Microsporidia are a model clade for studying intracellular parasitism, being well-known for their streamlined genomes and their extreme life history. Although microsporidia are highly diverse and ecologically important to a broad range of hosts, previous research on genome architecture has focused primarily on the mammal-infecting genus Encephalitozoon. Here, we expand that work, testing the universality of the patterns observed in Encephalitozoon by investigating and comparing variation in genetic and epigenetic architectures in the high-quality genome assemblies of several major microsporidia clades. Our comparison of nine genomes, including the first genome assemblies of Binucleata daphniae, Gurleya vavrai, and Conglomerata obtusa, and revised, improved assemblies of Glugoides intestinalis, Mitosporidium daphniae, and Ordospora colligata, found limited conservation of genetic and epigenetic architecture across all microsporidia, although many genomic characteristics, such as nucleotide composition and repeat content, were shared between genomes of the same or related clades. For example, rRNA genes were hypermethylated in most species, but their position close to chromosome ends was only found in the Encephalitozoon and its sister clade. GC-content varied widely, linked to genome size, phylogenetic position, and activity of repeat elements. These findings enhance our insight into genome evolution and, consistent with findings from other systems, suggest epigenetic modification as a regulatory mechanism of gene expression and repeat element activity in microsporidia. Our comparative genome analysis reveals high variation in genetic and epigenetic architecture among microsporidia, despite all of them adapting to a parasitic lifestyle within host cells.

In many species with sex chromosomes, the Y is a tiny chromosome. However, the dioecious plant Silene latifolia has a giant ~550-megabase Y chromosome, which has remained unsequenced so far. We used a long- and short-read hybrid approach to obtain a high-quality male genome. Comparative analysis of the sex chromosomes with their homologs in outgroups showed that the Y is highly rearranged and degenerated. Recombination suppression between X and Y extended in several steps and triggered a massive accumulation of repeats on the Y as well as in the nonrecombining pericentromeric region of the X, leading to giant sex chromosomes. Using sex phenotype mutants, we identified candidate sex-determining genes on the Y in locations consistent with their favoring recombination suppression events 11 and 5 million years ago.

This protocol was designed for DNA extraction of about 50 adult female Daphnia magna, it should also work for 1-150 animals but with adjusted reagent volumes. For achieving HMW DNA or maximizing yield, some modifications are indicated as substeps. Before DNA extraction, animals can be freed from microbes using antibiotics (https://www.evolution.unibas.ch/ebert/lab/daphnia_dna.htm) and should be dehydrated and snap-frozen with liquid nitrogen for archiving HMW DNA.

Genomic regions that play a role in parasite defense are often found to be highly variable, with the major histocompatibility complex serving as an iconic example. Single nucleotide polymorphisms may represent only a small portion of this variability, with Indel polymorphisms and copy number variation further contributing. In extreme cases, haplotypes may no longer be recognized as orthologous. Understanding the evolution of such highly divergent regions is challenging because the most extreme variation is not visible using reference-assisted genomic approaches. Here we analyze the case of the Pasteuria Resistance Complex in the crustacean Daphnia magna, a defense complex in the host against the common and virulent bacterium Pasteuria ramosa. Two haplotypes of this region have been previously described, with parts of it being nonhomologous, and the region has been shown to be under balancing selection. Using pan-genome analysis and tree reconciliation methods to explore the evolution of the Pasteuria Resistance Complex and its characteristics within and between species of Daphnia and other Cladoceran species, our analysis revealed a remarkable diversity in this region even among host species, with many nonhomologous hyper-divergent haplotypes. The Pasteuria Resistance Complex is characterized by extensive duplication and losses of Fucosyltransferase (FuT) and Galactosyltransferase (GalT) genes that are believed to play a role in parasite defense. The Pasteuria Resistance Complex region can be traced back to common ancestors over 250 million years. The unique combination of an ancient resistance complex and a dynamic, hyper-divergent genomic environment presents a fascinating opportunity to investigate the role of such regions in the evolution and long-term maintenance of resistance polymorphisms. Our findings offer valuable insights into the evolutionary forces shaping disease resistance and adaptation, not only in the genus Daphnia, but potentially across the entire Cladocera class.

Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years. Trans-species polymorphisms (TSP) in disease loci are thought to be caused by long-term host-parasite coevolution. Here, the authors identify consistent TSPs across three species of Daphnia and find several genes related to immunity function for resistance to a virulent bacterial pathogen.

Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about their ability to adapt. We explored the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A genome-wide association study (GWAS) further revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase (TPS) gene, suggesting that trehalose production facilitates salinity tolerance. Salinity-tolerant animals showed a positive correlation between water salinity and trehalose concentrations, while intolerant animals failed to produce trehalose. Animals with a non-functional TPS gene, generated through CRISPR-Cas9, supported the trehalose role in salinity stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionary mechanism known in bacteria, plants, and arthropods.

Two important characteristics of metapopulations are extinction-(re)colonization dynamics and gene flow between subpopulations. These processes can cause strong shifts in genome-wide allele frequencies that are generally not observed in "classical" (large, stable, and panmictic) populations. Subpopulations founded by one or a few individuals, the so-called propagule model, are initially expected to show intermediate allele frequencies at polymorphic sites until natural selection and genetic drift drive allele frequencies toward a mutation-selection-drift equilibrium characterized by a negative exponential-like distribution of the site frequency spectrum. We followed changes in site frequency spectrum distribution in a natural metapopulation of the cyclically parthenogenetic pond-dwelling microcrustacean Daphnia magna using biannual pool-seq samples collected over a 5-yr period from 118 ponds occupied by subpopulations of known age. As expected under the propagule model, site frequency spectra in newly founded subpopulations trended toward intermediate allele frequencies and shifted toward right-skewed distributions as the populations aged. Immigration and subsequent hybrid vigor altered this dynamic. We show that the analysis of site frequency spectrum dynamics is a powerful approach to understand evolution in metapopulations. It allowed us to disentangle evolutionary processes occurring in a natural metapopulation, where many subpopulations evolve in parallel. Thereby, stochastic processes like founder and immigration events lead to a pattern of subpopulation divergence, while genetic drift leads to converging site frequency spectrum distributions in the persisting subpopulations. The observed processes are well explained by the propagule model and highlight that metapopulations evolve differently from classical populations.

AbstractCollagen-like proteins (CLP) are commonly found in many pathogenic bacteria where they serve as adhesins to attach to host tissue. The repetition of the amino-acid pattern (Gly-Xaa-Yaa)nis the major feature of collagen and is essential to the formation of its stable triple helical structure. In theDaphnia magna–Pasteuria ramosasystem, a model system for studying antagonistic coevolution, a specific CLP in the virulent parasiteP. ramosaplays a pivotal role in host attachment, regulated by matching allele model. Recognizing the crucial role of CLPs in the infection process, we aimed to enhance our understanding ofP. ramosa-CLPs by sequencing high-quality genomes of two isolates, using long-read technology. An analysis of a CLP gene tree of representativeBacillotaspecies revealed a clear radiation of these genes inP. ramosa, which was not found in the closely relatedPasteuria penetrans. A comparison of the isolates reveals a high synteny, with the exception of a few duplications and inversions, mainly involving CLPs or transposases. Across isolates, we observed a recent burst of transposases as well as duplications of CLP genes. On average, CLP genes are well conserved between isolates, but the presence/absence of individual CLP genes is not fully shared, with 39 and 43 genes in the two isolates. Our findings suggest a rapid radiation of CLP genes combined with a birth and death process of the largeP. ramosa-CLP gene family, possibly driven by transposition and coevolution.ImportanceAlthough the host–pathogenDaphnia magna–Pasteuria ramosasystem has served as a model for coevolution, we have, to date, lacked high-quality genomic resources for the parasite, as is the case for many such systems. By presenting a complete assembly of two distinctP. ramosaisolates, our study addresses this lack and provides deeper insights into theP. ramosaCollagen Like Protein (CLP) family, essential proteins involved in attachment to the host. We discover that the rapid radiation of CLP genes inP. ramosaappears to be driven by transposition and coevolution, enabling the parasite to adapt to host resistance mechanisms. These insights improve our understanding of host–parasite interactions and pave the way for comparative genomic analyses to better understand the evolution of these genes. They also have broader implications for disease control and therapeutic development targeting pathogenic bacteria adhesion mechanisms.

The cuticles of arthropods provide an interface between the organism and its environment. Thus, the cuticle's structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the moulted cuticle of the aquatic crustacean Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology. We detected a total of 278 high-confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin-modifying enzymes as the most abundant protein groups in the cuticle proteome. Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle. Finally, cuticle protein genes were also clustered as tandem gene arrays in the D. magna genome. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing genome annotations and investigations on diverse topics such as the genetic basis of interactions with predators and parasites.

