Biparental immune priming in the broad-nosed pipefish (Syngnathus typhle)

All living organisms are permanently exposed to harmful parasites and pathogens. The immune system and with it sophisticated strategies arose to face challenging pathogens such as bacteria and viruses. One outstanding strategy is the transfer of immunity from mothers to offspring known as “trans-generational immune priming” (TGIP). Through the transfer of acquired immunological protection mothers can boost the immune defense of their offspring and facilitate resistance against bacteria present in the maternal environment. Commonly, TGIP is limited to females and was considered to be a maternal trait. Yet, in the sex-role reversed pipefish Syngnathus typhle fathers are closely connected to the embryos during male pregnancy over a placenta-like structure. This intimate physical connection between fathers and offspring may mechanistically facilitate additional paternal immune priming and provides an exclusive chance to study biparental investment into offspring immunity. In my thesis, I explored multi-generational biparental immune priming in the sex-role reversed pipefish S. typhle and discussed its evolutionary and ecological consequences as well as the mechanistic basis.Alle lebenden Organismen sind permanent allgegenwärtigen infektiösen Parasiten und Pathogenen ausgesetzt. Ausgelöst durch ihre schädliche Wirkung auf die Fitness ihres Wirtes evolvierten sich mit dem Immunsystem verschiedene Abwehrmechanismen, um einen Schutz zu gewährleisten. Eine raffinierte Strategie ist die Übertragung von erworbener Immunität von Müttern auf die Nachkommen über die Plazenta oder Muttermilch, bekannt als „generationsübergreifende Immunprägung". Mit der Weitergabe von erworbenem Immunschutz können somit Mütter die Immunabwehr ihrer Nachkommen in der frühen embryonalen Entwicklung gewährleisten. Zudem ermöglicht es den Nachkommen schneller eine Resistenz gegen die in der mütterlichen Umgebung vorhandenen Krankheitserreger aufzubauen. Daher wurde bisher davon ausgegangen, dass Immunprägung nur durch das weibliche Geschlecht verwirklicht wird. Doch in dem seltenen Fall der Grasnadel Syngnathus typhle ist die Rollenverteilung der Geschlechter vertauscht. Bei dieser besonderen Fischart brüten die Männchen während einer männlichen Schwangerschaft ihre Embryonen in einer Bruttasche und sind über eine plazenta-ähnliche Struktur so eng mit ihnen verbunden, dass mechanistisch eine väterliche Immunprägung möglich wäre. In meiner Doktorarbeit habe ich die Aspekte der biparentalen Immunprägung über zwei Generationen in der Grasnadel erforscht und ihre evolutionären und ökologischen Folgen, sowie mechanistische Grundlage näher untersucht

Biparental immune priming in the pipefish Syngnathus typhle

Highlights: • In a sex-role reversed pipefish, fathers have a major impact on offspring immunity. • Maternal effects are effective early in life but cease upon maturation. • Fathers provide long-term protection that comes with immunological specificity. • Long-term protection could be based on parental epigenetic traces. • Biparental immunological transfer comes with additive costs but lacks additive advantages. Abstract: The transfer of immunity from parents to offspring (trans-generational immune priming (TGIP)) boosts offspring immune defence and parasite resistance. TGIP is usually a maternal trait. However, if fathers have a physical connection to their offspring, and if offspring are born in the paternal parasitic environment, evolution of paternal TGIP can become adaptive. In Syngnathus typhle, a sex-role reversed pipefish with male pregnancy, both parents invest into offspring immune defence. To connect TGIP with parental investment, we need to know how parents share the task of TGIP, whether TGIP is asymmetrically distributed between the parents, and how the maternal and paternal effects interact in case of biparental TGIP. We experimentally investigated the strength and differences but also the costs of maternal and paternal contribution, and their interactive biparental influence on offspring immune defence throughout offspring maturation. To disentangle maternal and paternal influences, two different bacteria were used in a fully reciprocal design for parental and offspring exposure. In offspring, we measured gene expression of 29 immune genes, 15 genes associated with epigenetic regulation, immune cell activity and life-history traits. We identified asymmetric maternal and paternal immune priming with a dominating, long-lasting paternal effect. We could not detect an additive adaptive biparental TGIP impact. However, biparental TGIP harbours additive costs as shown in delayed sexual maturity. Epigenetic regulation may play a role both in maternal and paternal TGIP

