Amapeza and Nigripeza, new genera of Neotropical micropezid flies (Diptera, Micropezidae, Taeniapterinae).

The new Neotropical genera Amapeza and Nigripeza are described for seven new species and three named species previously treated as part of the genus Grallipeza Rondani. Amapeza includes A. amazonica (Enderlein) new combination, A. affinis (Hennig) new combination, A. hyaloptera (Hendel) new combination and the new species A. camelina A. plicata, and A. tricincta. Nigripeza includes N. obscura (Hennig) new combination and the new species N. cali, N. octa and N. spangleri.

Vertical stratification of insect abundance and species richness in an Amazonian tropical forest.

Tropical forests are among the most biodiverse biomes on the planet. Nevertheless, quantifying the abundance and species richness within megadiverse groups is a significant challenge. We designed a study to address this challenge by documenting the variability of the insect fauna across a vertical canopy gradient in a Central Amazonian tropical forest. Insects were sampled over two weeks using 6-m Gressitt-style Malaise traps set at five heights (0 m-32 m-8 m intervals) on a metal tower in a tropical forest north of Manaus, Brazil. The traps contained 37,778 specimens of 18 orders of insects. Using simulation approaches and nonparametric analyses, we interpreted the abundance and richness of insects along this gradient. Diptera, Hymenoptera, and Coleoptera had their greatest abundance at the ground level, whereas Lepidoptera and Hemiptera were more abundant in the upper levels of the canopy. We identified species of 38 of the 56 families of Diptera, finding that 527 out of 856 species (61.6%) were not sampled at the ground level. Mycetophilidae, Tipulidae, and Phoridae were significantly more diverse and/or abundant at the ground level, while Tachinidae, Dolichopodidae, and Lauxaniidae were more diverse or abundant at upper levels. Our study suggests the need for a careful discussion of strategies of tropical forest conservation based on a much more complete understanding of the three-dimensional distribution of its insect diversity.

A revision of the Australian species of Howickia Richards (Diptera: Sphaeroceridae: Limosininae).

Howickia Richards, 1951, the largest genus of Sphaeroceridae in Australia, is revised to include 43 species, including all wingless Australian species of the family. The following new species are described: H. acicula (TAS), H. auricoma (VIC), H. biantenna (TAS), H. cruspica (VIC), H. erythrocephala (NSW), H. flagella (TAS), H. grandisterna (TAS), H. holti (NSW), H. irwini (NSW), H. loligo (TAS), H. notechis (TAS), H. nuda (NSW), H. pertusa (TAS), H. pinnula (TAS), H. pseudovittata (NSW, QLD), H. robustaseta (NSW), H. tricolor (NSW), H. truemani (TAS), H. wamini (NSW, QLD, TAS, WA), and H. xanthocephala (QLD, NSW). The genera Apterobiroina Papp, 1979, Australimosina Papp, 2008, Bentrovata Richards, 1973, Monteithiana Richards, 1973, Popondetta Richards, 1973, and Otwayia Richards, 1973 are treated as new synonyms of Howickia. Howickia is redefined and placed in the context of a key to genera of Sphaeroceridae in Australia, the Australian species of Howickia are keyed, and a morphological phylogeny is given for the Australian species. The following new combinations are given: Howickia australis (Papp, 1979) comb. nov., Howickia cynthia (Richards, 1973) comb. nov., Howickia dealata (Richards, 1973) comb. nov., Howickia flavipes (Papp, 2021) comb. nov., Howickia flaviterga (Richards, 1973) comb. nov., Howickia flavithorax (Papp, 2021) comb. nov., Howickia regalis (Richards, 1973) comb. nov., Howickia sabina (Richards, 1973) comb. nov., Howickia truncata (Papp, 2021) comb. nov., Paralimosina nasalis (Richards, 1973) comb. nov. (formerly in Pseudocollinella Duda, 1924), Pseudopterogramma fenestrata (Richards, 1973) comb. nov. (originally as Leptocera (Limosina) Macquart, 1835, transferred to Biroina Richards, 1973 by Marshall in Rohácek et al. (2001)), and Pleuroseta dorrigonis (Richards, 1973) comb. nov. (formerly in Biroina). A new name, Howickia neoregalis nom. nov. is proposed for H. regalis Marshall, 2014, junior homonym of H. regalis (Richards, 1973). The following genera are newly recorded from Australia: Chaetopodella Duda, 1920, Paralimosina Papp, 1973, Pachytarsella Richards, 1963, Parapterogramma Papp, 2008, Philocoprella Richards, 1929, Piliterga Papp, 2008, Pterogrammoides Papp, 1972, Spelobia Spuler, 1924, and Spinilimosina Rohácek, 1983.

