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In many insect species, the ability of males to inhibit their mates from remating is an important component of fitness. This ability is also essential for the effective management of insect pests, including tephritid fruit flies, using the Sterile Insect Technique. Here we apply transcriptomics and proteomics to male reproductive tissues before and after mating to characterize components of semen that might mediate remating inhibition in Queensland fruit fly. We found 144 genes whose transcripts were enriched, or proteins expressed, in reproductive tissue and which also varied in amount after mating. Some were associated with testes, accessory glands and ejaculatory apodeme, but those from the ejaculatory apodeme were over-represented compared to those not enriched in reproductive tissue or mating responsive. These included 13 related genes clustered within one Mb on chromosome 5. Functional annotations implicated a broad range of biochemical processes in the genes/proteins enriched in reproductive tissue and mating responsive, with cuticle structure most commonly implicated among the subset of these that were apodeme-enriched and a kinase involved in vitellogenesis implicated for one of the 13 clustered genes. We did not find a homolog of the much studied Drosophila melanogaster Sex Peptide but comparative genomics indicated that some of the tissue-enriched, mating responsive genes/proteins were rapidly evolving in tephritids (including in the Queensland fruit fly lineage), suggesting recent adaptation to new functional niches. Our results provide a set of candidate mediators of remating inhibition for further functional testing., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Insects rely on odorant receptors (ORs) to detect and respond to volatile environmental cues, so the ORs are attracting increasing interest as potential targets for pest control. However, experimental analysis of their structures and functions faces significant challenges. Computational methods such as template-based modeling (TBM) and AlphaFold3 (AF3) could facilitate the structural characterisation of ORs. This study first showed that both models accurately predicted the structural fold of MhOR5, a jumping bristletail OR with known experimental 3D structures, although accuracy was higher in the extracellular region of the protein and binding mode of their cognate ligands with TBM. The two approaches were then compared for their ability to predict the empirical binding evidence available for OR-odorant complexes in two economically important fruit fly species, Bactrocera dorsalis and B. minax. Post-simulation analyses including binding affinities, complex and ligand stability and receptor-ligand interactions (RLIs) revealed that TBM performed better than AF3 in discriminating between binder and non-binder complexes. TBM's superior performance is attributed to hydrophobicity-based helix-wise multiple sequence alignment (MSA) between available insect OR templates and the ORs for which the binding data were generated. This MSA identified conserved residues and motifs which could be used as anchor points for refining the alignments., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non-synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range., (© 2024 John Wiley & Sons Ltd.)

Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) are sibling fruit fly species that are sympatric over much of their ranges. Premating isolation of these close relatives is thought to be maintained in part by allochrony-mating activity in B. tryoni peaks at dusk, whereas in B. neohumeralis, it peaks earlier in the day. To ascertain whether differences in pheromone composition may also contribute to premating isolation between them, this study used solid-phase microextraction and gas chromatography-mass spectrometry to characterize the rectal gland volatiles of a recently collected and a more domesticated strain of each species. These glands are typical production sites and reservoirs of pheromones in bactrocerans. A total of 120 peaks were detected and 50 were identified. Differences were found in the composition of the rectal gland emissions between the sexes, species, and recently collected versus domesticated strains of each species. The compositional variation included several presence/absence and many quantitative differences. Species and strain differences in males included several relatively small alcohols, esters, and aliphatic amides. Species and strain differences in females also included some of the amides but additionally involved many fatty acid esters and 3 spiroacetals. While the strain differences indicate there is also heritable variation in rectal gland emissions within each species, the species differences imply that compositional differences in pheromones emitted from rectal glands could contribute to the premating isolation between B. tryoni and B. neohumeralis. The changes during domestication could also have significant implications for the efficacy of Sterile Insect Technique control programs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Modern lipidomics has the power and sensitivity to elucidate the role of insects' lipidomes in their adaptations to the environment at a mechanistic molecular level. However, few lipidomic studies have yet been conducted on insects beyond model species such as Drosophila melanogaster . Here, we present the lipidome of adult males of another higher dipteran frugivore, Bactrocera tryoni . We describe 421 lipids across 15 classes of ester neutral lipids and phospholipids and ether neutral lipids and phospholipids. Most of the lipids are specified in terms of the carbon and double bond contents of each constituent hydrocarbon chain, and more ether lipids are specified to this degree than in any previous insect lipidomic analyses. Class-specific profiles of chain length and (un)saturation are broadly similar to those reported in D. melanogaster , although we found fewer medium-length chains in ether lipids. The high level of chain specification in our dataset also revealed widespread non-random combinations of different chain types in several ester lipid classes, including deficits of combinations involving chains of the same carbon and double bond contents among four phospholipid classes and excesses of combinations of dissimilar chains in several classes. Large differences were also found in the length and double bond profiles of the acyl vs. alkyl or alkenyl chains of the ether lipids. Work on other organisms suggests some of the differences observed will be functionally consequential and mediated, at least in part, by differences in substrate specificity among enzymes in lipid synthesis and remodelling pathways. Interrogation of the B. tryoni genome showed it has comparable levels of diversity overall in these enzymes but with some gene gain/loss differences and considerable sequence divergence from D. melanogaster .

