Confocal laser scanning microscopy as a valuable tool in Diptera larval morphology studies

Larval morphology of flies is traditionally studied using light microscopy, yet in the case of fine structures compound light microscopy is limited due to problems of resolution, illumination and depth of field, not allowing for precise recognition of sclerites’ edges and interactions. Using larval instars of cyclorrhaphan Diptera, we show the usefulness of confocal laser scanning microscopy (CLSM) for studying the morphological characters of immature stages by taking advantage of the autofluorescent properties of cephaloskeleton structures. We compare data obtained from killed but unprepared larvae with those from larvae prepared by clearing according to two commonly used methods, either with potassium hydroxide or with Hoyer’s medium. We also evaluated the CLSM application for examining already slide-mounted larvae stored in museum collections and those freshly prepared. Our results indicate that CLSM and 3D reconstruction are excellent for visualizing small, compound structures of cylrorrhaphan larvae cephaloskeleton, if appropriate clearing techniques, i.e. the application of KOH, are used. Maximum intensity projection of confocal data sets obtained from material freshly prepared and that stored in museum collection does not differ. Because of this and the fact that KOH is commonly used as a clearing method to examine the cephaloskeleton of Diptera larvae, it is possible, and highly recommended, to use slides already prepared with this method for re-examination by CLSM. We conclude that CLSM application can be an invaluable source of data for studies of larval morphology of Cyclorrhapha by way of taxonomic diagnoses, character identification and improvement in characters homologization.This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited

Morphology of the first instar larva of obligatory traumatic myiasis agents (Diptera: Calliphoridae, Sarcophagidae)

There are only three fly species that are obligate agents of traumatic myiasis of humans and livestock: a single species of flesh fly, Wohlfahrtia magnifica (Sarcophagidae), and two species of blow flies, Chrysomya bezziana and Cochliomyia hominivorax (Calliphoridae). The morphology of their first instar larvae is thoroughly and consistently documented here with light microscopy photographs and scanning electron microscopy micrographs. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, oral ridges, thoracic and abdominal spinulation, spiracular field, posterior spiracles and cephaloskeleton. New diagnostic features drawn from the cephaloskeleton and the spinulation of abdominal segments, including the anal pad, are discovered and extensively described. Earlier descriptions in the literature are revisited, and major discrepancies between these and the results of the current study are discussed. The present results allow clarification, correction and, especially, complementation of information provided by earlier authors. The relatively distant taxonomic position of all three species is evidence that obligatory myiasis has arisen independently, and the extensively similar morphology in the first instar larvae of Chrysomya bezziana, Cochliomyia hominivorax and W. magnifica in comparison to necrophagous species, especially the enhancement of the anterior part of the cephaloskeleton and the segmental spinulation, is therefore best interpreted as homoplasic adaptations to a life strategy as obligate vertebrate parasites. An identification key for first instar larvae of all obligatory traumatic myiasis agents of mammals is provided.Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited

Fungal Planet description sheets: 558–624

Novel species of fungi described in this study include those from various countries as follows: Australia: Banksiophoma australiensis (incl. Banksiophoma gen. nov.) on Banksia coccinea, Davidiellomyces australiensis (incl. Davidiellomyces gen. nov.) on Cyperaceae, Didymocyrtis banksiae on Banksia sessilis var. cygnorum, Disculoides calophyllae on Corymbia calophylla, Harknessia banksiae on Banksia sessilis, Harknessia banksiae-repens on Banksia repens, Harknessia banksiigena on Banksia sessilis var. cygnorum, Harknessia communis on Podocarpus sp., Harknessia platyphyllae on Eucalyptus platyphylla, Myrtacremonium eucalypti (incl. Myrtacremonium gen. nov.) on Eucalyptus globulus, Myrtapenidiella balenae on Eucalyptus sp., Myrtapenidiella eucalyptigena on Eucalyptus sp., Myrtapenidiella pleurocarpae on Eucalyptus pleurocarpa, Paraconiothyrium hakeae on Hakea sp., Paraphaeosphaeria xanthorrhoeae on Xanthorrhoea sp., Parateratosphaeria stirlingiae on Stirlingia sp., Perthomyces podocarpi (incl. Perthomyces gen. nov.) on Podocarpus sp., Readeriella ellipsoidea on Eucalyptus sp., Rosellinia australiensis on Banksia grandis, Tiarosporella corymbiae on Corymbia calophylla, Verrucoconiothyrium eucalyptigenum on Eucalyptus sp., Zasmidium commune on Xanthorrhoea sp., and Zasmidium podocarpi on Podocarpus sp. Brazil: Cyathus aurantogriseocarpus on decaying wood, Perenniporia brasiliensis on decayed wood, Perenniporia paraguyanensis on decayed wood, and Pseudocercospora leandrae-fragilis on Leandra fragilis. Chile: Phialocephala cladophialophoroides on human toe nail. Costa Rica: Psathyrella striatoannulata from soil. Czech Republic: Myotisia cremea (incl. Myotisia gen. nov.) on bat droppings. Ecuador: Humidicutis dictiocephala from soil, Hygrocybe macrosiparia from soil, Hygrocybe sangayensis from soil, and Polycephalomyces onorei on stem of Etlingera sp. France: Westerdykella centenaria from soil. Hungary: Tuber magentipunctatum from soil. India: Ganoderma mizoramense on decaying wood, Hodophilus indicus from soil, Keratinophyton turgidum in soil, and Russula arunii on Pterigota alata. Italy: Rhodocybe matesina from soil. Malaysia: Apoharknessia eucalyptorum, Harknessia malayensis, Harknessia pellitae, and Peyronellaea eucalypti on Eucalyptus pellita, Lectera capsici on Capsicum annuum, and Wallrothiella gmelinae on Gmelina arborea. Morocco: Neocordana musigena on Musa sp. New Zealand: Candida rongomai-pounamu on agaric mushroom surface, Candida vespimorsuum on cup fungus surface, Cylindrocladiella vitis on Vitis vinifera, Foliocryphia eucalyptorum on Eucalyptus sp., Ramularia vacciniicola on Vaccinium sp., and Rhodotorula ngohengohe on bird feather surface. Poland: Tolypocladium fumosum on a caterpillar case of unidentified Lepidoptera. Russia: Pholiotina longistipitata among moss. Spain: Coprinopsis pseudomarcescibilis from soil, Eremiomyces innocentii from soil, Gyroporus pseudocyanescens in humus, Inocybe parvicystis in humus, and Penicillium parvofructum from soil. Unknown origin: Paraphoma rhaphiolepidis on Rhaphiolepsis indica. USA: Acidiella americana from wall of a cooling tower, Neodactylaria obpyriformis (incl. Neodactylaria gen. nov.) from human bronchoalveolar lavage, and Saksenaea loutrophoriformis from human eye. Vietnam: Phytophthora mekongensis from Citrus grandis, and Phytophthora prodigiosa from Citrus grandis. Morphological and culture characteristics along with DNA barcodes are provided

Fungal Planet description sheets: 458–624

novel fungal species systematicsPeer reviewe