Actinomadura rubteroloni sp. nov. and Actinomadura macrotermitis sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29$^{T}$ and RB68T, were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29$^{T}$ were identified as Actinomadura rayongensis DSM 102126$^{T}$ (99.2 % similarity) and Actinomadura atramentaria DSM 43919$^{T}$ (98.7 %), and for strain RB68$^{T}$ was Actinomadura hibisca DSM 44148$^{T}$ (98.3 %). Digital DNA–DNA hybridization (dDDH) between RB29$^{T}$ and its closest phylogenetic neighbours, A. rayongensis DSM 102126$^{T}$ and A. atramentaria DSM 43919$^{T}$, resulted in similarity values of 53.2 % (50.6–55.9 %) and 26.4 % (24.1–28.9 %), respectively. Additionally, the average nucleotide identity (ANI) was 93.2 % (94.0 %) for A. rayongensis DSM 102126$^{T}$ and 82.3 % (78.9 %) for A. atramentaria DSM 43919$^{T}$. dDDH analysis between strain RB68$^{T}$ and A. hibisca DSM 44148$^{T}$ gave a similarity value of 24.5 % (22.2–27.0 %). Both strains, RB29$^{T}$ and RB68$^{T}$, revealed morphological characteristics and chemotaxonomic features typical for the genus Actinomadura, such as the presence of meso-diaminopimelic acid in the cell wall, galactose and glucose as major sugar components within whole-cell hydrolysates and the absence of mycolic acids. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Predominant menaquinones were MK-9(H$_{6}$) and MK-9(H$_{8}$) for RB29$^{T}$ and MK-9(H$_{4}$) and MK-9(H$_{6}$) for RB68T. The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C$_{18:0}$), 14-methylpentadecanoic acid (iso-C$_{16:0}$), hexadecanoic acid (C$_{16:0}$) and cis-9-octadecanoic acid (C$_{18:1}$ ω9c). Here, we propose two novel species of the genus Actinomadura: Actinomadura rubteroloni sp. nov. with the type strain RB29$^{T}$ (=CCUG 72668$^{T}$=NRRL B-65537$^{T}$) and Actinomadura macrotermitis sp. nov. with the type strain RB68$^{T}$ (=CCUG 72669$^{T}$=NRRL B-65538$^{T}$)

Comparative Genomics Reveals Prophylactic and Catabolic Capabilities of Actinobacteria within the Fungus-Farming Termite Symbiosis

Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth’s ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis. IMPORTANCE Actinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis

Microbial embryonal colonization during pipefish male pregnancy

While originally acquired from the environment, a fraction of the microbiota is transferred from parents to offspring. The immune system shapes the microbial colonization, while commensal microbes may boost host immune defences. Parental transfer of microbes in viviparous animals remains ambiguous, as the two transfer routes (transovarial vs. pregnancy) are intermingled within the maternal body. Pipefishes and seahorses (syngnathids) are ideally suited to disentangle transovarial microbial transfer from a contribution during pregnancy due to their maternal egg production and their unique male pregnancy. We assessed the persistency and the changes in the microbial communities of the maternal and paternal reproductive tracts over proceeding male pregnancy by sequencing microbial 16S rRNA genes of swabs from maternal gonads and brood pouches of non-pregnant and pregnant fathers. Applying parental immunological activation with heat-killed bacteria, we evaluated the impact of parental immunological status on microbial development. Our data indicate that maternal gonads and paternal brood pouches harbor distinct microbial communities, which could affect embryonal development in a sex-specific manner. Upon activation of the immune system, a shift of the microbial community was observed. The activation of the immune system induced the expansion of microbiota richness during late pregnancy, which corresponds to the time point of larval mouth opening, when initial microbial colonization must take plac