Divergence between populations in mating behaviour can function as a potent premating isolating mechanism and promote speciation. However, very few cases of inherited intraspecific variation in sexual signalling have been reported in tephritid fruit flies, despite them being a highly speciose family. We tested for such variation in one tephritid, the Queensland fruit fly, Bactrocera tryoni (Qfly). Qfly mating behaviour depends on volatiles secreted from male rectal glands but no role for the volatiles from female rectal glands has yet been reported. We previously detected over 100 volatile compounds in male rectal glands and identified over 30 of them. Similar numbers were recorded in females. However, many compounds showed presence/absence differences between the sexes and many others showed quantitative differences between them. Here we report inherited variation among 24 Qfly lines (23 isofemale lines established from recent field collections and one domesticated line) in the abundance of three esters, two alcohols, two amides, an aldehyde and 18 unidentified volatiles in male rectal glands. We did not find any compounds in female rectal glands that varied significantly among the lines, although this may at least partly reflect lower female sample numbers. Most of the 26 male compounds that differed between lines were more abundant in the domesticated line than any of the recently established isofemale lines, which concurs with other evidence for changes in mating behaviour during domestication of this species. There were also large differences in several of the 26 compounds among the isofemale lines, and some of these differences were associated with the regions from which the lines were collected. While some of the variation in different compounds was correlated across lines, much of it was not, implicating involvement of multiple genes. Our findings parallel reports of geographic variation in other Qfly traits and point to inherited differences in reproductive physiology that could provide a basis for evolution of premating isolation between ecotypes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Castro-Vargas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Narrow substrate ranges can impact heavily on the range of applications and hence commercial viability of candidate bioremediation enzymes. Here we show that an ester hydrolase from Nocardioides strain SG-4 G has potential as a bioremediation agent against various pollutants that can be detoxified by hydrolytic cleavage of some carboxylester, carbamate, or amide linkages. Previously we showed that a radiation-killed, freeze-dried preparation (ZimA) of this strain can rapidly degrade the benzimidazole fungicide carbendazim due to the activity of a specific ester hydrolase, MheI. Here, we report that ZimA also has substantial hydrolytic activity against phthalate diesters (dimethyl, dibutyl, and dioctyl phthalate), anilide (propanil and monalide), and carbamate ester (chlorpropham) herbicides under laboratory conditions. The reaction products are substantially less toxic, or inactive as herbicides, than the parent compounds. Tests of strain SG-4 G and Escherichia coli expressing MheI found they were also able to hydrolyse dimethyl phthalate, propanil, and chlorpropham, indicating that MheI is principally responsible for the above activities., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Rectal gland volatiles are key mediators of sexual interactions in tephritid fruit flies. We used solid-phase microextraction (SPME) plus gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID) to substantially expand rectal gland chemical characterisation of the Queensland fruit fly (Bactrocera tryoni (Diptera: Tephritidae); Qfly). The SPME GC-MS analysis identified 24 of the 30 compounds previously recorded from Qfly rectal glands, plus another 21 compounds that had not previously been reported. A few amides and fatty acid esters dominated the chromatograms of males and females respectively, but we also found other esters, alcohols and aldehydes and a ketone. The GC-FID analyses also revealed over 150 others, as yet unidentified, volatiles, generally in lesser amounts. The GC-FID analyses also showed 49 and 12 compounds were male- and female-specific, respectively, both in single sex (virgin) and mixed sex (mostly mated) groups. Another ten compounds were male-specific among virgins but undetected in mixed sex groups, and 29 were undetected in virgins but male-specific in mixed sex groups. The corresponding figures for females were four and zero, respectively. Most short retention time peaks (including a ketone and an ester) were male-specific, whereas most female-biased peaks (including five fatty acid esters) had long retention times. Our results indicate previously unsuspected diversity of rectal gland volatiles that might have pheromone functions in males, but far fewer in females., Competing Interests: The authors have declared that no competing interests exist.

Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. Here, we have developed a database of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, we then screened the data for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, we found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. We conclude that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Understanding the cumulative risk of chemical mixtures at environmentally realistic concentrations is a key challenge in honey bee ecotoxicology. Ecotoxicogenomics, including transcriptomics, measures responses in individual organisms at the molecular level which can provide insights into the mechanisms underlying phenotypic responses induced by one or more stressors and link impacts on individuals to populations. Here, fifth instar honey bee larvae were sampled from a previously reported field experiment exploring the phenotypic impacts of environmentally realistic chronic exposures of the pesticide imidacloprid (5 μg.kg -1 for six weeks) and the acaricide thymol (250 g.kg -1 applied via Apiguard gel in-hive for four weeks), both separately and in combination. RNA-seq was used to discover individual and interactive chemical effects on larval gene expression and to uncover molecular mechanisms linked to reported adult and colony phenotypes. The separate and combined treatments had distinct gene expression profiles which represented differentially affected signaling and metabolic pathways. The molecular signature of the mixture was characterised by additive interactions in canonical stress responses associated with oxidative stress and detoxification, and non-additive interactions in secondary responses including developmental, neurological, and immune pathways. Novel emergent impacts on eye development genes correlated with long-term defects in visual learning performance as adults. This is consistent with these chemicals working through independent modes of action that combine to impact common downstream pathways, and highlights the importance of establishing mechanistic links between molecular and phenotypic responses when predicting effects of chemical mixtures on ecologically relevant population outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Commonwealth Scientific and Industrial Research Organisation. Published by Elsevier B.V. All rights reserved.)

Females of many insect species are unreceptive to remating for a period following their first mating. This inhibitory effect may be mediated by either the female or her first mate, or both, and often reflects the complex interplay of reproductive strategies between the sexes. Natural variation in remating inhibition and how this phenotype responds to captive breeding are largely unexplored in insects, including many pest species. We investigated genetic variation in remating propensity in the Queensland fruit fly, Bactrocera tryoni, using strains differing in source locality and degree of domestication. We found up to threefold inherited variation between strains from different localities in the level of intra-strain remating inhibition. The level of inhibition also declined significantly during domestication, which implied the existence of genetic variation for this trait within the starting populations as well. Inter-strain mating and remating trials showed that the strain differences were mainly due to the genotypes of the female and, to a lesser extent, the second male, with little effect of the initial male genotype. Implications for our understanding of fruit fly reproductive biology and population genetics and the design of Sterile Insect Technique pest management programs are discussed., (© 2022. The Author(s).)

It is increasingly clear that pest species vary widely in their propensities to develop insecticide resistance. This review uses a comparative approach to analyze the key pest management practices and ecological and biochemical or genetic characteristics of the target that contribute to this variation. We focus on six heliothine species, three of which, Helicoverpa armigera , Heliothis virescens , and Helicoverpa zea , have developed resistances to many pesticide classes. The three others, Helicoverpa punctigera , Helicoverpa assulta , and Helicoverpa gelotopoeon , also significant pests, have developed resistance to very few pesticide classes. We find that host range and movement between alternate hosts are key ecological traits that influence effective selection intensities for resistance. Operational issues are also critical; area-wide, cross-pesticide management practices that account for these ecological factors are key to reducing selection intensity. Without such management, treatment using broad-spectrum chemicals serves to multiply the effects of host plant preference, preadaptive detoxification ability, and high genetic diversity to create a pesticide treadmill for the three high-propensity species.Without rigorous ongoing management, such a treadmill could still develop for newer, more selective chemistries and insecticidal transgenic crops.