Systematic relationships of the Taeniapterini (Diptera: Micropezidae, Taeniapterinae).

A molecular phylogeny for the Taeniapterinae is presented based on mitochondrial (cytochrome c oxidase subunit I, COI), ribosomal (16S rRNA and 28S rRNA) and nuclear (EF-1 and CAD) genes of 48 specimens including 40 species. Maximum likelihood and Bayesian inference were used to analyze the total concatenated dataset of 8769 bp. The results confirm that tribal classifications in Taeniapterinae are artificial and support the separation of Paragrallomyia Hendel and Taeniaptera sensu Ferro Marshall (2020).

UbiD domain dynamics underpins aromatic decarboxylation.

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.

The impact of and response to the COVID-19 pandemic on a hospital palliative care team.

London was at the forefront of the COVID-19 pandemic in the UK, with an exponential rise in hospital admissions from March 2020. This case study appraises the impact on and response of a hospital palliative care service based in a large inner-city teaching hospital. Referrals increased from a mean of 39 to 75 per week; deaths from 13 to 52 per week. Multiple actions were taken by the team to manage the surge in referrals, which have been categorised based on the 4S model: systems, space, stuff and staff. Several lessons are highlighted: need for flexible and responsive staffing over the 7-day week; implementing clear, accessible clinical guidance supported by ward-based teaching; benefits of integrating clinical practice with research; and the importance of maintaining team well-being and camaraderie to sustain change. Further evaluation is needed of the differential impact of changes made to inform service planning for future pandemics.

The In Vitro Production of prFMN for Reconstitution of UbiD Enzymes.

Prenylated flavin (prFMN) is a modified FMN cofactor, the isoalloxazine is extended by an additional six membered nonaromatic ring. The modification confers azomethine ylide characteristics on the oxidised prFMN, allowing it to support the reversible nonoxidative decarboxylation of unsaturated acids by the UbiD family of decarboxylases. In absence of a chemical synthesis route for prFMN, enzymatic production by the flavin prenyltransferase, UbiX, is required for in vitro reconstitution of prFMN-dependent enzymes. Here we provide an overview of the methods for producing prFMN in vitro using the flavin prenyltransferase UbiX, and the subsequent reconstitution and activation of UbiD enzymes.

Synopsis of Nothybidae (Diptera: Brachycera) from China, with redescription of Nothybus absens Lonsdale amp; Marshall.

The Nothybidae of China are reviewed, including Nothybus absens Lonsdale Marshall, 2016 which was previously known only from photographs, is here redescribed on the basis of male and female specimens. A key to Chinese species of Nothybidae is provided.

Structure and Mechanism of Pseudomonas aeruginosa PA0254/HudA, a prFMN-Dependent Pyrrole-2-carboxylic Acid Decarboxylase Linked to Virulence.

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the Pseudomonas aeruginosa virulence attenuation factor PA0254/HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMN-imidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on P. aeruginosa quorum sensing.

Stipulosina new genus (Diptera: Sphaeroceridae: Limosininae) from bamboo (Chusquea scandens) stipules in Ecuador.

The new genus Stipulosina (Sphaeroceridae: Limosininae) is described for four new species, S. amphibarba n. sp. (type species), S. barbistylus n. sp., S. ceroptistylus n. sp. and S. curvistylus n. sp., collected from bamboo stipules in the Ecuadorian Andes in 2002 and 2019. All four species are associated with stipules of the Andean bamboo Chusquea scandens Kunth but not with those of sympatric bamboo species.

New generic concepts for orphaned lineages formerly treated as part of the genus Ocyptamus Macquart, 1834 (Diptera, Syrphidae).

The large syrphid genus Ocyptamus Macquart has been subject to scrutiny in recent years, resulting in a more narrowly defined monophyletic Ocyptamus sensu stricto. Some of the species excluded from this narrower concept of the genus are in evolutionary lineages for which generic names are available, but other species are in lineages for which generic names do not exist. The current paper presents five new genera to correct this: Fragosa gen. nov. (former Ocyptamus stenogaster group), Hypocritanus gen. nov. (former Ocyptamus fascipennis group), Maiana gen. nov. (former Ocyptamus callidus group), Nuntianus gen. nov. (former Ocyptamus lepidus group) and Victoriana gen. nov. (former Ocyptamus melanorrhinus group). In addition to the new combinations related to these new genera, Argentinomyia aenea (Williston) comb. nov., Hybobathus pola (Curran) comb. nov., H. subchalybeus (Walker) comb. nov., H. wilhelmina (Doesburg) comb. nov., and Leucopodella nasuta (Williston) comb. nov. are moved out of Ocyptamus. Ocyptamus isthmus Thompson is a jun. syn. of Callostigma panamensis Curran stat. rev. Relevant morphological characters and their taxonomic and evolutionary implications are discussed.

A redefinition of Paragrallomyia Hendel (Diptera: Micropezidae, Taeniapterinae) and a revision of the P. albibasis complex.

A new diagnosis is provided for the genus Paragrallomyia, and the common and widely distributed species Paragrallomyia albibasis (Enderlein) and Paragrallomyia thiemei (Enderlein) are redefined as part of a group of 17 superficially similar Neotropical species. This group, here treated as the 'P. albibasis complex', includes P. albibasis, P. inpai (Albuquerque), P. teresacristinae (Albuquerque), P. thiemei, P. vulpes (Cresson) and 12 new species (P. aequorea, P. bifurcata, P. brasiliensis, P. caliensis, P. citrina, P. colombiana, P. diminuta, P. ecuadoriensis, P. nodulosa, P. pseudoalbibasis, P. quadrifurca and P. quaternaria). Paragrallomyia parens (Cresson) is newly recognized as a junior synonym of Paragrallomyia albibasis. Taeniaptera nigriceps Hennig and Taeniaptera vulpes Cresson are transferred to Paragrallomyia and 13 other species previously treated as Paragrallomyia are transferred to Poecilotylus Hennig (Grallomyia aeripennis Enderlein, Taeniaptera aliceae Albuquerque, Grallomyia dilutimacula Enderlein, Calobata latifascia Wulp, Grallomyia lauta Cresson, Grallomyia longifurca Hendel, Calobata nigritarsis Macquart, Calobata rufifacies Macquart, Grallomyia seiuncta Czerny, Tanypoda simillima Hendel, Grallomyia strigata Enderlein, Calobata tibialis Macquart, Calobata vittipennis Coquillett). Grallomyia lineata Enderlein is transferred from Taeniaptera to Poecilotylus. All named species remaining in Paragrallomyia are keyed out at least to the species complex level.

Revision of the genus Systellapha Enderlein (Micropezidae, Taeniapterinae).

The genus Systellapha Enderlein, currently treated as part of Grallipeza Rondani, is resurrected and redescribed to include S. ornatithorax Enderlein and two new species, S. digifurca and S. ornatifemur. The genus is diagnosed by its extremely broad palpus and characteristic silvery thoracic vittae, and further defined by characters of the male and female terminalia.

Successful sample preparation for serial crystallography experiments.

Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.

Revision of the Central American and Mexican species of the Curtonotum murinum species complex (Diptera: Curtonotidae).

The five Central American and Mexican species belonging to the Curtonotum murinum species complex are described as Curtonotum abrelatas Lindsay sp. nov., C. irksum Lindsay sp. nov., C. notatum Lindsay sp. nov., C. prolixum Lindsay sp. nov. and C. transitus Lindsay sp. nov. Photographic images, a distribution map of species and an identification key are provided.

Enzymatic control of cycloadduct conformation ensures reversible 1,3-dipolar cycloaddition in a prFMN-dependent decarboxylase.

The UbiD enzyme plays an important role in bacterial ubiquinone (coenzyme Q) biosynthesis. It belongs to a family of reversible decarboxylases that interconvert propenoic or aromatic acids with the corresponding alkenes or aromatic compounds using a prenylated flavin mononucleotide cofactor. This cofactor is suggested to support (de)carboxylation through a reversible 1,3-dipolar cycloaddition process. Here, we report an atomic-level description of the reaction of the UbiD-related ferulic acid decarboxylase with substituted propenoic and propiolic acids (data ranging from 1.01-1.39 Å). The enzyme is only able to couple (de)carboxylation of cinnamic acid-type compounds to reversible 1,3-dipolar cycloaddition, while the formation of dead-end prenylated flavin mononucleotide cycloadducts occurs with distinct propenoic and propiolic acids. The active site imposes considerable strain on covalent intermediates formed with cinnamic and phenylpropiolic acids. Strain reduction through mutagenesis negatively affects catalytic rates with cinnamic acid, indicating a direct link between enzyme-induced strain and catalysis that is supported by computational studies.

Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes.

The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMNreduced species to the active prFMNiminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.

Enzymatic Carboxylation of 2-Furoic Acid Yields 2,5-Furandicarboxylic Acid (FDCA).

The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO2 levels in vitro. We report the crystal structure of a thermophilic HmfF from Pelotomaculum thermopropionicum, revealing that the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrole compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors and improve our understanding of catalysis by members of the UbiD enzyme family.

The UbiX flavin prenyltransferase reaction mechanism resembles class I terpene cyclase chemistry.

The UbiX-UbiD enzymes are widespread in microbes, acting in concert to decarboxylate alpha-beta unsaturated carboxylic acids using a highly modified flavin cofactor, prenylated FMN (prFMN). UbiX serves as the flavin prenyltransferase, extending the isoalloxazine ring system with a fourth non-aromatic ring, derived from sequential linkage between a dimethylallyl moiety and the FMN N5 and C6. Using structure determination and solution studies of both dimethylallyl monophosphate (DMAP) and dimethyallyl pyrophosphate (DMAPP) dependent UbiX enzymes, we reveal the first step, N5-C1' bond formation, is contingent on the presence of a dimethylallyl substrate moiety. Hence, an SN1 mechanism similar to other prenyltransferases is proposed. Selected variants of the (pyro)phosphate binding site are unable to catalyse subsequent Friedel-Crafts alkylation of the flavin C6, but can be rescued by addition of (pyro)phosphate. Thus, retention of the (pyro)phosphate leaving group is required for C6-C3' bond formation, resembling pyrophosphate initiated class I terpene cyclase reaction chemistry.

Diptera of Canada.

The Canadian Diptera fauna is updated. Numbers of species currently known from Canada, total Barcode Index Numbers (BINs), and estimated numbers of undescribed or unrecorded species are provided for each family. An overview of recent changes in the systematics and Canadian faunistics of major groups is provided as well as some general information on biology and life history. A total of 116 families and 9620 described species of Canadian Diptera are reported, representing more than a 36% increase in species numbers since the last comparable assessment by JF McAlpine et al. (1979). Almost 30,000 BINs have so far been obtained from flies in Canada. Estimates of additional number of species remaining to be documented in the country range from 5200 to 20,400.