Cyanobacteria and Macroinvertebrate Relationships in Freshwater Benthic Communities beyond Cytotoxicity.

Cyanobacteria are harmful algae that are monitored worldwide to prevent the effects of the toxins that they can produce. Most research efforts have focused on direct or indirect effects on human populations, with a view to gain easy accurate detection and quantification methods, mainly in planktic communities, but with increasing interest shown in benthos. However, cyanobacteria have played a fundamental role from the very beginning in both the development of our planet's biodiversity and the construction of new habitats. These organisms have colonized almost every possible planktic or benthic environment on earth, including the most extreme ones, and display a vast number of adaptations. All this explains why they are the most important or the only phototrophs in some habitats. The negative effects of cyanotoxins on macroinvertebrates have been demonstrated, but usually under conditions that are far from natural, and on forms of exposure, toxin concentration, or composition. The cohabitation of cyanobacteria with most invertebrate groups is long-standing and has probably contributed to the development of detoxification means, which would explain the survival of some species inside cyanobacteria colonies. This review focuses on benthic cyanobacteria, their capacity to produce several types of toxins, and their relationships with benthic macroinvertebrates beyond toxicity.

Sarcophaga (Liosarcophaga) tibialis Macquart 1851 (Diptera: Sarcophagidae): micromorphology of preimaginal stages of a fly of medical and veterinary interest.

Sarcophagids are a large family of Diptera, with a worldwide distribution. They are related to decomposing organic matter and are very interesting for health science and in forensic cases since many species produce myiasis and occur in human corpses. This family is considered difficult to study, particularly with regard to their immature stages, to which little attention has been paid. Genus Sarcophaga Meigen, 1826 is composed of species of very similar morphology, making very difficult to distinguish. Knowledge of the immature stages of this genus is important because such stages occupy the greater part of the life cycle, so that establishing a basis for their identification will increase their usefulness in systematic and applied sciences. This contribution presents a detailed study of the morphological features, both external and internal, of the preimaginal stages of Sarcophaga (Liosarcophaga) tibialis Macquart, 1851, providing a taxonomical context for the correct identification of Liosarcophaga species of forensic interest in the Iberian Peninsula. Both light and scanning electron microscopy were applied. Complete descriptions of every stage are provided and illustrated, and their usefulness for species comparison, taking into account our uneven knowledge of morphologically immature stages of this subgenus, is indicated. Features of the cephalopharyngeal skeleton, such as the shape of the mouth hook and the intermediate and basal sclerites, and external morphology, such as the pattern of spinose band and anterior and posterior spiracles, proved useful for separating species. Finally, tentative identification keys based on light microscopy observation to distinguish S. (L.) tibialis from other species of forensic interest belonging to Liosarcophaga subgenus are proposed for every immature stage.

Micromorphology of immature stages of Sarcophaga (Liopygia) cultellata Pandellé, 1896 (Diptera: Sarcophagidae), a forensically important fly.

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.

Histological effects and localization of dissolved microcystins LR and LW in the mayfly Ecdyonurus angelieri Thomas (Insecta, Ephemeroptera).

The ability of microcystins (MCs), the main group of cyanotoxins, to affect the physiological processes and tissues of insects has received little attention. Fresh water dissolved MCs represent one of the main sources of cyanotoxins. In the experiment described herein, captured wild mayfly Ecdyonurus angelieri Thomas, 1968 larvae were exposed to 5 ppb of two distinct microcystins, MC-LR and MC-LW, in separate assays. Evidence of induced mortality, MCs bioaccumulation and severe histological damage affecting fat body and alterations in the tracheal system were evident. Our results reveal the acute sensitivity of the mayfly E. angelieri to MCS, which may serve as early indicators or cyanotoxins production and the quality of freshwater streams.

The first report of Telomerina flavipes (Meigen, 1830) (Diptera, Sphaeroceridae) in a forensic case, with redescription of its pupa.

This paper presents a forensic investigation that took place in the city of Murcia (SE Spain) and shows how the entomological specimens collected at the scene were extremely helpful for estimating the minimum post-mortem interval (PMImin). The occurrence of Telomerina flavipes (Meigen, 1830) (Diptera: Sphaeroceridae) is reported here for the first time in a forensic case. Additionally, the importance of other entomological evidence in this case is discussed. The first known images of the puparium are provided, as well as its redescription and that of the cephalopharyngeal skeleton recovered from the puparium.

Are the evidences of forensic entomology preserved in ethanol suitable for SEM studies?

In forensic practice, the use of arthropod evidences to estimate the postmortem interval is a very good approach when the elapsed time from death is long, but it requires the correct identification of the specimens. This is a crucial step, not always easy to achieve, in particular when dealing with immature specimens. In this case, scanning electronic microscopy (SEM) can be useful, but the techniques used to preserve specimens in forensic practice are usually different from those used to prepare specimens for SEM studies. To determine whether forensic evidences preserving techniques are also compatible with SEM analysis, we have compared specimens of all the immature stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae) preserved in 70% ethanol, with others prepared with aldehydic fixative techniques that are more appropriate for SEM studies. At the same time, two drying techniques have also been compared with both fixative techniques, the critical point drying and air-drying following with hexamethyldisilizane treatment (HMDS). Our results indicate that there are not basis against recommending the use of ethanol to preserve forensic entomological evidences and that both drying methods appear to offer good results for second and third instar larvae, although HMDS behaves better with eggs and pupae.

Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): a comparative study.

A comparative morphological study of preimaginal stages (larvae and pupae) of Calliphora vicina Robineau-Desvoidy, 1830 is presented. The entomological samples came from laboratory colonies bred under controlled environmental conditions (25°C and 60% relative humidity). In this study, a recently published technique to clear Diptera larvae for light microscopy and a standard protocol for scanning electron microscopy were used. For the morphological comparison of larval instars I, II and III, and pupae of C. vicina, different larval regions (cephalic, thoracic and abdominal, including anal division), as well the internal chitinised cephalopharyngeal skeleton, were considered separately. Our results focus on showing the changes observed throughout development for the most important structures in the cephalic region (sensilla of maxillary palpus, antennae and oral ridges), the thoracic region (the first segment and its anterior spinose band) and in the anal division of the abdominal region (posterior spiracles and shape of the papillae). In addition, some morphological structures are described or pictured for the first time, such as the ventral organ and the anterior spiracle of larva I and the antenna sensilla, Keilin's organ and wrinkled area of the anal division of all instars. The cephalopharyngeal skeleton is an important structure for the taxonomy of Diptera larvae in all instars, including Calliphoridae. Our observations in C. vicina indicate that an in-depth review of the sclerite composition is needed. Pupae and larvae stages can only be compared by following the segmentary spinose bands and the anal segment, where the morphology of the posterior spiracles and papillae can be observed, in some cases despite the reduced condition of the latter.

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy.

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.