Squalus acanthias, Linnaeus 1758) were investigated in the Adriatic Sea from mid February 2012 to mid July 2013 and in 2016. The whole sample consisted of 176 females and 150 males, ranging between 217–1025 mm and 219–875 mm, respectively. The individual age, which was estimated using a cross-sectioning technique of the second dorsal-fin spine, ranged from 0 to 13+ years for females and from 0 to 9+ years for males. Based on the length-at-age estimates, the Gompertz growth parameters were L∞ = 1130 mm, k = 0.18 and L∞ = 920 mm, k = 0.24 for females and males, respectively. The size at sexual maturity (L50) was 659 mm for females and 575 mm for males, corresponding to 7.5 and 5.5 years of age (A50), respectively. Mean biennial fecundity was approximately 11 embryos/female and 12 ripe oocytes/female. Mature males occurred during much of the sampling period, while mature females with nearly full-term embryos were exclusively recorded in May 2013 and July 2016. Monitoring of catches conducted in a sample port of the north Adriatic (Chioggia) over the past 20 years has shown fluctuating trends in landings, with peaks during the summer reproductive season.Pivotal life history traits concerning age structure and reproduction of the spiny dogfish ( Elasmobranchs are highly vulnerable to overfishing due to their life history characteristics that include long life span, late sexual maturity, large size at birth and low reproductive rates4. Fishery removes selectively larger individuals, causing a general decline in size. Considering the positive relationships between fecundity and female size, this causes a reduction in the reproductive potential (REF) and in the number of possible mating events per female5. Until now information on the status of population are missing and this did not allow developing sharks conservation measures in the Mediterranean Sea2.The Mediterranean Sea offers a unique perspective on fish population declines over historical timescales, as the resources have been highly exploited since a long time6. Critical key factors as size and age frequency distribution and fecundity, in relation- to female size and size/age of sexual maturity are, therefore, of priority importance to assess the population structure, dynamics and the reproductive potential of exploited stocks8.Life history traits awareness of overfished species is fundamental to understand the effects of overexploitation on the conservation of the species and to implement sustainable management strategieset al.9; Ferretti et al.10; Barausse et al.11). In the Adriatic Sea, accessory catches of many species of cartilagineous fish include Squalus acanthias, Mustelus mustelus and M. punctualtus, Raja asterias, R. clavata, R. miraletus, Torpedo spp., Scyliorhinus canicula, S. stellaris, Galeorhinus galeus, Alopias vulpinus and juveniles of Carcharhinus plumbeus12. Among them, the spiny dogfish (S. acanthias) is one of the most important valuable commercial species commonly caught by bottom13 and midwater pelagic trawl15 and passive nets17.The shallow northern-central part of the Adriatic Sea represents one of the most suitable fishing grounds of mixed multi-specific demersal fisheries in the Mediterranean, which have already caused a decrease in elasmobranchs diversity and abundance , we provided information on temporal trend of catches in the last two decades to infer the status of the Adriatic spiny dogfish population.2 goodness-of-fit test.The spiny dogfish were collected monthly off Chioggia and Ancona harbours, which are located in two different areas of the Adriatic Sea Fig. . A totalAll specimens were frozen at −20 °C until dissection in the laboratory. Each specimen was measured to the nearest mm from the tip of the snout to the tip of the upper lobe of the caudal fin  and weighed to the nearest gram . Sex was established by recording presence/absence of claspers.27.The abdomen of each individual was cut with a knife from anus to throat, and the gonads were removed and weighed. Gonads were macroscopically examined to determine the maturity stage for males and females, following the description provided by the Report of the Workshop on Sexual Maturity Staging of ElasmobranchsIn 150 males, sexual maturity was determined by clasper length and calcification , testes shape and amount of sperm stored in the testes/epididymis/sperm sac. Five maturity stages were considered: M1 (immature), M2 (developing), M3a (spawning capable), M3b (actively spawning), M4 (regressing). At the Chioggia harbour, before the specimens being sold, 407 additional males beyond the 150 processed in the laboratories were staged by direct examination of claspers length and calcification; those with flaccid claspers shorter than pelvic fins were classified as immature, while the others with rigid claspers longer than pelvic fins as mature.In 176 females, sexual maturity was determined from the size of ova and the state of uteri. Seven maturity stages were considered: F1 (immature), F2 (developing), F3a (capable to reproduce), F3b (early pregnancy), F3c (mid pregnancy), F3d (late pregnancy), F4a (regressing), F4b (regenerating).50) was assessed for both sexes by fitting a logistic model to the proportion of mature specimens (i.e. at stages 3–4) per size class.Size at sexual maturity (TLmean), which is the percentage of gonad mass (GM) to total body mass (TM), was calculated for each maturity stage both for males and females, in which it was possible to extract and weight the gonads, as a measure of reproductive investment.The mean gonadosomatic index . A linear regression was used to determine the relationship between fecundity and size of mature females.28. For each spine, the number of annuli was determined by counting the alternating translucent and opaque zones visible on the spine cross-section29 using a light microscope (Leica DM4000B) at 4x magnification. Assuming that the annuli were laid down yearly, the age of fish was estimated by counting all the translucent zones. The final individual age was then calculated in months, based on the date of capture and presumed common birthday (June). Two readings were performed by two different readers one week apart. The precision of age readings was estimated by calculating the average percentage error (APE) and the coefficient of variation (CV)31.For age determination, the second dorsal fin spine was removed by cutting horizontally just above the vertebrae. Spine cross-sections were obtained following the procedure described by Soldat32 was calculated for the best model selection. Statistical analyses were performed using the Growth II software .To describe the growth pattern, the von Bertalanffy and the Gompertz growth models were initially fitted to the length-at-age data estimated for each sex. Growth parameters and 95% confidence intervals were estimated by applying a non-linear regression analysis, and the Akaike Information Criterion (AIC)50).Age of maturity was assessed both for females and males from the resulting growth-curve knowing the size of maturity  and TL is the total length (mm). Isometric growth (i.e. b = 3) departure was tested using a t-Student test. The allometric indices (b) calculated for males and females were compared by applying an F test.The length-mass relationship was estimated for each sex using the function TM = aTL33, which hosts the major fleet of the northern Adriatic Sea34. Total commercial landings, expressed in biomass of landed specimens, of spiny dogfish were analyzed for the last two decades (1997–2016), since before 1997 the spiny dogfish was registered with other shark species . Total fishing capacity  of the Chioggia trawlers was used as a proxy of fishing effort, because estimates of fishing effort were not available. Therefore, annual CPUEs were estimated by dividing total landings (t) per total fishing capacity.Fishery data include official landing statistics of the Chioggia harborMean monthly landings across years were also calculated to investigate monthly tons variations.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All specimens collected were died at the time of sampling.χ2 = 2.07 test, P = 0.15).Overall 176 females and 150 males of the spiny dogfish were collected during the sampling period. Size ranged from 217 to 1025 mm  for females and from 219 to 875 mm  for males  = 0.00022, P = 0.98).The b-values were significantly higher than 3 in both sexes, indicating a positive allometric growth , with no difference between them (F∞ exp(−exp(−k(t − l))), where L∞ is the asymptotic length, k is the growth rate, t is the age and l is the age at the inflection point, was the best model describing the spiny dogfish body growth. The theoretical asymptotic length (L∞) were 920 mm (95% CI = 815–1024) and 1130 mm (95% CI = 986–1273) for males and females, with an instantaneous growth rate (k) of 0.24 year−1 (95% CI = 0.19–0.29) and 0.18 year−1 (95% CI = 0.14–0.22), respectively. Indices of age precision between readers and within readings of the same reader were both relatively low , indicating good consistency (or reproducibility) of age estimates.Age was successfully determined for all the specimens caught. The estimated age range was between 0 and 13+ years and between 0 and 9+ years for females and males, respectively Fig. . Based o50) was assessed for 557 males  and 157 females. Size at sexual maturity was different in males and females  was calculated for 18 females and 37 males, from which it was possible to remove gonads at different maturity stages. GSI values consistently increased with gonad maturation, being 0.2%, 1.5%, 1.6% and 1.7% in maturity stages M1, M2, M3a and M3b, and 0.3%, 0.5%, 3.0%, 6.4%, 5.1% and 13.3% in stages F1, F2, F3a, F3b, F3c and F3d, respectively.During the sampling period, pregnant females with nearly full-term embryos  were exclusively found in May 2013 and July 2016, whereas mature males with sperm coming out at pressure were present all the year round. Ovarian fecundity, based on the number of ripe oocytes ranging from 15 to 40 mm , was counted in both ovaries of 14 mature females, and ranged from 6 to 18, with a mean of 12 ± 