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

Strepsiptera and triungula in Cretaceous amber

The first definitive strepsipteran is reported from the Cretaceous, named Cretostylops engeli, n.gen., n.sp., which is an adult male in amber from the mid-Cretaceous (approximately Cenomanian) of northern Myanmar (Burma). A triungulin from the Late Cretaceous (Campanian, c. 80 myo) of Manitoba, Canada is possibly a strepsipteran. The triungulin is described in detail but its morphology does not conform to any known clade of Recent strepsipterans. Other Cretaceous triungula reported here are in Burmese amber and are probably of the family Rhipiphoridae (Coleoptera), and bizarre (possibly coleopteran) triungula in mid-Cretaceous (Turonian, c. 90 myo) amber from New Jersey, USA. Phylogenetic analysis confirms the primitive position of Cretostylops among families of Strepsiptera, but it is not as primitive as Protoxenos in Eocene Baltic amber. Protoxenos and Cretostylops are still too highly modified to address the controversial relationships of Strepsiptera among insect orders, but the generalized structure of the mandible is inconsistent with the hypothesis that this order is the sister group to Diptera or closely related to Mecopterida. Phylogeny of living and Recent Strepsiptera suggests an origin of the order in the Early Cretaceous or Late Jurassic, which is also inconsistent with this order being a sister group to the much older Diptera

Strepsiptera and triungula in Cretaceous amber

The first definitive strepsipteran is reported from the Cretaceous, named Cretostylops engeli, n.gen., n.sp., which is an adult male in amber from the mid-Cretaceous (approximately Cenomanian) of northern Myanmar (Burma). A triungulin from the Late Cretaceous (Campanian, c. 80 myo) of Manitoba, Canada is possibly a strepsipteran. The triungulin is described in detail but its morphology does not conform to any known clade of Recent strepsipterans. Other Cretaceous triungula reported here are in Burmese amber and are probably of the family Rhipiphoridae (Coleoptera), and bizarre (possibly coleopteran) triungula in mid-Cretaceous (Turonian, c. 90 myo) amber from New Jersey, USA. Phylogenetic analysis confirms the primitive position of Cretostylops among families of Strepsiptera, but it is not as primitive as Protoxenos in Eocene Baltic amber. Protoxenos and Cretostylops are still too highly modified to address the controversial relationships of Strepsiptera among insect orders, but the generalized structure of the mandible is inconsistent with the hypothesis that this order is the sister group to Diptera or closely related to Mecopterida. Phylogeny of living and Recent Strepsiptera suggests an origin of the order in the Early Cretaceous or Late Jurassic, which is also inconsistent with this order being a sister group to the much older Diptera

Focusing on morphology: Applications and implications of confocal laser scanning microscopy (Diptera: Campichoetidae, Camillidae, Drosophilidae)

Volume: 107Start Page: 323End Page: 33

Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans

The utility of cuticular autofluorescence for the visualization of copepod morphology by means of confocal laser scanning microscopy (CLSM) was examined. Resulting maximum intensity projections give very accurate information on morphology and show even diminutive structures such as small setae in detail. Furthermore, CLSM enables recognition of internal structures and differences in material composition. Optical sections in all layers and along all axes of the specimens can be obtained by CLSM. The facile and rapid preparation method bears no risk of artefacts or damage occurring to the preparations and the visualized specimens can be used for later analyses allowing for the investigation of irreplaceable type specimens or parts of them. These features make CLSM a very effective tool for both taxonomical and ecological studies in small crustaceans; however, the maximum thickness of the specimens is limited to a few hundred micrometers. Three-dimensional models based on CLSM image stacks allow observation of the preparations from all angles and can permit, improve and speed up studies on functional morphology. The visualization method described has a strong potential to become a future standard technique in aquatic biology due to its advantages over conventional light microscopy and scanning electron microscopy

Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster.

International audienceThe evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sex-linked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterized by intra-species variation in hybridization signal size, appeared on the fourth chromosome related to heterochromatic rearrangements. Nucleolar material is thought to be required for sex chromosome pairing and disjunction in a variety of organisms including Drosophila. Thus, either remnant sequences, possibly intergenic spacer repeats, are still present in the sex chromosomes which have lost their NORs (as observed in D. simulans and D. sechellia), or an alternative mechanism has evolved