Collagen-like proteins (CLP) are commonly found in many pathogenic bacteria where they serve as adhesins to attach to host tissue. The repetition of the amino-acid pattern (Gly-Xaa-Yaa)n is the major feature of collagen and is essential to the formation of its stable triple helical structure. In the Daphnia magna – Pasteuria ramosa system, a model system for studying antagonistic coevolution, a specific CLP in the virulent parasite P. ramosa plays a pivotal role in host attachment, regulated by matching allele model. Recognizing the crucial role of CLPs in the infection process, we aimed to enhance our understanding of P. ramosa -CLPs by sequencing high-quality genomes of two isolates, using long-read technology. An analysis of a CLP gene tree of representative Bacillota species revealed a clear radiation of these genes in P. ramosa , which was not found in the closely related Pasteuria penetrans . A comparison of the isolates reveals a high synteny, with the exception of a few duplications and inversions, mainly involving CLPs or transposases. Across isolates, we observed a recent burst of transposases as well as duplications of CLP genes. On average, CLP genes are well conserved between isolates, but the presence/absence of individual CLP genes is not fully shared, with 39 and 43 genes in the two isolates. Our findings suggest a rapid radiation of CLP genes combined with a birth and death process of the large P. ramosa -CLP gene family, possibly driven by transposition and coevolution. Importance Although the host–pathogen Daphnia magna – Pasteuria ramosa system has served as a model for coevolution, we have, to date, lacked high-quality genomic resources for the parasite, as is the case for many such systems. By presenting a complete assembly of two distinct P. ramosa isolates, our study addresses this lack and provides deeper insights into the P. ramosa Collagen Like Protein (CLP) family, essential proteins involved in attachment to the host. We discover that the rapid radiation of CLP genes in P. ramosa appears to be driven by transposition and coevolution, enabling the parasite to adapt to host resistance mechanisms. These insights improve our understanding of host–parasite interactions and pave the way for comparative genomic analyses to better understand the evolution of these genes. They also have broader implications for disease control and therapeutic development targeting pathogenic bacteria adhesion mechanisms.

Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about ability to adapt. We explore the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A GWAS revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase ( TPS ) gene, suggesting that trehalose production facilitates salinity tolerance. We found a positive correlation between water salinity and trehalose concentrations in tolerant animals, while intolerant animals failed to produce trehalose. Using CRISPR/Cas9, a silenced TPS gene supported the role of trehalose under salt stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionarily conserved mechanism known in plants and bacteria, but not in metabolic-active animals.Highlights ### Competing Interest StatementThe authors have declared no competing interest.

We have sequenced, assembled, and analyzed the nuclear and mitochondrial genomes and transcriptomes of Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa, two prosobranch snail species native to New Zealand that together span the continuum from estuary to freshwater. These two species are the closest known relatives of the freshwater species Potamopyrgus antipodarum-a model for studying the evolution of sex, host-parasite coevolution, and biological invasiveness-and thus provide key evolutionary context for understanding its unusual biology. The P. estuarinus and P. kaitunuparaoa genomes are very similar in size and overall gene content. Comparative analyses of genome content indicate that these two species harbor a near-identical set of genes involved in meiosis and sperm functions, including seven genes with meiosis-specific functions. These results are consistent with obligate sexual reproduction in these two species and provide a framework for future analyses of P. antipodarum-a species comprising both obligately sexual and obligately asexual lineages, each separately derived from a sexual ancestor. Genome-wide multigene phylogenetic analyses indicate that P. kaitunuparaoa is likely the closest relative to P. antipodarum. We nevertheless show that there has been considerable introgression between P. estuarinus and P. kaitunuparaoa. That introgression does not extend to the mitochondrial genome, which appears to serve as a barrier to hybridization between P. estuarinus and P. kaitunuparaoa. Nuclear-encoded genes whose products function in joint mitochondrial-nuclear enzyme complexes exhibit similar patterns of nonintrogression, indicating that incompatibilities between the mitochondrial and the nuclear genome may have prevented more extensive gene flow between these two species.

In some species, the Y is a tiny chromosome but the dioecious plant Silene latifolia has a giant ∼550 Mb Y chromosome, which has remained unsequenced so far. Here we used a hybrid approach to obtain a high-quality male S. latifolia genome. Using mutants for sexual phenotype, we identified candidate sex-determining genes on the Y. Comparative analysis of the sex chromosomes with outgroups showed the Y is surprisingly rearranged and degenerated for a ∼11 MY-old system. Recombination suppression between X and Y extended in a stepwise process, and triggered a massive accumulation of repeats on the Y, as well as in the non-recombining pericentromeric region of the X, leading to giant sex chromosomes.One-Sentence Summary This work uncovers the structure, function, and evolution of one of the largest giant Y chromosomes, that of the model plant Silene latifolia , which is almost 10 times larger than the human Y, despite similar genome sizes.### Competing Interest StatementThe authors have declared no competing interest.