3.85 ripe oocytes/female. It showed a positive relationship with size of mature females, as summarized in the following equation  showed a fluctuating trend in the last two decades. Three main peaks were observed in 1998, 2002 and 2014, each followed by a steep decline afterwards. Mean monthly landings (tons) across years showed three main peaks in January, April and June-July, just before the closure of fishing activities in August in the whole area Fig. .Figure 7et al.35) and elsewhere . A more recent study indicated that age estimates based on external growth bands were overestimated compared to those obtained by sectioned vertebrae37. As a results, a pletora of different growth curves and longevity estimates has been reported for this species, only partially due to different growth models used or different sampling areas .The present study reports key information on the life history traits of spiny dogfish in the northern-central Adriatic Sea, focusing the attention on age determination and reproductive biology. The growth bands formed on the external surface of the second dorsal-fin spine have been generally used to estimate individual age of this species, both in the Mediterranean Sea 37 and a bigger size and age at maturity 45 have been reported. The lack of significant sex ratio bias commonly recorded from different areas indicates that spiny dogfish are generally characterized by balanced populations .The wide sampling area of the northern-central Adriatic Sea covered by fishing fleets operating in Chioggia and Ancona harbour, and the relatively long sampling period spanning almost three years, make us confident that the fish size range sampled was representative of the spiny dogfish really present in the basin. Therefore, the maximum age estimated in this study likely represented the life span of this species in the Adriatic Sea. On the other hand, the long life span and the late sexual maturity reported by Gračan 48; Capapé and Reynaud49) and outside  the Mediterranean Sea analyzed GSI related to size finding GSI values higher for females especially during advanced gestation stages. Females dogfish seem to invest a lot of energy to produce gonadic products as they attain maturity.Data on individual maturity stages and relevant GSI enabled us to describe the reproductive cycle of spiny dogfish in the Adriatic Sea. The GSI of immature specimens was comparable between sexes, accounting for less than 0.5% of total body mass. At sexual maturity, GSI of males increased until reaching approximately 2% of total body mass, remaining roughly the same across the mature gonad stages. Conversely, GSI of females at sexual maturity exhibited a steady increase, attaining for more than 13% of total body mass in pregnant females with fully formed embryos. Other authors inside .Both ovarian and uterine fecundity resulted to be positively correlated with females size, as larger females have commonly more space to bear pups57, size and age at sexual maturity was different between sexes of spiny dogfish, females maturing later and at a bigger size than males. Size at sexual maturity reported for this species was rather different among different localities within and outside the Mediterranean Sea  of mature specimens caught in the previous years. The complete recovery of the resource at initial levels took almost ten years, which is roughly the period necessary to reach sexual maturity for females (7.5 years) plus the gestation (2 years). Difference in mean tons monthly landed across years showed 3 different peaks, may be due to different fishing areas exploited during the year and to the migratory behavior of the species during the reproductive season and different growth stages.Taking into account the shallow waters of the northern-central Adriatic Sea continental shelf (maximum depth less than 50 m) consisting of sandy-muddy sea bed entirely accessible to towing gears and the low reproductive potential of spiny dogfish in terms of gestation time and individual fecundity, the risk that fishing may reduce the stock of breeding animals below the limit beyond which the stock is no longer able to recover is real and cannot be underestimated. As a consequence, the reduction of spiny dogfish bycatch is an urgent matter and mitigation measures should be promptly adopted, fishermen should be trained on releasing live sharks, nursery/mating areas should be identified and closed during specific times of the year. Finally, awareness campaigns should be launched to reduce shark meat consumption, mainly considering that Italy is globally one of the most important importer of it.


Prompt identification of the causative pathogen of an infectious disease is essential for the choice of treatment or preventive measures. In this perspective, nucleic acids purified from the brain tissue of a dog succumbed after severe neurological signs were processed with the MinION  sequencing technology. Canine distemper virus (CDV) sequence reads were detected. Subsequently, a specific molecular test and immunohistochemistry were used to confirm the presence of CDV RNA and antigen, respectively, in tissues. This study supports the use of the NGS in veterinary clinical practice with potential advantages in terms of rapidity and broad-range of molecular diagnosis. Rapid identification of the etiologic agent of an infectious disease is essential for setting up treatment and preventive measures. In general, pathogen identification is performed by direct diagnostic tests which normally include amplification of target nucleic acids by PCR-based assays. Although these approaches are highly specific and often validated, they suffer a number of limitations, including the difficulties of testing for the plethora of rare pathogens that might be expected to cause a given pathology and their inability to identify new or unexpected pathogens, eventually originating from cross-species jumps. Therefore, the availability of other more rapid, broad-range techniques has become more and more important in the milieu of laboratory diagnosis of infectious diseases.3. Recently the release of the MinION , a novel portable real-time NGS sequencer, enables the application of sequencing for rapid diagnosis with inexpensive sample preparation even in low-throughput laboratories10.In this context one of these novel and intriguing technologies is certainly represented by next generation sequencing (NGS), even more when performed for direct identification of pathogens from clinical samples. Genome sequencing directly from biological samples can provide insights into how viruses transmit and spread, and it has been successfully applied to both virus discovery and diagnostics12.Canine distemper (CD) is a highly contagious disease which involves mainly young dogs and other susceptible carnivores with high morbidity and mortality rates. CD infection is characterized by lympho-, neuro- and epithelio-tropism resulting in systemic infection with severe clinical signs and deathMorbillivirus of the family Paramyxoviridae. CDV is an enveloped, negative-sense, single-stranded RNA virus. Like other paramyxoviruses, the virus contains six structural proteins, termed nucleocapsid (N), phospho (P), large (L), matrix (M), hemagglutinin (H) and fusion (F) protein, and two accessory non-structural proteins (C and V) that were found as extra-transcriptional units within the P gene13. Circulating CDV strains are grouped in geographical lineages based on the genetic correlation with the H protein encoding gene. Phylogenetic and molecular evolutionary analyses of CDV have revealed that mutations affecting the binding site of the H protein for virus entry receptors are associated with the occurrence of disease emergence in novel host species14. In Italy, CDV has been detected in several occasions with different viral strains belonging to separate lineages circulating in the field22. Generally, in Italy CDV circulation in domestic dogs is under control as for the availability of valid vaccines on the market. However, occasional outbreaks occur, and these outbreaks are generally related with the illegal trade of infected dogs from Eastern Europe23. The last large CDV outbreak in Italy was caused by a strain belonging to the Arctic lineage (prototype CDV2784/2013). It occurred in 2013 involving primarily the Abruzzi region, an area hosting several natural parks with significant animal biodiversity. In this outbreak, distemper caused overt disease in unvaccinated domestic and shepherd dogs, Apennine wolves (Canis lupus) and other wild carnivores living in natural areas of the Abruzzi and neighboring regions, including Molise. CDV circulation was still evidenced in the following years in wild and domestic animals22. Direct diagnosis of CDV infection in animals showing neurological signs is primarily performed by molecular assays detecting the viral genome in the cerebrospinal fluid or alternatively in the urine24.The causative agent is canine distemper virus (CDV), a member of the genus In this manuscript, we described the identification and characterization of a CDV strain from the brain tissue of a dog by using the MinION sequencing Nanopore technology.Data were produced by 460 active channels of the flow cell. A total number of 88.357 reads 89.313.926 bp) were obtained from the cDNA sequencing run. By assessing a quality cut-off (>Q7), a total number of 87.221 reads of sequencing reads was selected. The average length of the reads was 1.011 nt. Of these 1.886, the 96,4% was classified as eukaryota, the 2,4% as bacteria and the 1,2% as viruses Fig. . A total6 bp wereCDV confirmed the presence of RNA belonging to CDV in both tissues. Threshold cycles (CT) were 24 and 22 from brain and lung, respectively. Histological lesions and visualization of CDV antigen are showed in Fig. CDV.A specific molecular test was performed after the identification of CDV by MinION. The rtPCR22 and CDV2784/2013 (Arctic lineage)25 which have been recently identified in central and southern Italy, sequence identity ranges from 90.89% to 95.48% (Table The whole genome sequence of CDV1747/ITL2018 (cell passage 3 onto