analysis in MINITAB, version 17 (Minitab Inc., State College, Pennsylvania) and the LT 30 (the lethal temperature resulting in 30% mortality of a test population) was elucidated. The LT 30 was chosen to act as a discriminating temperature for subsequent neuropeptide testing since it enabled detection of directional effects of subsequent neuropeptide treatment, but primarily in the direction of interest i.e.

which neuropeptides significantly increased mortality in the species of interest.

Peptide analogue treatment and testing at the discriminating temperature

Pre-reproductive anholocyclic adult aphids of M. persicae were treated with neuropeptide analogues using the microinjection method detailed above. Following microinjection treatment, individuals were returned to microcages containing excised leaves of the host plant at densities of approximately 20-30 per microcage and allowed to recover for 24 h at the culture temperature. Following the 24 h recovery period, adults were placed within plastic 0.5mL Eppendorf tubes at densities of ten adults per tube to a total of 30 for each species x neuropeptide treatment group (i.e. 3 biological replicates of 10 per replicate). Eppendorf tubes were then placed within glass boiling tubes held within the alcohol bath pre-set to the desired discriminating temperature. Pieces of cotton wool were used to stopper the boiling tubes to limit air circulation and to ensure a more stable internal temperature within the tubes.

Adults were held at the desired exposure temperature for 1 h. Following exposure, adults

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were allowed to recover at the culture temperature in microcages containing excised leaves of the host plant and survival was assessed after 48 h. The procedure was repeated for each species x peptide analogue treatment group.

Statistical analyses were performed using R Software (R Development Core Team, 2013). A generalised linear model (GLM) with binomial family was fitted to survival data with analogue 'Treatment' (peptide analogue), treatment 'Type' (test vs. control), and analogue treatment x treatment type interaction as factors.

Results

Receptor mapping assay using fluorescently labelled neuropeptides

A fluorescent ligand-receptor binding assay was employed to map specificity of binding of Kinin and CAPA-1 within M. persicae and M. rosae. Fluorophore-labelled kinin (kinin-F) and CAPA-1 (CAPA-1-F) revealed the neuropeptides to bind to circular muscles along the aphid gut (Figure 1). Kinin-F binding to longitudinal muscles in the hindgut region of the gut was further suggested (data not shown). Both the kinin-F and CAPA-1-F signals were displaced by excess unlabelled peptide in the ligand competition assay, thus confirming specificity of binding. Additional labelling with rhodamine phalloidin acted to confirm the gut muscle as the site of binding (Figure 1c). Interestingly, specific kinin-F and CAPA-1-F binding of the gut musculature was not evident under low magnification (x10) (Figure 2).

The presence of smaller cells, running the length of the gut, were detected as a site of kinin-F binding (Figure 1b and 2 indicated by white arrows), although were not a site of CAPA-1-F binding (Figure 1a). In addition, CAPA-1-F specific binding was detected in a region of the aphid midgut (stomach) closest to the foregut (Figure 3) and may represent enterocytes, although further testing is required to confirm this.

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Receptor mapping of the M. persicae brain and ventral nerve cord (VNC) revealed kinin-F staining apparent in a bilateral symmetrical 'ladder' of neuronal clusters (2-3 neurons) and a set of baso-lateral neurons in the suboesophageal ganglion (Figure 4a). Staining was also apparent in symmetrical pairs of neurons/neuronal clusters in the ventro-to dorso-lateral protocerebrum. Little to no kinin-F staining was observed in the VNC with the exception of a set of cells in the most distal tip of the abdominal ganglion (Figure 4b). In contrast, no specific staining with kinin-F was observed in the brain or VNC of M. rosae. Labelling with CAPA-1-F revealed no sites of receptor binding in either the brain (Figure 4c) or the VNC of both species (Figure 4d).

Desiccation stress

The CAP2b analogues 1895 and 2129 significantly increased desiccation / starvation mortality in both species (Table 2, Figure 5). Here, treatment with 1895 acted to reduce the LTime 50 by 3.5 h and 9.6 h in M. persicae and M. rosae respectively, and median survival by 4.0 h and 10.5 h respectively (Table 2). Treatment with 2129 acted to reduce the LTime 50 by 7.1 h and 11.6 h in M. persicae and M. rosae respectively, and median survival by 9.8 h and 12.8 h respectively (Table 2). Neither 1895 nor 2129 impacted survival under non-stress conditions (ANOVA DF = 2 , F = 0.00, p = 0.999) with 1 out of 32 aphids treated with 1895, 1 out of 31 aphids treated with 2129, and 1 out of 29 aphids from the control group dying within 7 days post treatment. None of the kinin analogues significantly affected desiccation/starvation mortality in either species (Table 2).

Cold stress

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A survival curve was calculated for M. persicae (Figure 6) and the LT 30 (discriminating temperature) calculated as -9.7°C. There was a significant effect of 'Type' (control vs. treatment) on the cold stress survival of M. persicae following cold shock at the discriminating temperature of -9.7°C (GLM DF = 1, χ 2 = 5.9844, p = 0.014), indicating that all analogues are efficient at increasing the mortality of test aphids under conditions of cold stress. However, there was no effect of the factor 'Treatment' (peptide analogue) on aphid cold stress survival (GLM DF = 9, χ 2 = 7.8355, p = 0.551), indicating that all analogues appear equivalent in their effect, with no analogue having a stronger effect than another.

Interestingly, treatment with analogue 2139-AC implies a reverse effect on aphid survival (Figure 7), acting to increase survival relative to the control. However, further examination restricted to this case against its control proved non-significant (GLM DF = 1, χ 2 = 7.8355, p = 0.3771). It must be concluded that all studied analogues increase the mortality of aphids under conditions of cold stress, with the exception of 2139-Ac, although no individual peptide is significantly more powerful in its effect.

Discussion

Neuropeptides are regulators of critical life processes in insects and, due to their high specificity, hold great potential in the drive for target-specific and environmentally friendly insecticidal agents. [START_REF] Audsley | G protein coupled receptors as targets for next generation pesticides[END_REF] In pursuit of the development of neuropeptidomimetic-based insecticides, the current study mapped kinin (kinin-F) and CAPA (CAPA-1-F) neuropeptide binding sites within M. persicae and M. rosae to determine the location of neuropeptide receptors and thus the targets of neuropeptide action. A total of 10 kinin and CAP2b/PK (active on the CAPA receptor) biostable analogues were subsequently assayed for targetinsect-specificity and an ability to reduce aphid pest fitness under a range of environmental stressors.

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Receptor mapping employing fluorescently labelled kinin revealed the gut musculature as a main target for kinin activity in both M. persicae and M. rosae, as previously shown for M. persicae. [START_REF] Halberg | Tracing the evolutionary origins of insect renal function[END_REF] Additional areas in the brain and VNC were also indicated in M. persicae. In the pea aphid, Acyrthosiphon pisum, it is thought that kinin regulates gut motility, digestive enzyme release, fluid cycling and nutrient transport across the gut. [START_REF] Smagghe | Antifeedant activity and high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) induced by biostable insect kinin analog[END_REF] Indeed, kinin analogues have shown great potential in the laboratory for their aphicidal properties, acting as antifeedant agents during artificial diet trials on the pea aphid (A. pisum). [START_REF] Smagghe | Antifeedant activity and high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) induced by biostable insect kinin analog[END_REF] Interestingly, whilst kinin analogues, including the present analogue 1728, have displayed prior antifeedant potential, none of the kinin analogues in the current study acted to reduce aphid fitness under desiccation and starvation stress conditions.

As with receptor mapping of kinin activity, receptor mapping with fluorescently labelled CAPA revealed the muscles of the aphid midgut as the target for neuropeptide binding, with no CAPA receptor binding detected in the aphid brain or VNC. However, in contrast to the kinin analogues, CAP2b/PK analogues displayed greater promise in stress tolerance assays, with analogues 1985 (2Abf-Suc-FGPRLa) and 2129 (2Abf-Suc-ATPRIa) acting to expedite aphid (M. persicae and M. rosae) mortality under conditions of desiccation and starvation stress. Furthermore, all tested analogues (kinin and CAP2b/PK), with the exception of 2139-Ac, enhanced M. persicae mortality under cold stress conditions, although were all considered equivalent in the strength of their effect.

Neuropeptides of the CAPA family have roles in the stimulation of fluid secretion in Malpighian (renal) tubules [START_REF] Davies | CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP[END_REF] and, more recently, have been linked to desiccation and cold tolerance in Drosophila. [START_REF] Terhzaz | Insect capa neuropeptides impact desiccation and cold tolerance[END_REF] Unlike most insects, aphids lack Malpighian tubules; [START_REF] Douglas | Nutritional physiology of aphids[END_REF] organs with

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vital roles in osmoregulation, detoxification and immunity. [START_REF] Beyenbach | The developmental, molecular, and transport biology of Malpighian tubules[END_REF][START_REF] Jing | The molecular correlates of organ loss: The case of insect Malpighian tubules[END_REF] Due to this secondary loss of Malpighian tubules in the aphids, key osmoregulatory roles have been reassigned to other organs, particularly the aphid gut. [START_REF] Jing | The molecular correlates of organ loss: The case of insect Malpighian tubules[END_REF] Receptor mapping assays performed in the current study offer support to this, highlighting the presence of CAPA receptors along the aphid gut and implicating the gut as a primary target for CAPA neuropeptide action. The role of CAPA neuropeptides in osmoregulation further offers explanation for the relative effectiveness of the CAP2b analogues tested in expediting aphid mortality under conditions of desiccation stress.

Conclusion

Kinin and CAPA (CAPA-1) binding was confirmed in the aphids M. persicae and M. rosae, with the musculature of the gut the primary target of neuropeptide action. In-vivo assays Staining is abrogated when outcompeted with unlabelled 10 -5 M CAPA-1 (not shown).

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CAPA-1-F, red; DAPI, blue. Scale bars = 50 um. Accepted Article

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  was synthesized and coupled to Alexfluor488 resulting in fluorescent kinin (Alexa-488-C 5maleimide-CNSVVLGKKQRFHSWGamide). The same rationale was used for the production of CAPA-1 (GANMGLYAFPRVamide) and labelled CAPA-1-F with the

  double distilled water (DDH 2 0) to a concentration of 1 x 10 -5 M. Neuropeptide analogues were diluted in DDH 2 0 to the following concentrations: kinin analogues 1728 (2.5 x 10 -5 M), 2139 (3.5 x 10 -5 M), 2139-Ac (3.5 x 10 -5 M); CAP2b/PK analogues 1895 (3.5 x 10 -5 M), 1896 (3.5 x 10 -5 M), 1902 (3.5 x 10 -5 M), 2089 (3.9 x 10 -5 M), 2123 (1.0 x 10 -5 M), 2125 (1.0 x 10 -5 M), 2129 (2.0 x 10 -5 M). Once at the desired concentration, neuropeptide solutions were administered to test aphids at an injection volume of 9nl based on total haemolymph volume, to produce an approximate 1:20 dilution of injection volume to haemolymph.Injections were performed using a pulled glass needle and a Nanoject II Auto-Nanoliter Injector (Drummond Scientific Company, Broomall, Pennsylvania). A vehicle control was set up for each treatment / day of experiments to account for variation in needle pulling. For this, control aphids were injected with 9nl of DDH 2 0 and subsequently exposed to the same experiments as aphids receiving the neuropeptide treatment. Neuropeptide treated and vehicle control aphids were subsequently used in the stress bioassays detailed below.2.5 Desiccation / starvation tolerance bioassayAnholocyclic adults of mixed age of either M. persicae or M. rosae were selected from the stock cultures and treated with a native neuropeptide or neuropeptide analogue via microinjection using the method detailed above. Treated and vehicle control aphids were allowed to recover on excised leaves of the host plant for 1 h before being placed in an empty ventilated microcage (L = 4 cm, Ø = 9.5 cm) at densities of 10 per cage. In total, 30-40 aphids were treated for each neuropeptide treatment group and a further 30-40 for the associated vehicle control group (i.e. 3-4 biological replicates of 10). From the point of placement in the microcage (taken as 0 h), aphid survival was checked every hour during Accepted Article daylight hours and approximately every 4 h during night-time hours until the final aphid died. Survival data were subsequently analysed using a Log-rank (Mantel-Cox) test in GraphPad Prism version 7.0. LTime 50 (the time taken to kill 50% of the test population) values were calculated via Probit Analysis in Minitab 17 (Minitab Inc., State College, Pennsylvania).

revealed

  CAP2b/PK analogues to be the most effective in expediting aphid mortality under conditions of desiccation and starvation stress. In highlighting the PRXamide neuropeptide family more generally, and the structures of promising CAP2b/PK analogues more specifically, this research will feed into the development of second and third generation analogues and ultimately drive forward the development of neuropeptidomimetic-based insecticidal agents. Current testing of new generation analogues is focusing on mode of application to elucidate the best method of delivery to apply neuropeptide-based insecticides in the field.

Figure 1

 1 Figure 1 Aphid intestine (distal midgut and proximal hindgut) stained with 10 -7 M CAPA-1-F

Figure 2

 2 Figure 2 Myzus persicae intestine (distal midgut and proximal hindgut) stained with 10 -7 M kinin labelled with alexafluor488 (A) and then out-competed with 10 -5 M unlabelled kinin

Figure 3

 3 Figure 3 Myzus persicae stomach (midgut) stained with 10 -7 M CAPA-1-F labelled with

Figure 4 (

 4 Figure 4 (A) Unstained Myzus persicae CNS, demonstrating baseline autofluorescent levels

Figure 5

 5 Figure 5 Effect of CAP2b and kinin analogue treatment on the survival of Myzus persicae (1)

Figure 6

 6 Figure 6 Survival curve calculated via Probit analysis of Myzus persicae pre-reproductive

Figure 7

 7 Figure 7 Mean ± standard error proportion survival of M. persicae when treated with

Acknowledgements

This work was funded by the European Union's Horizon 2020 Research and Innovation programme under grant agreement No. 634361 (SD/JATD) (nEUROSTRESSPEP). The authors are grateful to the Smagghe laboratory, Ghent University, for providing M. persicae.

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Thanks also to Angela Douglas, Cornell University, for discussion on aphid internal morphology.

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Table 2 The effect of neuropeptide analogue treatment via microinjection on the desiccation and starvation tolerance of M. persicae and M. rosae. Neuropeptide analogues were administered to a final concentration of x 10 -5 M. Survival is shown as both a median survival (h) ± IQR and an LTime 50 (h). Values in bold significantly increased desiccation / starvation mortality in relation to a vehicle control group. Example survival curves are displayed in Figure 5.

Myzus persicae

Documented effect