Microsporidia are intracellular parasitic fungi whose genomes rank among the smallest of all known eukaryotes. A number of outstanding questions remain concerning the evolution of their large-scale variation in genome architecture, responsible for genome size variation of more than an order of magnitude. This genome report presents the first near-chromosomal assembly of a large-genome microsporidium, Hamiltosporidium tvaerminnensis. Combined Oxford Nanopore, Pacific Biosciences (PacBio), and Illumina sequencing led to a genome assembly of 17 contigs, 11 of which represent complete chromosomes. Our assembly is 21.64 Mb in length, has an N50 of 1.44 Mb, and consists of 39.56% interspersed repeats. We introduce a novel approach in microsporidia, PacBio Iso-Seq, as part of a larger annotation pipeline for obtaining high-quality annotations of 3,573 protein-coding genes. Based on direct evidence from the full-length Iso-Seq transcripts, we present evidence for alternative polyadenylation and variation in splicing efficiency, which are potential regulation mechanisms for gene expression in microsporidia. The generated high-quality genome assembly is a necessary resource for comparative genomics that will help elucidate the evolution of genome architecture in response to intracellular parasitism.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of mitochondrial genome architecture and landscapes of recombination. We used PacBio sequencing to re-assemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus . These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by assembly of short sequencing reads and likely corresponding to a large non-coding region commonly present in mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy region (LSC) and a small single-copy region (SSC) separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa–SSC–IRb structure revealed the occurrence of “flip-flop” recombination, apparently mediated by the IRs. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus , as well as extensive inter-molecular recombination between SNPs in the LSC region. Together, these observations suggest that mitochondrial inheritance is not strictly maternal in these snails. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of such architecture, whether and how recombination mediates mitochondrial genome evolution, and the role of genome architecture in driving cytoplasmic genome biology and the maintenance of cytoplasmic genomes.

Background: The disease severity index (DSI) for inflammatory bowel disease (IBD) combines measures of disease phenotype, inflammatory activity, and patient-reported outcomes. We aimed to validate the DSI and assess its utility in predicting a complicated IBD course.Methods: A multicenter cohort of adults with IBD was recruited. Intraclass correlation coefficients (ICCs) and weighted Kappa assessed inter-rater reliability. Cronbach's alpha measured internal consistency of DSI items. Spearman's rank correlations compared the DSI with endoscopic indices, symptom indices, quality of life, and disability. A subgroup was followed for 24 months to assess for a complicated IBD course. Area under the receiver operating characteristics curve (AUROC) and multivariable logistic regression assessed the utility of the DSI in predicting disease progression.Results: Three hundred and sixty-nine participants were included (Crohn's disease [CD], n = 230; female, n = 194; mean age, 46 years [SD, 15]; median disease duration, 11 years [interquartile range, 5-21]), of which 171 (CD, n = 99; ulcerative colitis [UC], n = 72) were followed prospectively. The DSI showed inter-rater reliability for CD (ICC 0.93, n = 65) and UC (ICC 0.97, n = 33). The DSI items demonstrated inter-rater agreement (Kappa > 0.4) and internal consistency (CD, alpha > 0.59; UC, alpha > 0.75). The DSI was significantly associated with endoscopic activity (CDn=141, r = 0.65, P < .001; UCn=105, r = 0.80, P < .001), symptoms (CDn=159, r = 0.69, P < .001; UCn=132, r = 0.58, P < .001), quality of life (CDn=198, r = -0.59, P < .001; UCn=128, r = -0.68, P < .001), and disability (CDn=83, r = -0.67, P < .001; UCn=52, r = -0.74, P < .001). A DSI of 23 best predicted a complicated IBD course (AUROC = 0.82, P < .001) and was associated with this end point on multivariable analyses (aOR, 9.20; 95% confidence interval, 3.32-25.49).Conclusions: The DSI reliably encapsulates factors contributing to disease severity and accurately prognosticates the longitudinal IBD course.

