Comparison of cleaning methods for delicate insect specimens for scanning electron microscopy.

The objective of the present study was to compare cleaning methods for delicate insect specimens for investigations with scanning electron microscopy (SEM). As typical specimens we used aquatic larvae of mosquitoes, springtails, larvae of mayflies and caterpillars because they are very fragile and large parts of their body consist of soft tissue. Additionally their cuticle is very often covered with dirt, soil particles or other materials. Cleaning with ultrasonic sound, as the most common cleaning method used for SEM, will destroy fragile insects. Therefore we tested different procedures to remove the dirt particles. In a first approach we compared cleaning with Potassium hydroxide (KOH), Proteinase K, and Triton X in aquatic larvae of flies, which were available in numbers and kept under the same conditions. As our results showed that the treatment with KOH gives the best results we treated in a second approach springtails, larvae of mayflies and caterpillars only with KOH. The springtails and caterpillars were largely free of particles after treatment with KOH; however, the larvae of mayflies were still covered with remnants of diatoms and precipitates of calcium carbonate of the algae. KOH dissolves organic impurities, on the other hand silicon dioxide and lime crusts are not solved. With this limitation, treatment with KOH is a simple technique for routine use as cleaning method for fragile insect specimens for SEM.

Catching prey with the antennae - The larval head of Corethrella appendiculata (Diptera: Corethrellidae).

The larval cephalic morphology of Corethrella appendiculata Grabham, 1906 is described and documented in detail. The observed features are compared to conditions found in Chaoboridae, Culicidae, and other culicomorph families. The function of antennae, mouthparts and associated muscles is interpreted based on the morphological results. The prey catching mechanism is compared to what occurs in other predaceous larvae of Culicomorpha. The cephalic larval morphology is discussed with respect to homology and possible phylogenetic implications. The horizontal frontoclypeal antennal grooves and the lateral rows of strongly developed bristles are likely larval autapomorphies of Corethrellidae. The presence of raptorial antennae is a highly unusual apomorphy shared with Chaoboridae. The systematic position of Corethrellidae remains ambiguous.

Comparative thoracic anatomy of the wild type and wingless (wg1cn1) mutant of Drosophila melanogaster (Diptera).

Genetically modified organisms are crucial for our understanding of gene regulatory networks, physiological processes and ontogeny. With modern molecular genetic techniques allowing the rapid generation of different Drosophila melanogaster mutants, efficient in-depth morphological investigations become an important issue. Anatomical studies can elucidate the role of certain genes in developmental processes and point out which parts of gene regulatory networks are involved in evolutionary changes of morphological structures. The wingless mutation wg1 of D. melanogaster was discovered more than 40 years ago. While early studies addressed the external phenotype of these mutants, the documentation of the internal organization was largely restricted to the prominent indirect flight muscles. We used SEM micrographs, histological serial sections, µ-computed tomography, CLSM and 3D reconstructions to study and document the thoracic skeletomuscular system of the wild type and mutant. A recently introduced nomenclature for the musculature of neopteran insects was applied to facilitate comparisons with closely or more distantly related taxa. The mutation is phenotypically mainly characterized by the absence of one or both wings and halteres. The wing is partly or entirely replaced by duplications of mesonotal structures, whereas the haltere and its associated muscles are completely absent on body sides showing the reduction. Both the direct and indirect mesothoracic flight muscles are affected by loss and reorientation of bundles or fibers. Our observations lead to the conclusion that the wingless mutation causes a homeotic transformation in the imaginal discs of wings and halteres with a direct effect on the development of skeletal structures and an indirect effect on the associated muscular system.

The larval head of Exechia (Mycetophilidae) and Bibio (Bibionidae) (Diptera).

Exechia and Bibio have retained several plesiomorphic groundplan features of Diptera and Bibionomorpha, including a fully exposed and sclerotized head capsule, the transverse undivided labrum, the absence of movable premandibles, and undivided mandibles without combs. The fusion of the hypostomal bridge with the head capsule and largely reduced antennae are derived features shared by both taxa. The absence of teeth at the anterior hypostomal margin is a potential autapomorphy of Bibionomorpha. A basal position of Anisopodidae is suggested by a number of plesiomorphies retained in this family. Apomorphies of Bibionomorpha excluding Anisopodidae are the reduction of tentorial elements, the partial fusion of the labrum and clypeus, one-segmented antennae, the absence of a separate submental sclerite, the loss of the labial palpus, and the reduction of the pharyngeal filter apparatus. Head structures of Bibio are largely unmodified. The subprognathous orientation is one of few autapomorphic features. In contrast, the mouthparts of Exechia are highly modified in correlation with the specialized food uptake. The rasping counterrotating movements of maxillae and mandibles with teeth oriented in opposite directions are carried out by strongly developed extensors and flexors of the paired mouthparts. The modified labium mechanically supports the "drill head" formed by the mandibles und maxillae. The necessary stability of the head capsule is provided by the hypostomal bridge which also compensates the far-reaching reduction of the tentorium.

The adult head morphology of the hessian fly Mayetiola destructor (Diptera, Cecidomyiidae).