Vero.DogSLAMtag-cells) has been obtained by an additional MinION sequencing run. Data were produced using 453 active channels of the flow cell. A total number of 131.930 reads were obtained from the cDNA sequencing run. By assessing a quality cut-off (>Q7), a total number of 129.354 reads (129.779.778 bp) of sequencing reads was selected. The average of the length of the reads was 961 nt. A total number of 5.164 reads, representing the 100% of the virus-assigned reads, were classified as canine distemper virus Fig. . GenomicThis study was planned and performed to assess the feasibility of the MinION sequencing technology in routine diagnostics. In this occasion, CDV infection was identified in the brain tissue of a dog imported to Italy from Cuba. The dog died after severe neurological infection, which was evidenced two days after the arrival from Cuba.The MinION, performed with nucleic acids purified from the infected brain tissue, was able to provide indication of CDV infection. A specific molecular assay and IHC were then performed to confirm the presence of CDV in tissues. The same NGS platform was also used to obtain the whole genome sequence of the isolate. The involved strain, CDV1747/ITL2018, has been shown to be different from CDV strains which have been demonstrated to circulate in central/southern Italy in recent years.CDV. Although this aspect is undeniably true, the intention of this study is to promote efforts to validate NGS into routine diagnostics in order to get as soon as possible the identification of the etiological agent of a given disease and potentially opportunistic pathogens as previously demonstrated to occur in the field26 with a broad-range diagnostic method. Undeniably, our study has some drawbacks. One of them may be represented by the low number of CDV reads originating from the MinION run which may apparently underestimates its use into diagnostic according to the adopted protocol. This could be explained by the fact that we did not perform any viral enrichment. In this regard, novel techniques for viral enrichment from stools, environmental and tissue samples have been recently described for viral metagenomics studies with second-generation sequencing technologies29. As for today, further efforts are still necessary to assess protocols for virome preparation for third-generation sequencing platforms. Indeed, although Theuns and colleagues7 described a viral enrichment protocol from stool samples for MinION sequencing5, a specific viral enrichment from tissue samples combined with MinION still needs to be explored.One could reasonably argue that the clinical signs were already satisfactory to address the molecular diagnosis of CDV by existing direct assays, including rtPCR30, Schmallenberg virus31, Hepatitis C virus from clinical biopsy samples32, a novel mink astrovirus33, viruses from human stool samples34, porcine epidemic diarrhea virus from swine feces35, classical and atypical Bluetongue virus serotypes from blood samples39, feline morbillivirus from urine40 and viruses responsible for respiratory tract infections41. Moreover, recently our group combined successfully SISPA and MinION sequencing for the identification of bovine enterovirus from a cell-culture isolate6. By the way, several enrichment strategies from tissues and combination of MinION sequencing are currently under investigation by our team. Furthermore, we did not perform live basecalling and diagnostic indications were provided after the sequencing run was stopped without live acquisition of raw sequence data. The local WIMP analysis, indeed, was completed after 13 hours starting from the load of the flow cell with cDNA. However, the retrospective analysis showed that the first read came out within the first 20 minutes of the sequencing run. Interestingly, when the sample was re-processed by the live pipeline  the first sequence read was assigned to CDV after 40 minutes from the beginning of the sequencing run. This would suggest that if used as point-of-care device, solid identification of CDV would have been acquired within less than 1 hour starting from the beginning of the sequencing run.On the other hand, in this study we performed the SISPA amplification before the sequencing run. SISPA is an established protocol which permitted, through random primer amplification, the identification and characterization of several viruses, including different avian RNA virusesCDV was negative when used as first diagnostic attempt? The only valid strategy for diagnosis would have been the one which includes the testing of purified nucleic acids for all the plethora of pathogens known to cause the clinical signs observed in that dog. Reasonably, this approach would have required a longer period to get, eventually, a valid response. This aspect combined with the high chance of unavailability of specific tests, contributes to the relatively high proportion of cases that remain undiagnosed. Therefore, we attempted to use the MinION sequencing platform to get diagnostic indications. In this case, acquired raw CDV reads were not identical to the deposited consensus sequence. This can be easily explained by the fact that reads originated by the second MinION run, with nucleic acids purified from the viral isolate, were further processed with two bioinformatics, GraphMap and Pilon, tools. These tools fix the errors which occurred during the assembly of sequence reads. Further support to the credibility of the final consensus sequence has been provided by the Sanger sequencing of the H protein encoding gene of the occurring strain, either from the isolate or directly from the tissue specimens. Indeed, Sanger sequencing evidenced 100% of nt identity with the sequence obtained by the second MinION run. Nevertheless, raw reads were indispensable and satisfactory to identify the involved viral species and to get diagnostic indications.Conversely, what could have happened if rtPCRThe MinION platform is a unique, highly portable, single molecule real time sequencer currently available. This technology recently emerged to reduce the sequencing costs and to simplify the library preparation procedures. The hallmark of the MinION technology is certainly represented by the fact that this platform can be routinely used to investigate viral and bacterial outbreaks also in low throughput laboratories and, importantly, in field conditions with a real time acquisition of raw data. Further validation experiments are currently ongoing to validate the use of MinION in the daily routine with different biological matrices. Overall, these data demonstrate that MinION is affordable and able to detect CDV quickly and with enough accuracy, supporting its potential for virus surveillance also in field conditions.th, 2017 was legally introduced from Cuba to Italy  on February 19th, 2018. The dog was vaccinated for rabies  on November 10th, 2017. On February 21st, 2018, two days after the arrival in Italy, the dog showed labored breathing and severe neurological signs including disorientation, lack of alertness, convulsive movements of the head and paws  born on July 442) were inoculated with lung and brain homogenates and incubated at 37  °C with 5% CO2 for a maximum of 5–7 days until cytopathic effect (CPE) was observed. Plates showing no CPE were further passaged up to passage 3.Vero cells stably expressing canine SLAM  and homogenized with a TissueLyser II  for 5 min following manufacturer’s guidelines.6.The homogenate was centrifuged and 200 μl of the supernatant was used for nucleic acid purification using High Pure Viral Nucleic Acid Kit  and metagenomic analysis by MinION . Nucleic acid elution was divided in two aliquots to perform RNA and DNA sequencing in two different runs as described in a previous study from our group with some modificationsLikewise, the supernatant of the Vero.DogSLAMtag-cells CDV isolate (passage 3) was collected and processed for RNA purification .43 with some modifications to obtain cDNA40. FR26RV-N (GCCGGAGCTCTGCAGATATCNNNNNN) and FR20RV (GCCGGAGCTCTGCAGATATC) primers were used for SISPA protocol43. Reverse transcriptase and PCR were carried out by using the SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and Pfu Ultra II Fusion HS DNA Polymerase (Agilent), respectively.RNA samples were processed by means of the Sequence Independent Single Primer Amplification (SISPA) methodDNA and amplified cDNA were quantified by Qubit dsDNA HS assay  and used for library preparation by Low input genomic DNA by PCR Barcoding  following manufacturer’s guidelines. Briefly, DNA ends were end-repaired and dA-tailed using the NEBNext End Repair/dA tailing module . PCR adapters were ligated onto the prepared ends using the Blunt/TA Ligase Master Mix  and a unique barcode was added by PCR. The amplification program consisted of a first 3 min step at 95 °C followed by 14 cycles with the following conditions: 95 °C for 15 sec, 56 °C for 15 sec and 65 °C for 6 min. The program ended with 1 step at 65 °C for 6 min. Sequencing adapters were added prior library loading on the Flow cell MIN106, R9 version (Oxford Nanopore Technologies). All purification steps were carried out using AMPure XP beads  according the SQK-LWB001 sequencing protocol. For sequencing, the NC_48hr_sequencing_FLO-MIN106_SQK-LBW001 program was run on MinKNOW Software v.1.4.2. The sequencing run was stopped after 11 hours form starting sequencing.43 was then initiated in the EPI2MEAgent Software (version 2.47.537208) for analyzing brain tissue originated-reads produced by Albacore. Briefly, WIMP tool enables the comparison in real time of the raw reads against the NCBI Organism Database; this tool classifies and identifies the species by looking at the k-mers produced. WIMP platform required 10 minutes of analysis for classification.Fast5 raw MinION files were processed locally by using the Albacore software (version 2.1.10) to obtain fastq files output with a quality cut-off (>Q7). The local base calling allowed to obtain fastq files within less than 2 hours of processing. “What is in my pot” (WIMP) workflow44) aligner and using Pilon software (version 1.22). Specifically, we used the default parameters for Graphmap and a combination of samtools, bcftools, vcfutils and internal scripts to extract the consensus sequence from the alignment. Then, consensus sequence was refined by Pilon. The final sequence and ORFs were also manually inspected using the Ugene software (version 1.29.045). Quality check analysis of raw reads was performed by using the Nanoplot (version 1.17.2).Reads derived from the CDV isolate sequencing run, after Albacore processing, were further demultiplexed, and trimmed by using Porechop (version 0.2.3). To obtain a draft sequence by mapping, the closest reference sequence was identified performing Blastn search (version 0.5.2). The consensus sequence was obtained using the GraphMap  were stained with haematoxylin and eosin (HE). Additional tissue sections were also subjected to immunohistochemistry (IHC) using primary antibody specific for CDV  anti CDVNP; VMRD™, Pulmann WA). Labeling was performed by using a biotinylated goat anti-mouse Ab and subsequently by indirect streptavidin-biotin method . Antigen retrieval was performed by heat treatment in citrate buffer pH 6.0, 0.01 M at 121 °C for 5 min. Sections were visualized with 3,3 diaminobenzidine solution , and finally counterstained with Mayer’s hematoxylin. CDV-positive lung tissue sections of dogs available in house were used as positive controls, whereas the specificity of the immune-labeling was verified with an unrelated Ab.Purified nucleic acids from brain and lung were screened by rtPCR47 and sequenced by dideoxy Sanger method, performed onto 3130xl Genetic Analyzer . Sequence reads obtained by Sanger were assembled with DNAStar software package . Representative H protein nucleotide (nt) sequences of canine distemper virus strains were retrieved from GenBank . Phylogenetic analysis was carried out by Mr Bayes program49 implemented within the software package Geneious . Bayesian inference was performed using four “chains” run over one million generations (with the first 2000 trees discarded as ‘burn-in) and supplying statistical support with subsampling over 200 replicates. jModelTest50 was used to identify the most appropriate model of evolution. The identified program settings for all partitions, under the Akaike Information Criteria, included six-character states , a proportion of invariable sites, and a gamma distribution of rate variation across sites (GTR + I + G). H gene sequence of Phocine distemper virus was used as outgroup.RNAs purified from the CDV isolate (passage 3) and tissue specimens were used for RT-PCR and Sanger sequencing of the H protein encoding sequence. Amplicons were obtained using RH-3 and RH-4 primersAll methods, including necropsy and sampling, were carried out in accordance with internal guidelines and regulations of IZSAM. All experimental protocols, including handling and processing of infectious tissues, were approved by the Ethical Committee of IZSAM.Video S1Supplementary Information


Ricin and abrin are ribosome-inactivating proteins leading to inhibition of protein synthesis and cell death. These toxins are considered some of the most potent and lethal toxins against which there is no available antidote. Digital holographic microscopy (DHM) is a time-lapse, label-free, and noninvasive imaging technique that can provide phase information on morphological features of cells. In this study, we employed DHM to evaluate the morphological changes of cell lines during ricin and abrin intoxication. We showed that the effect of these toxins is characterized by a decrease in cell confluence and changes in morphological parameters such as cell area, perimeter, irregularity, and roughness. In addition, changes in optical parameters such as phase-shift, optical thickness, and effective-calculated volume were observed. These effects were completely inhibited by specific neutralizing antibodies. An enhanced intoxication effect was observed for preadherent compared to adherent cells, as was detected in early morphology changes and confirmed by annexin V/propidium iodide (PI) apoptosis assay. Detection of the dynamic changes in cell morphology at initial stages of cell intoxication by DHM emphasizes the highly sensitive and rapid nature of this method, allowing the early detection of active toxins. Ricinus communis and Abrus precatorius, respectively. The ease and low cost of isolation make these proteins potent biothreat agents.Ribosome inactivating proteins (RIPs) irreversibly damage ribosomes , leadingCurrently, there are two main approaches for assessing ricin activity in noncell-based assays: measuring protein synthesis inhibition ,6,7,8 ancaspase-3. This has been demonstrated in several cancer cell lines as well as mammalian lymphoid tissues [Cell death induction by apoptosis, necrosis, and autophagy is characterized by a distinct set of temporal morphological, biochemical, and gene expression features ,22. Thes tissues ,24.There are several commercial in vitro cell-based assays for assessing cell viability by the loss of membrane integrity. Such assays include nuclear staining of nonviable cells by trypan blue or propidium iodide dying ,26 or meDigital holographic microscopy (DHM) is a time-lapse, label-free, and noninvasive imaging technique that can provide both quantitative and qualitative phase information on morphological features such as cellular area, shape, thickness, volume and confluence ,30,31,32In this study, we present the feasibility of a cell-based assay for ricin and abrin intoxication using DHM. In contrast to current methodologies, DHM is noninvasive, rapid and able to monitor changes in cell morphology dynamically, allowing early detection of low dose intoxication. The method described herein may be applied for early and sensitive detection of these as well as other toxins and substances.In order to characterize morphological changes mediated by ricin exposure, HeLa cells were seeded in six-well plates at different confluence levels (10–30%) and incubated for 4–6 h for cell adhesion. Following incubation, cells were exposed to ricin at 100 ng/mL and digital holograms of four different areas in each well were recorded at 10 min time intervals for 20 h.The resulting digital holographic images were used to measure and define changes in various morphological and optical features of treated versus untreated cells. Images of treated cells depicted in To ascertain whether these changes are attributed specifically to ricin intoxication, neutralizing polyclonal antibodies against ricin were added to cells concomitantly with toxin administration. As shown in p < 0.05) in morphological features compared to untreated cells for the two cell lines. These results summarize several independent experiments (n = 3), with some variation in terms of cell initial confluence and adhesion times before toxin was administered. The same trend of morphological changes was observed for both cell lines compared to HeLa cells. Vero cells were less sensitive to 100 ng/mL ricin compared to HeLa cells, which manifested in a significant delay in morphology change detection. The only exception was ECV, which was significantly reduced in Vero cells within 4 to 7 h compared to 14–15 h in HeLa cells. In order to verify whether the observed morphological changes during intoxication of HeLa and Vero cells are related to cell death, an established viability assay using AlamarBlue, was performed in a dose–response assay. As shown in In order to define the suitable cell line in terms of assay sensitivity, as well as other technical aspects, the Vero cell line was chosen in lieu of the fact that this cell line (similar to HeLa cells) is widely used in the study of ricin intoxication and neutralization ,41. TablThe differences in structural features during toxic exposure were visualized using scanning electron microscopy B. Five hSince ricin and abrin share high structure homology as well as the same biological activity, we tested whether their toxic effect in vitro will be similar. To determine if this is the case, a comparison of the toxic effect of ricin and abrin (100 ng/mL) was performed. As expected, the same trend in morphological changes was observed, with no significant differences in time ranges A,B. As wThus far, we demonstrated that DHM allows for morphological analysis that can identify significant changes induced by ricin/abrin intoxication by analyzing the entire field of view. The cell populations analyzed are comprised of cells undergoing all stages of ricin intoxication, including early and late apoptotic cells. As a consequence, each parameter is an average of a heterogenic cell population. Using the annexin V and PI apoptotic assay we could evaluate the early and late apoptotic cell populations reaches ~15% within 8 h post 100 ng/mL ricin exposure . The apo4/mL, 3 mL) in six well plates, following toxin administration without preincubation for cell adhesion. Digital holograms of four different areas in each well were recorded at 10 min time intervals. In a visual inspection of the preadherent cells, sedimentation and spreading occur within 3 h, both in treated and untreated cells  is a time-lapse, label-free, and noninvasive imaging technique that can provide both qualitative and quantitative phase information on morphological features of cells. Detection by DHM of morphological features characteristic of early stages of cell death  can provIn this study, we employed DHM to evaluate the morphological changes of cell lines during ricin and abrin intoxication. We demonstrated that the effect of ricin and abrin intoxication on HeLa and Vero cell lines is characterized by a decrease in cell confluence which was also observed by monitoring less cell divisions and a reduction of total cell counts compared to untreated cells. In addition, a decrease in morphological parameters such as cell area, perimeter, irregularity, and ECV, and an increase in optical thickness and roughness were also observed. These effects are directly attributed to ricin or abrin intoxication as they were inhibited by the addition of neutralizing antibodies against ricin or abrin