Outbred laboratory mice (Mus musculus) are readily available and have high fecundity, making them a popular choice in biomedical research, especially toxicological and pharmacological applications. Direct high throughput genome sequencing (HTS) of these widely used research animals is an important genetic quality control measure that enhances research reproducibility. HTS data have been used to confirm the common origin of outbred stocks and to molecularly define distinct outbred populations. But these data have also revealed unexpected population structure and homozygosity in some populations; genetic features that emerge when outbred stocks are not properly maintained. We used exome sequencing to discover and interrogate protein-coding variation in a newly established population of Swiss-derived outbred stock (J:ARC) that is closely related to other, commonly used CD-1 outbred populations. We used these data to describe the genetic architecture of the J:ARC population including heterozygosity, minor allele frequency, LD decay, and we defined novel, protein-coding sequence variation. These data reveal the expected genetic architecture for a properly maintained outbred stock and provide a basis for the on-going genetic quality control. We also compared these data to protein-coding variation found in a multiparent outbred stock, the Diversity Outbred (J:DO). We found that the more recently derived, multiparent outbred stock has significantly higher interindividual variability, greater overall genetic variation, higher heterozygosity, and fewer novel variants than the Swiss-derived J:ARC stock. However, among the novel variants found in the J:DO stock, significantly more are predicted to be protein-damaging. The fact that individuals from this population can tolerate a higher load of potentially damaging variants highlights the buffering effects of allelic diversity and the differing selective pressures in these stocks. While both outbred stocks offer significant individual heterozygosity, our data provide a molecular basis for their intended applications, where the J:DO are best suited for studies requiring maximum, population-level genetic diversity and power for mapping, while the J:ARC are best suited as a general-purpose outbred stock with robust fecundity, relatively low allelic diversity, and less potential for extreme phenotypic variability.

Introduction: Bleeding is a known complication of EUS-guided cystgastrostomy (CG). We report 2 patients with prior surgical CG who underwent EUS-CG for recurrent pseudocyst (PC) complicated by severe gastrointestinal (GI) bleeding from a pseudoaneurysm requiring additional interventions. Case Description/Methods: Patient 1: 70-yr M with necrotizing pancreatitis s/p surgical CG. Referred to our center with symptomatic recurrent pseudocyst (Figure 1, 1a). EUS-CG was performed using 15mm LAMS + 7 fr double pigtail plastic stent (DPPS) (Figure 1, 1b). No intra-procedural bleeding. Two days later patient had hematochezia and Hb dropped by 3 gms/dL. CT angiogram (CTA) no bleed, PFC smaller, stents in place. Patient remained stable, sent home, but returned 2 days later with recurrent hematochezia. CTA with actively bleeding pseudoaneurysm from SMA branch close to LAMS (Figure 1, 1c). Embolization performed, bleeding controlled (Figure 1, 1d). LAMS was removed, DPPS left in place (Figure 1, 1e). CT 2 months later without residual cyst, patient doing well. Patient 2: 65-yr M with severe necrotizing pancreatitis s/p surgical CG 1 yr ago. Referred to our center with symptomatic recurrent pseudocyst (Figure 1, 2a). EUS-CG using 15mm LAMS +7 Fr DPPS done, patient went home next day. Five days later, readmitted with hematemesis, anemia, hypotension. CT no bleed, stents in place. EGD revealed LAMS+DPPS in place, active bleeding noted from cyst cavity (Figure 1, 2b). Repeat CTA done, actively bleeding pseudoaneurysm (Figure 1, 2c). Successful embolization done (Figure 1, 2d), but patient continued with intermittent bleeding requiring transfusions. Repeat EGD, active oozing from within cyst cavity. 15cc of novel PuraStat gel used to fill pseudocyst cavity with complete hemostasis (Figure 1, 2e). LAMS removed, a 7-Fr x 4cm DPPS placed into cyst cavity. Patient stabilized, discharged home after 2 days. CT scan 6 weeks later, no residual pseudocyst, patient doing well. Discussion: Surgical CG is rarely performed in the era of EUS-CG with LAMS. Our patients needed EUS-CG for recurrent PC post-surgery. Severe bleeding within 1 week post EUS-CG is unusual. Both patients had nearly identical presentation, bleeding etiology, imaging findings and clinical course. Our experience suggests that patients who undergo LAMS based EUS-CG after initial surgical CG may be at higher risk for bleeding. Initial CTAs may be negative. IR embolization is effective. Endoscopists should keep these caveats in mind when managing such patients.Figure 1.: Endoscopic and fluoroscopic images demonstrating interventions performed in both cases. 1,1a: CT revealing 10 cm pancreatic pseudocyst. 1,1b: Endoscopic view of cystgastrostomy with LAMS and DPPS. 1,1c: IR angiogram revealing 7 mm pseudoaneurysm in close proximity to the LAMS. 1,1d: IR coil embolization of the SMA jejunal branch pseudoaneurysm. 1,1e: LAMS removed and DPPS left in place across the cystgastrostomy. 1, 2a: CT scan showing recurrent 8 x 8 x 7cm pancreatic pseudocyst Image 2b: Oozing of fresh blood through and around the LAMS. 1, 2c: IR angiogram revealing actively bleeding GDA pseudoaneurys. 1, 2d: IR coil embolization of the GDA pseudoaneurysm. 1, 2e: PuraStat gel used to achieve hemostasis within the pseudocyst cavity.