Marine microalgae are a diverse group of microscopic eukaryotic and prokaryotic organisms capable of photosynthesis. They are important primary producers and carbon sinks but their physiology and persistence are severely affected by global climate change. Powerful experimental evolution technologies are being used to examine the potential of microalgae to respond adaptively to current and predicted future conditions, as well as to develop resources to facilitate species conservation and restoration of ecosystem functions. This review synthesizes findings and insights from experimental evolution studies of marine microalgae in response to elevated temperature and/or pCO 2 . Adaptation to these environmental conditions has been observed in many studies of marine dinoflagellates, diatoms and coccolithophores. An enhancement in traits such as growth and photo-physiological performance and an increase in upper thermal limit have been shown to be possible, although the extent and rate of change differ between microalgal taxa. Studies employing multiple monoclonal replicates showed variation in responses among replicates and revealed the stochasticity of mutations. The work to date is already providing valuable information on species' climate sensitivity or resilience to managers and policymakers but extrapolating these insights to ecosystem- and community-level impacts continues to be a challenge. We recommend future work should include in situ experiments, diurnal and seasonal fluctuations, multiple drivers and multiple starting genotypes. Fitness trade-offs, stable versus plastic responses and the genetic bases of the changes also need investigating, and the incorporation of genome resequencing into experimental designs will be invaluable., (© 2021 John Wiley & Sons Ltd.)

Background: Bactrocera tryoni and Bactrocera neohumeralis mate asynchronously; the former mates exclusively around dusk while the latter mates during the day. The two species also differ in the colour of the post-pronotal lobe (callus), which is predominantly yellow in B. tryoni and brown in B. neohumeralis. We have examined the genetic relationship between the two characters in hybrids, backcrosses and multigeneration hybrid progeny., Results: Our analysis of the mating time of the parental species revealed that while B. tryoni mate exclusively at dusk, B. neohumeralis females pair with B. neohumeralis males during the day and with B. tryoni males at dusk. We found considerable variance in mating time and callus colour among hybrid backcross individuals of both sexes but there was a strong although not invariant trend for callus colour to co-segregate with mating time in both sexes. To genetically separate these two phenotypes we allowed the interspecific F1 hybrids to propagate for 25 generations (F25) without selection for mating time or callus colour, finding that the advanced hybrid population had moved towards B. tryoni phenotypes for both traits. Selection for day mating in replicate lines at F25 resulted in significant phenotypic shifts in both traits towards B. neohumeralis phenotypes in F26. However, we were unable to completely recover the mating time profile of B. neohumeralis and relaxation of selection for day mating led to a shift back towards dusk mating, but not yellow callus colour, by F35., Conclusion: We conclude that the inheritance of the two major species-defining traits is separable but tightly linked and involves more than one gene in each case. It also appears that laboratory conditions select for the B. tryoni phenotypes for mating time. We discuss our findings in relation to speciation theory and the likely effects of domestication during the generation of mass release strains for sterile insect control programmes.

Background: The highly polyphagous Queensland fruit fly (Bactrocera tryoni Froggatt) expanded its range substantially during the twentieth century and is now the most economically important insect pest of Australian horticulture, prompting intensive efforts to develop a Sterile Insect Technique (SIT) control program. Using a "common garden" approach, we have screened for natural genetic variation in key environmental fitness traits among populations from across the geographic range of this species and monitored changes in those traits induced during domestication., Results: Significant variation was detected between the populations for heat, desiccation and starvation resistance and wing length (as a measure of body size). Desiccation resistance was correlated with both starvation resistance and wing length. Bioassay data for three resampled populations indicate that much of the variation in desiccation resistance reflects persistent, inherited differences among the populations. No latitudinal cline was detected for any of the traits and only weak correlations were found with climatic variables for heat resistance and wing length. All three stress resistance phenotypes and wing length changed significantly in certain populations with ongoing domestication but there was also a strong population by domestication interaction effect for each trait., Conclusions: Ecotypic variation in heat, starvation and desiccation resistance was detected in Australian Qfly populations, and these stress resistances diminished rapidly during domestication. Our results indicate a need to select source populations for SIT strains which have relatively high climatic stress resistance and to minimise loss of that resistance during domestication.