Cover illustration. Mayetiola destructor is a major pest of wheat in Europe, North Africa and North America. In this issue of the Journal of Morphology, Schneeberg et al. (pp. 1299-1311) investigate the adult head structures of the cecidomyiid fly and compared their findings with evolutionarily less successful families within Bibionomorpha. The cover image shows a histological cross section of the head of Mayetiola destructor (Diptera, Cecidomyiidae).

The adult head morphology of the hessian fly Mayetiola destructor (Diptera, Cecidomyiidae).

The adult head of the Hessian fly Mayetiola destructor was examined and described in detail. Morphological features are evaluated with respect to phylogenetic implications and possible effects of miniaturisation. Preserved groundplan features of Diptera are the orthognathous orientation of the head, the vestiture of small microtrichia (possible autapomorphy), filiform antennae inserted frontally between the compound eyes, the presence of a clypeolabral muscle (possible autapomorphy), the presence of labellae (autapomorphy), and the presence of only one premental retractor. Potential synapomorphies of the groups assigned to Bibionomorpha are the origin of M. tentorioscapalis medialis on the frons and the loss of M. craniolacinialis. Further apomorphies of Cecidomyiidae identified in Mayetiola are the unusually massive anterior tentorial arm, the absence of the labro-epipharyngeal food channel, the absence of the lacinia, and the presence of antennal sensilla connected by a seta, a feature not known from any other group of Diptera. The very large size of the compound eyes (in relation to the entire head surface) and the complete loss of ocelli are possible effects of miniaturization. The large size of the brain (in relation to the cephalic lumen), the unusual shape of the optic lobes, and the absence of the frontal ganglion as a separate structure are probably also linked with size reduction.

The skeletomuscular system of the larva of Drosophila melanogaster (Drosophilidae, Diptera): a contribution to the morphology of a model organism.

The morphological features of the third instar larva of the most important insect model, Drosophila melanogaster, are documented for the first time using a broad spectrum of modern morphological techniques. External structures of the body wall, the cephaloskeleton, and the musculature are described and illustrated. Additional information about other internal organs is provided. The systematic implications of the findings are discussed briefly. Internal apomorphic features of Brachycera and Cyclorrhapha are confirmed for Drosophila. Despite the intensive investigations of the phylogeny of the megadiverse Diptera, evolutionary reconstructions are still impeded by the scarcity of anatomical data for brachyceran larvae. The available morphological information for the life stages of three insect model organisms -D. melanogaster (Diptera, Drosophilidae), Manduca sexta (Lepidoptera, Sphingidae) and Tribolium castaneum (Coleoptera, Tenebrionidae) - is addressed briefly. The usefulness of a combination of traditional and innovative techniques for an optimized acquisition of anatomical data for different life stages is highlighted.

The larvae of Nymphomyiidae (Diptera, Insecta) - ancestral and highly derived?

Larval head structures of Nymphomyia dolichopeza were examined and described in detail. The conditions are compared to those of other dipteran representatives. Our results support the monophyly of Nymphomyiidae. Potential apomorphies are dimorphic crochets on the abdominal prolegs and the complete loss of the tentorium. Possible synapomorphies of Nymphomyiidae and Deuterophlebiidae could be the rows of spatulate macrosetae covering the ventral surface of the labrum-epipharynx, the presence of distinct teeth along the anterior premento-hypopharyngeal margin, the absence of labral microtrichia and some other affinities concerning the life history of the two groups. A clade Blephariceromorpha is also supported by some larval features. Potential synapomorphies of Nymphomyiidae, Deuterophlebiidae and Blephariceridae are the vestigial M. labroepipharyngalis, the absence of a movable premandible, crochet-tipped prolegs, the complete loss of spiracles and non-retractable anal papillae. A clade Nymphomyiidae and Chironomidae is only weakly supported by characters of the larval head. The anteriorly serrate and posteriorly fused hypostoma is a potential apomorphic character. Our results support neither phylogenetic affinities between Nymphomyiidae and Axymyiidae nor a sistergroup relationship between Nymphomyiidae and the remaining Diptera. However, a comprehensive cladistic analysis is not presented in our study.

Adult head structures of Deuterophlebiidae (Insecta), a highly derived "ancestral" dipteran lineage.

Adult head structures of Deuterophlebia coloradensis were examined, 3-dimensionally reconstructed and described. The results are compared to those of other representatives of basal dipteran lineages, primarily Nymphomyia dolichopeza (Nymphomyiidae) and Edwardsina gracilis (Blephariceridae). The head structures are extremely simplified. The labrum, mandibles, maxillae and labium are completely reduced. Only eight pairs of muscles are present. These modifications are possibly linked with the extremely short life span and non-feeding of adults. Possible synapomorphies of Deuterophlebiidae + Nymphomyiidae are the loss of all mouthparts, the elongation of the terminal antennal segment, and the loss of M. tentoriobuccalis anterior. An alternative placement of Deuterophlebiidae as sister group of Blephariceridae is only suggested by the origin of M. tentorioscapalis posterior on the vertex. Blephariceromorpha (Deuterophlebiidae, Nymphomyiidae, Blephariceridae) is only weakly supported by features of the adult head. The missing frontoclypeal and clypeolabral suture and the origin of M. tentorioscapalis on the head capsule are potential autapomorphies. Our results do not support a sister group relationship between Deuterophlebiidae and the remaining Diptera. A reliable reconstruction of basal dipteran relationships is impeded by missing morphological data for many potential key taxa.