respectively.The morphological changes analyzed by the DHM are related to the total cell population. To exclude the contribution of dead cells from the analysis we used the cell tracking application of the DHM. This allowed us to examine the effect of ricin on live cells exclusively. Detection of morphological changes in presumed live cells upon early stages of intoxication reflects a rapid and sensitive method for assessing toxin activity. It was found that HeLa cells exhibited earlier significant morphological changes following intoxication and a significant reduction in cell viability, when compared to Vero cells.Shortening the preparation time of the assay by exposing preadherent HeLa cells to toxins increased assay sensitivity, with an earlier onset of detectable morphological changes compared to fully adhered cells. A further reduction of toxin concentrations was performed in a dose–response assay (0.1–100 ng/mL), showing clear detection of morphological changes within 4 to 6 h for 10 ng/mL toxins. Significant changes in confluence and phase-shift parameters were also observed in 0.3 ng/mL intoxication within 15 h, defining the limit of detection of the assay. The effect of cell adhesion on drug- induced apoptosis was well described in several studies ,43,44. DThe addition of anti-ricin polyclonal antibodies induced a complete inhibition of ricin-induced morphological changes, while the use of the monoclonal antibodies (either directed against subunit A or subunit B) induced only a partial inhibition of cell confluence and phase-shift and complete inhibition of all other parameters. These variations may originate from the fact the polyclonal antibodies are composed of antibodies that target different epitopes simultaneously, increasing the neutralization effect.The DHM in our assay was able to detect single cells for analysis only at confluence levels of under 70%. Above this level, an inconsistency in consecutive frame analyze was observed, hampering the analysis after 20 h of incubation (due to cell replication). In order to detect changes in HeLa and Vero cells with a low dose of toxins—less than 0.3 ng/mL—prolonged intoxication times are crucial. To that end, cells should be initially cultured at less than 10% confluence. Another option, not tested in this study, is to limit cell replication .The characterization of toxin-mediated morphology changes of single cells, detected by DHM, was also examined with shiga toxin , resultiPhototoxicity has the ability to increase the intoxication effect of ricin, as was demonstrated by Song et al.  using flCell based assays for ricin toxicity measure cell survival as the intoxication end point, usually within 48 to 72 h postexposure ,17. As rC. difficile toxins [Overall, our results emphasize the benefits of using DHM for the detection of ricin and abrin activity in cell lines as a rapid and sensitive assay. Application of cell morphology detection in high throughput screening is used by researchers in academia and pharma in order to address cell death determination in drug discovery ,50, advee toxins .The ability of DHM technology to detect early changes in cellular features upon toxin-mediated cell death processes makes it suitable for toxin activity assays, and is comparable, if not superior, to established methods.Purified ricin and abrin were prepared as described previously ,55. Antiv/v) fetal bovine serum and 1% L-glutamine (200 nM) and incubated at 37 °C in 5% CO2 humidified incubator.HeLa and Vero cell lines were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% , and a motorized stage. Digital holographic microscopy, as a quantitative phase-contrast imaging method, is a form of optical interferometry, which detects the phase delay related to the light passing through the tested object. Images were converted from wavelength interaction to cells’ representation by a computer algorithm . This computation enables to obtain information on various morphological and optical parameters. Two different threshold settings prior to segmentation of cells were used for analyzing the experiments: Otsu thresholding (OT) [5) were seeded in 6-well plates at different degrees of confluence (10–30%) with or without incubation for 4 to 6 h for cell adhesion. Following incubation, cells were exposed to ricin or abrin at 1–100 ng/mL in a 3 mL volume. The neutralizing effect of anti-ricin and anti-abrin antibodies was assessed by incubating toxins with 20 μg MH1 or MH75 monoclonal antibodies or 1:100 dilution of polyclonal antibodies. Cells were monitored continuously, in time-lapse mode (every 10 min), in multiple locations in each well using a high precision motorized stage. Quantified parameters obtained from DHM in each frame were analyzed as changes relative to time zero from each area and presented as mean ± SE for all the monitored areas in each well. In this work we focused on significant changes observed during intoxication in several parameters: phase-shift (phase delay related to the light passing through the tested object), confluence, area, irregularity (determines the deviation of the cell region from the circular shape), perimeter, and roughness (indication on the smoothness of the surface of the cell). As the refractive index of the cell and the surrounding medium cannot be measured, the presented cell thickness and volume values are expressed as the optical thickness and effective-calculated volume (ECV).Digital holographic microscopy was performed using a Holomonitor M4 microscope . The Holomonitor M4 is a label-free cell analyzer for adherent cells equipped with X20 magnification objective (NA-0.4), a 0.8 mW HeNe laser   and miniing (OT) . While t4/well and then treated with 10–100 ng/mL of ricin or abrin for 3–17 h. AlamarBlue reagent was added to each well up to 10% of tissue culture medium and incubated for an additional 3 at 37 °C. Cell viability was determined by measuring absorbances at 562 nm and 600 nm.The cytotoxic effects of purified ricin and abrin on HeLa and Vero cells were determined by AlamarBlue viability assay . Briefly, cells were cultured in 96-well plates overnight at a density of approximately 104/well HeLa cells were cultured overnight in 24-wells plates for cell adhesion. Following cell adhesion, toxins (10–100 ng/mL) were added to cells and apoptosis measurements were performed after 4–8 h. In order to assess the effect of cell adhesion on toxin susceptibility, HeLa cells were treated with trypsin for cell detachment and washed in a fresh growth medium prior to toxin administration. Apoptosis induction was compared between the preadherent and fully adherent cells. Untreated preadherent/adhered cells were used as a control.Apoptosis induction with ricin and abrin in HeLa cells was assessed by APC Annexin V/PI apoptosis kit  and analyzed using a BD Fortesa Flow Cytometer from Becton Dickinson . In general, 5 × 102 flasks at 37 °C and 5% CO2. The cells were detached using trypsin and then seeded in 24 well plates at ~50,000 cells/well on top of polylysine-coated cover slips. The cells were allowed to attach and treated with ricin for 5 h at 37 °C. After incubation, the cells were washed with phosphate-buffered saline (PBS) once and a fixative buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer  was added. Cells were washed in PBS and 1% osmium tetraoxide was added for 1 h at room temperature (RT) Cells were washed in DDW and dehydration was carried out in a gradient of ethanol concentrations. After dehydration, the ethanol was substituted with CO2 in a Critical Point Drying  machine and, finally, the cover slips containing cells were coated with 2 nm iridium. The cells were imaged using QuantaFEG SEM  operated at 6 kV in high vacuum mode using an ET detector.HeLa cells were grown in 75 cmt-test. p value ≤ 0.05 was considered to be significant.All experiments were repeated three times and data was analyzed using GraphPad Prism5 software . Results are expressed as mean ± standard error. Statistical significance was determined by 2-tailed unpaired Student’s


Phormia regina (Meigen)  is one of the most abundant carrion flies in North America. Calliphorids are important in agriculture and animal production, veterinary sciences, forensics and medical entomology. While the role of flies in the epidemiology of human and animal diseases is an active area of research, little is known about the microorganisms associated with these insects. We examined the diversity of wild-caught black blow fly endogenous  and exogenous  microbial communities using 16S rRNA gene sequencing. Overall, 27 phyla, 171 families and 533 genera were detected, and diversity was significantly higher (P < 0.05) on external body surfaces. At the genus level, Dysgonomonas, Ignatzschineria, Acinetobacter, Vagococcus, Myroides, and Wohlfahrtiimonas were predominant. Cloning and sequencing of nearly full-length fragments of the 16S rRNA gene showed that some of the species identified are known to be pathogenic to humans, animals, and plants. Myroides odoratimimus and Acinetobacter radioresistens are well-known, multi-drug resistant bacteria. These results provide a snapshot of the microbial communities harbored by adult black blow flies and call for more comprehensive studies to better characterize the role these flies may play in the transmission of pathogenic microorganisms.The black blow fly,  In calyptrate flies, calliphorids represent about 1,500 described species and account for 8% of species diversity3. They are saprophagous/necrophagous insects, breeding in decaying organic matter of plant and animal origins and are of medical6, veterinary/agricultural7, and forensic importance9. For example, adult calliphorids are known to be the first insects to colonize animal and human remains and are frequently used in the estimation of the minimum postmortem interval (minPMI) in medico-legal investigations10. Several species of blow flies, such as the primary screwworm, Cochliomyia hominivorax (Coquerel) and the Australian sheep blow fly, Lucilia cuprina (Wiedmann) , are notorious myiasis (flystrike) agents in livestock causing hundreds of millions of dollars in economic losses11.Blow flies  are a group of medium to large flies with a worldwide distribution, usually harboring bright or metallic colors12, while their synanthropic nature (association with human environments) as well as strong flight abilities13, allow them to potentially disseminate those organisms over long distances into human habitat; they have the potential of infecting different substrates through contact by just landing on a surface but also by regurgitation of their gut content and defecation16. In addition, it has also been shown that blow flies can carry and mechanically transmit several bacterial pathogens, including Burkholderia pseudomallei17, Providencia rettgeri, Morganella morganii, Klebsiella spp. and Staphylococcus aureus18; Escherichia coli20; and Helicobacter pylori21.The presence of blow flies in microbe-rich substrates, such as manure, carrion and corpses, confer them a high probability of encountering/interacting with a large variety of microorganisms such as bacteriaPhormia regina (Meigen) , the focus of this paper, is a Holarctic fly and is distributed throughout the Northern Hemisphere22. In the USA, P. regina is typically a cold weather fly, as it is more abundant during the “chilled” summer months in the Northern part of the US while being more frequent in the cooler months of spring and fall in the South24. However, high population densities of P. regina have previously been recorded during the summer in Kansas25 and California26. Phormia regina has been of interest since it is one of the most abundant carrion flies in North America27 and is one of the primary insects used to estimate the minimum postmortem interval (minPMI) in forensic entomology28. Phormia regina is also listed by the U.S. Food and Drug Administration (FDA) as one of the 22 pests (the “Dirty 22”) that could potentially spread food-borne pathogens29 and is an important research model for the study of fly physiology32. Several studies have been conducted on P. regina morphology33, genomics27, biology and ecology36, including aspects of the interactions that exist between this species and bacteria37. However, studies that investigated the microbial communities in free-flying, wild-type adult P. regina using 16S rRNA gene sequencing are absent. Weatherbee et al.37 investigated the internal microbiome of P. regina collected from swine carcasses but focused on third-stadium larvae. Russell et al.38 investigated the presence of antibiotic-resistant bacteria in adult Calliphorids collected from piglet cadavers introduced into a forest environment but only identified the flies to the family level. Also, the study was based on culture-dependent techniques.The black blow fly, In the present study, we investigated the endogenous and exogenous bacterial microbiomes of wild-caught adult black blow flies collected in a suburban environment using high-throughput 16S rRNA gene amplicon sequencing. We hypothesized that (i) the microbial communities from these two regions of the fly body would be different, (ii) bacterial diversity on the outside will be higher compared to the gut and (iii) the flies collected will harbor known pathogenic bacteria.39.Adult blow flies (mixed sexes) were collected at the Lake Wheeler Road experiment station at NC State University (NCSU), Raleigh, North Carolina in November 2017. This field station contains several animal production systems including a feed mill laboratory, equine education center, poultry waste management facility, etc.) and plant-related units . This experiment station is located close to the main NCSU campus and urban areas in the city. Blow flies are strong fliers and highly adapted to evasion making it difficult to collect them individually in flight or after they have landed, therefore, an effective alternative approach is trapping. Flies were collected at a dump site  using a Captivator fly trap jug  that contained 900 ml of a Flies-Be-Gone fly attractant solution  Fig. . The juget al.40. Briefly, the pooled exogenous PBS wash (1 mL) was lyophilized and reconstituted with 200 µL of TNE buffer. For endogenous sampling of the microbiome, each fly was transferred to a sterilized screw cap 2 mL microcentrifuge tube  containing 10 sterilized 3 mm solid glass beads  each and was homogenized in 200 µL TNE buffer using the FastPrep FP120 system  for 30 s. For lysis, we added 160 µL of lysis buffer 1 . The samples were then incubated at 37 °C for 1 h. After which 200 µl of lysis buffer 2  was added, and the samples were further incubated at 56 °C for 1 h. The use of the whole body to sample the endogenous microbiome allowed us to account for bacteria present internally but localized to organs other than the gut. DNA was isolated through phenol-chloroform extraction and ethanol precipitation, and the resulting DNA was resuspended in 100 µl of molecular grade water. The DNA samples were further purified using the Wizard DNA cleanup system . The DNA quality and quantity were assessed using a NanoDrop 1000 Spectrophotometer . The total genomic DNA was normalized to 50-100 ng/µl and stored at −40 °C until PCR amplification.Prior to endogenous microbial DNA extraction, each adult fly was surface sterilized using 70% ethanol (30 s) followed by 1% bleach in water (30 s) and then washed 5 times with sterile water. The final washes were pooled together for subsequent verification of sterility. DNA extraction from internal (12 endogenous samples) and external body (12 exogenous samples) extracts was modified from Ponnusamy 41. The 16S rRNA sequencing libraries were constructed according to the Illumina’s 16S rRNA metagenomics sequencing library preparation protocol . During library preparation, the amplicon products were cleaned following each PCR round using magnetic beads from the AxyPrep Mag PCR Clean-up kit , and the size of the amplicons was verified each time on a 1.5% agarose gel. One endogenous sample failed to generate PCR amplicons and was excluded. Illumina sequencing libraries were constructed for all 23 remaining samples (11 endogenous and 12 exogenous), and amplicon DNA concentration was measured with Quant-iT PicoGreen . Final libraries were pooled in equimolar amounts. Illumina sequencing (250-bp paired-ends) was performed at the Microbiome Core Facility, School of Medicine, University of North Carolina, Chapel Hill, NC, USA.To characterize the bacterial communities present in the samples (see S1 File), the hypervariable V4 region of the 16S rRNA gene was targeted using the 515 F (GTGCCAGCMGCCGCGGTAA) and 806 R (GGGACTACHVGGGTWTCTAAT) primers42. Illumina paired-end sequence reads were first demultiplexed to assign the sequences to their sample of origin using the QIIME2 demux emp-paired command and then quality filtered using the DADA243 algorithm as a QIIME2 plugin. Through the DADA2 pipeline, the paired-end reads were joined together, denoised, and chimeras, as well as residual PhiX reads, were removed. This results in the identification of all unique bacterial sequences called amplicon sequence variants (ASVs) which are technically equivalent to 100% OTUs but without errors45. Primers sequences were trimmed off the reads, but the sequences were not truncated to increase the chances of joining the paired-end reads efficiently. The 16S rRNA gene sequence variants (representative sequences) were assigned taxonomic classification using a pretrained Naïve Bayes classifier which was trained on the Greengenes database 46. Taxonomy was assigned using the Qiime feature-classifier classify-sklearn command. To calculate the statistical difference between the exogenous and endogenous groups, relative abundances of taxa with abundance higher than 1%  were arcsine square root transformed, and a Welch two-sample t-test47 was run using abundances from each sample as replicates. Relative abundance data were imported from QIIME2 into the R statistical software48 for running the t-tests.The sequencing data were processed using the Quantitative Insights Into Microbial Ecology 2 (QIIME2) program49. To avoid biases due to sample-based variations in library sizes and to retain all of our samples in the subsequent analyses, each sample was rarefied to a depth of 18,000 sequences per sample. Alpha diversity was estimated by the Shannon index (integrates both richness and evenness)50, the number of observed OTUs (ASVs), and Faith’s phylogenetic index51. The statistical significance of alpha diversity between groups (exogenous and endogenous samples) was inferred using pairwise Kruskal-Wallis H-tests. Beta diversity was calculated using the weighted UniFrac52 metric, and a PERMANOVA test was run to determine if there was any statistical difference between the two treatment groups. The principal coordinate analysis (PCoA) result was visualized using EMPEROR53.Prior to computing diversity metrics, the representative sequences obtained after DADA2 were aligned and masked to remove gaps, and a mid-point rooted phylogenetic tree was constructed using the QIIME2 FastTree 2 plugin54 following a protocol previously described in Ponnusamy et al.55. DNA from four fly samples that contained the highest abundances of the targeted taxa were used , a nearly full-length fragment of the 16S rRNA gene was amplified using the universal bacterial primers, 27 f and 1492r56. The sequences were aligned using the ClustalW program. Due to sequencing errors and low-quality reads towards the ends of the reads when using Sanger sequencing, a less error-bearing 16S rRNA gene DNA sequence was reconstructed by joining three DNA contigs (500-600 bp each) together using a minimum 100 bp overlap between the sequences. First, 16S rRNA gene sequences were screened for vector contamination using the NCBI VecScreen program and sequences that were identified to be of vector origin were removed. The final version of each 16S rRNA gene clone sequence was then blasted against the NCBI GenBank database using BLASTn. The 16S rRNA gene sequences were blasted against the NCBI 16S ribosomal RNA database on February 6, 2019. Clones with DNA sequences sharing 99-100% query coverage and at least 99% identity with GenBank