Streptomyces smaragdinus sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis

The taxonomic position of a novel aerobic, Grampositive actinobacteria, designated strain RB5(T), was determined using a poly phasic approach. The strain, isolated from the gut of the fungusfarming termite Macrotermes natalensis, showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5(T) was Streptomyces polyrhachis DSM 42102(T) (98.87 %). DNA-DNA hybridization experiments between strain RB5(T) and S. polyrhachis DSM 42102(T) resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5(T) contained iota iota diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside and phosphatidylserine. The menaquinone profile showed hexaand octahydrogenated menaquinones containing nine isoprene units [MK-9(H-6) and MK-9(H-8)]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12methyltridecanoic acid (isoC(14:0)) 12-methyltetradecanoic acid (anteiso-C-15:0), 13-methyltetradecanoic acid (isoC(15:0)) and 14-methylpentadecanoic acid (isoC(16:0)). Here, we propose a novel species of the genus Streptomyces - Streptomyces smaragdinus with the type strain RB5(T) (=VKM Ac-2839(T)=NRRL B65539(T))

Nocardia macrotermitis sp. nov. and Nocardia aurantia sp. nov., isolated from the gut of the fungus-growing termite Macrotermes natalensis

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20$^{T}$ and RB56$^{T}$, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860$^{T}$ (98.9 %) and Nocardia nova DSM44481$^{T}$ (98.5 %) for RB20$^{T}$ and Nocardia takedensis DSM 44801$^{T}$ (98.3 %), Nocardia pseudobrasiliensis DSM 44290$^{T}$ (98.3 %) and Nocardia rayongensis JCM 19832$^{T}$ (98.2 %) for RB56$^{T}$. Digital DNA–DNA hybridization (DDH) between RB20$^{T}$ and N. miyunensis JCM12860$^{T}$ and N. nova DSM 44481$^{T}$ resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56$^{T}$ and N. takedensis DSM44801$^{T}$ and N. pseudobrasiliensis DSM44290$^{T}$ showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56$^{T}$ and N. rayongensis JCM19832$^{T}$ resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia , such as the presence of meso-diaminopimelic acid (A$_{2}$pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C$_{16 : 0}$), 10-methyloctadecanoic acid (10-methyl C$_{18 : 0}$) and cis-9-octadecenoic acid (C$_{18 : 1}$ ω9c). We propose two novel species within the genus Nocardia : Nocardia macrotermitis sp. nov. with the type strain RB20$^{T}$ (=VKM Ac-2841$^{T}$=NRRL B65541$^{T}$) and Nocardia aurantia sp. nov. with the type strain RB56$^{T}$ (=VKM Ac-2842$^{T}$=NRRL B65542$^{T}$)

The Innate Immune Response of Atlantic Salmon (Salmo salar) Is Not Negatively Affected by High Temperature and Moderate Hypoxia