Competing Interests: The authors declare no competing interest.

The cuticular layer of the insect exoskeleton contains diverse compounds that serve important biological functions, including the maintenance of homeostasis by protecting against water loss, protection from injury, pathogens and insecticides, and communication. Bactrocera tryoni (Froggatt) is the most destructive pest of fruit production in Australia, yet there are no published accounts of this species' cuticular chemistry. We here provide a comprehensive description of B. tryoni cuticular chemistry. We used gas chromatography-mass spectrometry to identify and characterize compounds in hexane extracts of B. tryoni adults reared from larvae in naturally infested fruits. The compounds found included spiroacetals, aliphatic amides, saturated/unsaturated and methyl branched C 12 to C 20 chain esters and C 29 to C 33 normal and methyl-branched alkanes. The spiroacetals and esters were found to be specific to mature females, while the amides were found in both sexes. Normal and methyl-branched alkanes were qualitatively the same in all age and sex groups but some of the alkanes differed in amounts (as estimated from internal standard-normalized peak areas) between mature males and females, as well as between mature and immature flies. This study provides essential foundations for studies investigating the functions of cuticular chemistry in this economically important species.

The Queensland fruit fly, Bactrocera tryoni, is a major pest of Australian horticulture which has expanded its range in association with the spread of horticulture over the last ~ 150 years. Its distribution in northern Australia overlaps that of another fruit fly pest to which some authors accord full species status, Bactrocera aquilonis. We have used reduced representation genome-wide sequencing to genotype 359 individuals taken from 35 populations from across the current range of the two taxa, plus a further 73 individuals from six of those populations collected 15-22 years earlier. We find significant population differentiation along an east-west transect across northern Australia which likely reflects limited but bidirectional gene flow between the two taxa. The southward expansion of B. tryoni has led to relatively little genetic differentiation, and most of it is associated with a move into previously marginal inland habitats. Two disjunct populations elsewhere in Australia and three on Melanesian islands are each clearly differentiated from all others, with data strongly supporting establishment from relatively few founders and significant isolation subsequently. Resequencing of historical samples from one of the disjunct Australian populations shows that its genetic profile has changed little over a 15-year period, while the Melanesian data suggest a succession of 'island hopping' events with progressive reductions in genetic diversity. We discuss our results in relation to the control of B. tryoni and as a model for understanding the genetics of invasion and hybridisation processes.

F 420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F 420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F 420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis , phosphoenolpyruvate rather than 2-phospholactate stimulates F 420 biosynthesis. Analysis of F 420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F 420 -0. To determine the structural basis of dehydro-F 420 -0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F 420 -0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F 420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F 420 , and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F 420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F 420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F 420 , determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F 420 ., (Copyright © 2020 Grinter et al.)

Coral reefs worldwide are suffering mass mortalities from marine heat waves. With the aim of enhancing coral bleaching tolerance, we evolved 10 clonal strains of a common coral microalgal endosymbiont at elevated temperatures (31°C) for 4 years in the laboratory. All 10 heat-evolved strains had expanded their thermal tolerance in vitro following laboratory evolution. After reintroduction into coral host larvae, 3 of the 10 heat-evolved endosymbionts also increased the holobionts' bleaching tolerance. Although lower levels of secreted reactive oxygen species (ROS) accompanied thermal tolerance of the heat-evolved algae, reduced ROS secretion alone did not predict thermal tolerance in symbiosis. The more tolerant symbiosis exhibited additional higher constitutive expression of algal carbon fixation genes and coral heat tolerance genes. These findings demonstrate that coral stock with enhanced climate resilience can be developed through ex hospite laboratory evolution of their microalgal endosymbionts., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/ p -cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K m of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes. IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia , that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/ p -cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans., (Copyright © 2020 American Society for Microbiology.)