sequences were assigned to that specific phylotype. Multiple alignments were performed using the ClustalW algorithm. The phylogenetic relationship between these clones and a selection of other closely related species was reconstructed using the kimura two-parameter model57 and neighbour-joining algorithms as implemented in the MEGA7 software package.For each of the four colonies, each targeting a different taxon, the DNA sequences obtained using the M13F and two internal 16S rRNA gene primers were imported into the Molecular Evolutionary Genetics Analysis (MEGA version 7.0) softwareP. regina extracts for each fly separately were subjected to Illumina paired-end sequencing. A total of 10,622,697 16S rRNA gene sequences were obtained. After quality filtering using DADA2, 7,684,848 reads remained with an average of 334,124 reads . The remaining sequences were clustered into 2,452 sequence variants and assigned to 27 phyla, 171 families and 533 genera (with some further classified into species).DNA extraction from pooled washes (from surface sterilization) yielded no bands on agarose gel after PCR amplification and thus was not sequenced. A total of 23 16S rRNA gene libraries from exogenous and endogenous P < 0.001)    suggests that our sequencing depth of 18,000 was sufficient to capture the majority of taxa present in the samples  Fig. . Similar01) Fig.  as well 01) Fig.  were sigP = 0.001).Bacterial community difference between the two groups (β-diversity of exogenous vs. endogenous communities) was tested using the weighted Unifrac metric. This analysis revealed that the majority of the variation in bacterial diversity across the samples could be attributed to where the samples originated from Fig. . PrincipDysgonomonas (36.8%), Ignatzschineria (20.6%), Acinetobacter (10.0%), Vagococcus (3.8%), Myroides . In the exogenous samples, Dysgonomonas (16.9%), Ignatzschineria (24.9%), Acinetobacter (19.2%), Vagococcus (5.6%), Myroides (2.2%) and Wohlfahrtiimonas (1.8%) were the most abundant. The endogenous samples harbored Dysgonomonas (58.5%), Ignatzschineria (15.9%) and Vagococcus (1.8%), as the most abundant genera. Myroides (0.01%) and Acinetobacter (0.01%) were almost absent from the inside samples while Wohlfahrtiimonas had a very marginal relative abundance (0.3%) , Bacteroidetes (38.9%), Firmicutes (8.5%) and Actinobacteria 1.1%)  bacteria  found on some flies not found on others (see S2 File).We also compared the bacterial diversity between individuals for the 7 predominant taxa found on the outside of the flies, the relative abundance of http://blast.ncbi.nlm.nih.gov/). The nearly full-length reconstructed 16S rRNA gene clones had an average length of 1496 bp. The 16S rRNA gene clone sequence “Clone A1” matched to the species Ignatzschineria ureiclastica strain FFA (NR_116121.1) and Ignatzschineria larvae DSM 13226 (NR_025381.1) with 99% similarity. BLASTn searches with “Clone B9” affiliation to the species Acinetobacter radioresistens with 99% similarity to both the strains NBRC 102413 (NR_114070.1) and FO-1 (NR_026210.1). The first three highest BLAST matches for “Clone C8” were 16S rRNA gene sequences from Dysgonomonas capnocytophagoides strain JCM 16697 (NR_113133.1) (93%), Dysgonomonas capnocytophagoides strain CDC F9047 (NR_044778.1) (93%) and Dysgonomonas alginatilytica strain HUA-2 (NR_137388.1) (92%). The first four top hits after BLAST searches for “Clone D17” were: Paraburkholderia rhizoxinica strain HKI 454 (NR_102769.1) (94%), Paraburkholderia rhizoxinica strain HKI 454 (NR_042393.1) (94%), Burkholderia rinojensis strain A396 (NR_118637.1) (94%) and Paraburkholderia endofungorum strain HKI 454 (NR_042584.1) (94%).All 30 16S rRNA cloned colonies that were Sanger sequenced using the M13 primers matched to their respective targeted taxa when the clone sequences were blasted against the NCBI database (http://rdp.cme.msu.edu/)58. The 16S “CLONE A1” was placed within the genus Ignatzschineria  and more similar to Ignatzschineria larvae  within the Acinetobacter genus  and was more closely related to both Dysgonomonas capnocytophagoides and Dysgonomonas macrotermitis  and external body surfaces (exogenous) of et al.59 demonstrated that both the number and variety of bacteria isolated from external surfaces of filth flies were significantly higher compared to the gut. One possible reason for higher diversity on the fly surface is the availability of high microbe-laden feeding/breeding sites; black blow flies are known for example to frequently visit human and/or animal dung60. In addition, blow flies, house flies, and flesh flies have adhesive pads between the tarsal claws of their legs called pulvilli which are covered in fine hairs or “tenant setae”. The setae have been shown to secrete an adhesive substance to increase surface area for attachment to smooth surfaces but are also deemed to retain many microorganisms on the legs63. Microorganisms also attach to other body parts such as the wings, the mouthparts and abdomen62.Our findings revealed the number of observed ASVs (OTUs), Shannon diversity, and Faith’s phylogenetic diversity were all significantly higher in the outside samples representing DNA extracted from the surface of each fly. This demonstrates that diversity was higher on the external surface of the fly’s body compared to the gut and therefore the flies harbored more different bacterial species on the outside of their bodies. This result was not unexpected and has been shown previously in flies. For example, using culture-dependent techniques, Adeyemi Phormia regina). Trapping of a single species mitigated potential contamination of our target flies by bacteria associated with other flies.Although the transfer of bacteria between flies during the trapping period cannot be ruled out, there was no indication that the trapping method was an issue. Alpha diversity and bacterial community composition analyses showed that the exogenous microbiomes were not homogenous between the flies; variability was determined to have resulted from differences in bacterial composition and taxonomic abundances between samples. Additionally, minor taxa (abundances <1%) included several bacterial species that were detected on some flies while absent (relative abundance of 0%) on others, suggesting that the exogenous samples were independent. Further, the timing (November) and the methods by which trapping was conducted permitted the collection of a single species .64, fruit flies65, flesh flies66 and other blow flies67. It is worth noting that the overall average relative abundances of the last four families were essentially driven by outside samples as members from those families were barely detected in inside samples (abundances ranging between 0 and 0.15%). No Moraxellaceae was detected inside. A similar trend was also observed by Weatherbee et al.37 who investigated the internal microbial communities of third-instar specimens of P. regina as well as the external microbiome of the larval mass (larva aggregate composed of multiple fly species including P. regina) present on swine carcasses at different time points. At 132 and 156 h after carcass placement, where P. regina larvae were the most predominant species , several taxa at the family level identified outside were not detected within the larvae. These results suggest that both the immature and adult stages of P. regina may selectively control the gut bacteria either through digestion or other unknown mechanisms. Several families (Bacteroidaceae and Bifidobacteriaceae) identified among the 10 most abundant families in the swine carcass study were marginally present in our samples while Porphyromonadaceae, the most abundant family taxon found internally in this study, was not present and/or abundant within P. regina larvae. These differences may be due to several factors including developmental stage-related differences , the geographic locations of the studies (Indiana vs. North Carolina) as well as the feeding sources available to the flies and different bacteria or differences in bacteria abundance in the available feeding sources. All those factors have been shown to significantly impact the microbiomes of several insects68.Overall, Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria were the predominant phyla in our samples. This result is in agreement with findings in housefliesDysgonomonas, Ignatzschineria, Acinetobacter, Vagococcus, Myroides, and Wohlfahrtiimonas were the most predominant taxa in our study. These genera, even though their relative abundance may vary, have been isolated from various insect species before. The majority of them, however, are frequently detected together (in 16S-based NGS studies) and often times in high abundances in insects associated with filth and most particularly in saprophagous and/or necrophagous insects/flies70. This result may be due to similar ecological niches in those insects and their overlapping range of feeding sources, an adaptation of these bacterial taxa to physicochemical conditions of the guts of these insects and to the role that these microorganisms may be playing in host nutrition and/or fitness.At the genus level, Dysgonomonas, the genus with the highest abundance has been isolated from a burying beetle Nicrophorus vespilloides (Herbst) (Coleoptera: Silphidae)69, a bombardier beetle Brachinus elongatus (Chaudoir) (Coleoptera: Carabidae)45, a wood-feeding termite Reticulitermes speratus (Kolbe) (Isoptera: Rhinotermitidae)71, the common house fly Musca domestica (Diptera: Muscidae)64, the oriental latrine fly Chrysomya megacephala 67, the Australian sheep blow fly, Lucilia cuprina (Wiedmann) and the common green bottle fly Lucilia sericata (Meigen) . Similarly, results from analyses of this genus isolated from termites, dung beetles, black soldier flies and mosquitoes suggest that Dysgonomonas is implicated in the breakdown of carbohydrates namely cellulose and hemicellulose as well as the production of vitamin B1276. Whether Dysgonomonas plays similar roles in the black blow fly is yet to be confirmed. However, the high abundance of this genus in internal samples in this study