Climate change is predicted to increase water temperatures and decrease oxygen levels in freshwater and marine environments, however, there is conflicting information regarding the extent to which these conditions may impact the immune defenses of fish. In this study, Atlantic salmon were exposed to: (1) normoxia (100–110% air saturation) at 12°C; (2) an incremental temperature increase (1°C per week from 12 to 20°C), and then held at 20°C for an additional 4 weeks; and (3) “2” with the addition of moderate hypoxia (~65–75% air saturation). These conditions realistically reflect what farmed salmon in some locations are currently facing, and future conditions in Atlantic Canada and Europe, during the summer months. The salmon were sampled for the measurement of head kidney constitutive anti-bacterial and anti-viral transcript expression levels, and blood parameters of humoral immune function. Thereafter, they were injected with either the multi-valent vaccine Forte V II (contains both bacterial and viral antigens) or PBS (phosphate-buffer-saline), and the head kidney and blood of these fish were sampled at 6, 12, 24, and 48 h post-injection (HPI). Our results showed that: (1) neither high temperature, nor high temperature + moderate hypoxia, adversely affected respiratory burst, complement activity or lysozyme concentration; (2) the constitutive transcript expression levels of the anti-bacterial genes il1β, il8-a, cox2, hamp-a, stlr5-a, and irf7-b were up-regulated by high temperature; (3) while high temperature hastened the peak in transcript expression levels of most anti-bacterial genes by 6–12 h following V II injection, it did not affect the magnitude of changes in transcript expression; (4) anti-viral (viperin-b, mx-b, and isg15-a) transcript expression levels were either unaffected, or downregulated, by acclimation temperature or V II injection over the 48 HPI; and (5) hypoxia, in addition to high temperature, did not impact immune transcript expression. In conclusion, temperatures up to 20°C, and moderate hypoxia, do not impair the capacity of the Atlantic salmon's innate immune system to respond to bacterial antigens. These findings are surprising, and highlight the salmon's capacity to mount robust innate immune responses (i.e., similar to control fish under optimal conditions) under conditions approaching their upper thermal limit

The transcriptomic responses of Atlantic salmon (Salmo salar) to high temperature stress alone, and in combination with moderate hypoxia

Background Increases in ocean temperatures and in the frequency and severity of hypoxic events are expected with climate change, and may become a challenge for cultured Atlantic salmon and negatively affect their growth, immunology and welfare. Thus, we examined how an incremental temperature increase alone (Warm & Normoxic-WN: 12 → 20 °C; 1 °C week− 1), and in combination with moderate hypoxia (Warm & Hypoxic-WH: ~ 70% air saturation), impacted the salmon’s hepatic transcriptome expr\ession compared to control fish (CT: 12 °C, normoxic) using 44 K microarrays and qPCR. Results Overall, we identified 2894 differentially expressed probes (DEPs, FDR < 5%), that included 1111 shared DEPs, while 789 and 994 DEPs were specific to WN and WH fish, respectively. Pathway analysis indicated that the cellular mechanisms affected by the two experimental conditions were quite similar, with up-regulated genes functionally associated with the heat shock response, ER-stress, apoptosis and immune defence, while genes connected with general metabolic processes, proteolysis and oxidation-reduction were largely suppressed. The qPCR assessment of 41 microarray-identified genes validated that the heat shock response (hsp90aa1, serpinh1), apoptosis (casp8, jund, jak2) and immune responses (apod, c1ql2, epx) were up-regulated in WN and WH fish, while oxidative stress and hypoxia sensitive genes were down-regulated (cirbp, cyp1a1, egln2, gstt1, hif1α, prdx6, rraga, ucp2). However, the additional challenge of hypoxia resulted in more pronounced effects on heat shock and immune-related processes, including a stronger influence on the expression of 14 immune-related genes. Finally, robust correlations between the transcription of 19 genes and several phenotypic traits in WH fish suggest that changes in gene expression were related to impaired physiological and growth performance. Conclusion Increasing temperature to 20 °C alone, and in combination with hypoxia, resulted in the differential expression of genes involved in similar pathways in Atlantic salmon. However, the expression responses of heat shock and immune-relevant genes in fish exposed to 20 °C and hypoxia were more affected, and strongly related to phenotypic characteristics (e.g., growth). This study provides valuable information on how these two environmental challenges affect the expression of stress-, metabolic- and immune-related genes and pathways, and identifies potential biomarker genes for improving our understanding of fish health and welfare

Polyhalogenation of isoflavonoids by the termite-associated Actinomadura sp. RB99

The National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2018R1A2B2006879); the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRS), the School for Microbial Communication (JSMC, DFG), the Deutsche Forschungsgemeinschaft (DFG, German Science Foundation) (BE 4799/3-1, CRC1127 project A6) and Project-ID 390713860 “Balance of the Microverse,” (Germany’s Excellence Strategy, EXC 2051).https://pubs.acs.org/journal/jnprdfhj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog