Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.

The Sarcophagidae are one of the most numerous groups of Diptera in the world, consisting of many species of forensic interest, whose immature stages are useful in the estimation of postmortem interval. The immature stages of some species of forensic importance still remain unknown or undescribed, like in the case of Sarcophaga (Liopygia) cultellata Pandellé, 1986, a species restricted to the Iberian Peninsula, south of France and north of Italy, which shares a ecological niche with species of the same subgenus, e.g., Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830) and Sarcophaga (Liopygia) crassipalpis Macquart, 1839, making it necessary to lay the groundwork for a proper specific differentiation before it can be successfully applied in forensic practice. This study provides the first micromorphological description of all preimaginal stages of S. (L.) cultellata using light microscopy and scanning electron microscopy (SEM), the results of which allow the morphology of the main features to be followed during the immature life cycle. We propose a combination of features for distinguishing Liopygia from other sarcophagid subgenera, based on the current level of morphological knowledge of immature stages. S. (L.) cultellata can be differentiated from S. (L.) argyrostoma and S. (L.) crassipalpis in every immature stage by both light microscopy and SEM. The presence of tegumental warts and a fan-shaped anterior spiracle with a single row of 15-18 respiratory papillae allow distinguishing the third instar larvae of S. (L.) cultellata from other Sarcophaga species described hitherto by SEM. Identification keys based on light microscopy observations are provided, covering all the immature stages of Liopygia subgenus occurring in the Iberian Peninsula.

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Piophila Fallén, 1810 is a genus of small flies composed of two species: Piophila casei (P. casei ) (Linnaeus, 1758), worldwide distributed, and Piophila megastigmata (P. megastigmata ) McAlpine, 1978, recently referred in the Palaearctic Region, from the Iberian Peninsula. Both species share ecological niche and are very interesting for forensic purposes, since they are present in carrion in advance stages of decay and have been found to be related to human corpses. The immature stages of P. megastigmata have ever been described, so this paper gives the ultrastructural morphologies of all preimaginal stages of P. megastigmata studied by light microscopy and scanning electron microscopy (SEM). Particular attention is given to pseudocephalon features—antenna, maxillary palps, facial mask, etc.—cephalopharyngeal skeleton, anterior and posterior spiracles, tegumentary sculpturing, and anal division among others. A comparative analysis of the main distinguishing features is made in order to understand how those features evolve along the developmental process, while larvae II and III are morphologically similar to each other, the larva I shows particular features. Larvae of all stages and pupae are easily distinguishable from other Diptera of forensic importance just based on the presence of trichoid sensilla associated to respiratory slits, instead of peristigmatig tufts, as well as on thewell-known disposition of anal papillae. The shapes of both dorsal edge at the basal part of mouthhook and dorsal bridge of cephalopharyngeal skeleton, and the tegumental ornamentationmay be considered as good features to distinguish the Piophila species, especially for P. megastigmata and P. casei . At the SEM level, shape, number, and arrangement of oral combs, oral ridges, sensilla of maxillary palpus, papillae of anterior spiracle, scales of spiracular field, and posterior spiracles represent good features to distinguish P. megastigmata from P. casei, but further studies will be necessary in West-Paleartic specimens of latter species. The key for identifying third instar larvae of forensically important Piophilidae in the Iberian Peninsula has been updated to include P. megastigmata.

Contrary to what was assumed regarding the presence of respiratory proteins in insects, a functional hemocyanin was recently found in larvae and adults of the stoneflies species Perla marginata, whereas in the close species Perla grandis, hemocyanin functionality was deduced from sequence data. In order to verify if the presence of this ancient trait is widespread within the order and to investigate why stoneflies have maintained it, we have extended the search for hemocyanin to species of other Plecoptera families. In particular, we assessed the presence of hemocyanin in the larval stage of nine Plecoptera species, belonging to six of the seven families of the European stonefly-fauna, and analyzed its potential functionality as deduced by sequence data. We cloned and sequenced the corresponding cDNAs and studied their expression with RT-PCR technique. Moreover, we performed homology studies using the deduced amino acid sequences. On the basis of our analysis, we hypothesized a functional role of the hemocyanin only for two species: Dinocras cephalotes and Isoperla grammatica (Perloidea). In all the investigated Nemouroidea and in Siphonoperla torrentium (Perloidea), this protein may have been lost. Larval size, life-cycle length, trophic role and environmental induction are discussed as possible explanations of these different physiological requirements.