points to that direction.Ignatzschineria77 and Wohlfahrtiimonas78 were both originally described in the larvae of a parasitic fly, the spotted flesh fly Wohlfahrtia maginifica (Schiner) (Diptera: Sarcophagidae) a major myasis agent in animals66. Phylogenetically, they are two related genera but belong to two different lineages. These two genera have been isolated from other flesh flies79, house flies64, blow flies70 and carrion beetles69. Little is known about the role that members of these genera play in insects. However, they both exhibit strong chitinase activity, and Toth et al.77 have suggested a possible role of Ignatzschineria in metamorphosis. The role that they play in adult insects needs investigation. The genus Vagococcus was previously isolated from fruit flies80, house flies64, blow flies70, red imported fire ants, Solenopsis invicta81 and burying beetles69. They are deemed to produce antiviral metabolites in Aedes albopictus82, but nothing is known about their role in Phormia regina.Acinetobacter was almost exclusively located externally (abundance inside was 0.01%). In fact, Acinetobacter spp. are widespread in natural environments and thus are frequently recovered from soil, polluted water, plants, animals and humans83. However, they have also been isolated from the guts of different insects85. For example, Coon et al.86, showed that the inoculation of Acinetobacter spp. restored larval development in axenic mosquito larvae while Minard et al.87 suggested they aided in the digestion of plant nectar consumed by Aedes albopictus mosquitoes. It was also suggested that they may help detoxify defensive plant compounds for tropical herbivorous beetles88. They also secrete antiparasitic compounds in the guts of several insects89. The presence of this genus on the outer body of the flies is probably of environmental origin; they may have been picked up mechanically from a contaminated source and may not have a function role in fly biology. It would be interesting to investigate if the bacteria could sustain the black blow fly gut environment and what possible role those bacteria could play.Myroides odoratimimus, previously known as Flavobacterium odoratum90, was also exclusively observed in outside samples (abundance inside was 0.01%). Like some of the previous taxa described in this section, M. odoratimimus are ubiquitous in natural environments and are commonly found in soil, fresh and marine water, meat-processing plants and insects91. Myroides spp. are well known for their ability to secrete antimicrobial substances90, and thus it is possible that they help protect insects that harbor them against other pathogenic bacteria.P. regina, the absence of some taxa on the outside of the fly that are found internally and the fact that the most abundant taxa found inside are also present on the fly’s surface suggests that the microbial communities associated with black blow flies are acquired from the environment probably from feeding/breeding sites. Also, based on previous fly research, it is likely that the internal bacteria present may contribute to the host’s development. Most of the bacterial taxa identified in the P. regina microbiome contained known pathogens (discussed in more detail below).Based on our findings in Myroides odoratimimus) thus limiting the depth of analysis of the results. A literature search revealed that some of those genera are well known to be pathogenic microorganisms. To improve taxonomic resolution and to verify the detection of potential pathogens in our samples, the first four identified taxa in terms of relative abundance  were further targeted by developing nearly full length 16S rRNA gene clones and sequencing them using Sanger sequencing. The results showed some of the most abundant taxa in our samples may be new bacterial taxa and also confirmed the existence of potential human, animal and plant pathogens in our fly samples.Most reads (89%) obtained from Illumina sequencing were classified down to the genus level even for the most sequence-rich taxa . Therefore, the bacterium corresponding to that sequence constitutes a distinct lineage with no closely related allied 16S rRNA gene sequences and probably represents a novel genus in the family Burkholderiaceae, order Burkholderiales, class Betaproteobacteria in the phylum Proteobacteria. The genus Burkholderia sensus lato (s.l.) is the original genus described by Yabuuchi et al.92. Since then, the taxonomy of this genus has been evaluated several times with the addition of several new taxa, and today it is subdivided into 5 different groups or “sub-genera”94. The most interesting issue is that during the phylogenetic tree reconstruction, we made sure to include at least one member from each of the five groups (based on type species present in RDP), but Clone D17 was placed as a sister group to Burkholderia and Pandoraea with strong support . No assumptions can be made at the current stage about the pathogenicity of this potentially new genus.Our results suggest that some of our samples contained a new genus in Burkholderiaceae: Ten percent of the reads was classified as Betaproteobacteria, a class in the phylum Proteobacteria. BLASTn searches for “Clone D17” which was targeting that class for better identification revealed that the clone sequence was 94% similar to two strains of Dysgonomonas species: The taxa previously classified as Dysgonomonas was targeted with Clone C8. The sequence from this clone had 93% similarity with two strains of Dysgonomonas capnocytophagoides and 92% similarity with a strain of Dysgonomonas alginatilytica based on BLASTn searches. Phylogenetically, it clustered with all the other Dysgonomonas  but was more closely related to both Dysgonomonas capnocytophagoides and Dysgonomonas macrotermitis with strong statistical support . These results suggest that this molecular isolate represents a potential new species within the genus Dysgonomonas, family Porphyromonadaceae, order Bacteroidales, class Bacteroidia in the phylum Bacteroidetes. Dysgonomonas capnocytophagoides, formerly known as CDC group DF-3 (Center for Disease Control group dysgonic fermenter-3) is an opportunistic bacterium associated with diarrhea and bacteremia96. Dysgonomonas macrotermitis, on the other hand, was only recently isolated from a fungus-growing termite97 and has not been associated with any disease so far. More work is needed to better characterize the bacterium represented by Clone C8 (this study).Our results indicate that our samples contained a new Ignatzschineria species: The sequence from “Clone A1” matched to the species Ignatzschineria ureiclastica and Ignatzschineria larvae with 99% similarity based on BLASTn searches conducted in February 2019. Clone A1 clustered together with all the species in the genus Ignatzschineria  in the phylogenetic analyses but was more closely related to Ignatzschineria larvae  which can cause bacteremia98. This finding is indicative of a potential new species in the genus Ignatzschineria, family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria within the phylum Proteobacteria.New Acinetobacter radioresistens: Both BLASTn searches and phylogenetic analyses revealed the sequence of Clone B9 belonging to the bacterium, A. radioresistens. It was originally isolated from cotton and soil99. Like several other members of the genus Acinetobacter, this species has emerged as an opportunistic human pathogen. It can cause septicemia100, nosocomial bloodstream infection and community-acquired infections in immune-compromised patients102 and has the potential to damage human epithelial kidney cells through the production of outer membrane vesicles (OMVs)103. A. radioresistens have also been shown recently to cause nosocomial bacterial infection outbreaks in research animals104. Another concerning fact about this species is its ability to withstand harsh environmental conditions as well as its strong and well-established resistance to several antimicrobials. In fact, A. radioresistens is highly resistant to desiccation and radiation105 and is known to be resistant to Carbapenem, a class of antibiotic normally highly effective against multidrug-resistant bacteria107.Myroides odoratimimus: This bacterium is also an opportunistic pathogen causing a variety of diseases/infections of which some are life-threatening. M. odoratimimus can cause bacteremia, endocarditis, pericarditis, pneumonia and septic shock, urinary tract infections, calcaneal ulcer and several other important bacteria-related infections in humans111. This species can also cause lethal infections in fish112.Pseudomonas viridiflava (relative abundance of 0.7%), a multi host plant pathogen. P. viridiflava causes foliar and stem necrosis as well as stem and root rot in a wide range of plants such as tomato, melon and eggplant115. Finally, in insects, Wohlfahrtiimonas chitiniclastica, the most frequently identified species of the genus Wohlfahrtiimonas is known to cause human infections66.In addition to the taxa reported here as the most abundant (≥1%), it is important to note the detection in our samples of Myroides odoratimimus, Acinetobacter radioresistens and Pseudomonas viridiflava on the fly body of which several are also resistant to several antibiotics.In summary, this study provides, for the first time, a snapshot of the bacterial communities harbored both internally and externally by free-flying, black blow flies. The results revealed that the flies harbored a higher diversity of bacterial species on their body surfaces probably owing to their ecology and behavior. Even though the majority of the most abundant bacterial taxa identified in this study have also been reported in other flies/insects, the presence of many potentially new taxa further confirms the necessity to conduct microbiome studies in insects in general but more importantly in synanthropic insects that breed in microbe-rich substrates such human and animal dung. Arguably, the most intriguing fact is the identification of known human, animal and plant pathogens such as Supporting Information.Supporting Information.2Supporting Information.3