Epidemic mortality of the sponge Ircinia variabilis (Schmidt, 1862) associated to proliferation of a Vibrio bacterium.

In recent years, several episodes of mass mortality of sessile epibenthic invertebrates, including sponges, have been recorded worldwide. In the present study, we report a disease event on Ircinia variabilis recorded in September 2009 along the southern Adriatic and Ionian seas (Apulian coast), with the aim to quantify the mortality incidence on the sponge population, to investigate the effect of the disease on the sponge tissues and to assess whether the disease is associated with vibrios proliferation. The injured sponges showed wide necrotic areas on the surface or disruption of the body in several portions. Necrotic areas were whitish and often were covered with a thin mucous coat formed by bacteria. In the most affected specimens, sponge organisation resulted partial or complete loss, with the final exposure of the dense skeletal network of spongine fibres to the environment. The results of microbiological cultural analysis using in parallel Marine Agar 2216 and thiosulphate/citrate/bile salts/sucrose agar demonstrated that, in affected specimens, vibrios represented 15.8 % of the total I. variabilis surface culturable bacteria. Moreover, all the isolated vibrios, grown from the wide whitish areas that characterize the surface of the diseased sponges, were identified, and their assignment to the Vibrio rotiferianus was consistent with phylogenetic analysis and data of morphological, cultural and biochemical tests. Studies on V. rotiferianus have shown that its pathogenicity, with respect to various aquatic organisms, is higher than that of Vibrio harveyi. The factors triggering the disease outbreak in Ircinia variabilis populations remain unclear. At present, we can hypothesize the involvement in the disease of a synergetic mechanism that, under stressful physiological conditions (high temperature, elevated nutrients and reduced water flow), induces sponge pathogens, in our case V. rotiferanius, to become virulent, making sponges unable to control their proliferation. Additional studies are needed to understand the etiological processes as well as the factors involved in sponges recovering from this epidemic event allowing them to face mass mortality. A drastic reduction of sponge-specific representatives could have marked a negative impact on the environmental health on account of their role in the sea remediation processes as filter-feeding organisms.

Collagenic architecture and morphotraits in a marine basal metazoan as a model for bioinspired applied research.

In some Porifera (Demospongiae: Keratosa), prototypes of the connective system are almost exclusively based on collagenic networks. We studied the topographic distribution, spatial layout, microtraits, and/or morphogenesis of these collagenic structures in Ircinia retidermata (Dictyoceratida: Irciniidae). Analyses were carried out on a clonal strain from sustainable experimental mariculture by using light and scanning electron microscopy. Histology revealed new insights on the widely diversified and complex hierarchical assemblage of collagenic structures. Key evolutionary novelties in the organization of sponge connective system were found out. The aquiferous canals are shaped as corrugate-like pipelines conferring plasticity to the water circulation system. Compact clusters of elongated cells are putatively involved in a nutrient transferring system. Knob-ended filaments are characterized by a banding pattern and micro-components. Ectosome and outer endosome districts are the active fibrogenetic areas, where exogenous material constitutes an axial condensation nucleus for the ensuing morphogenesis. The new data can be useful to understand not only the evolutionary novelties occurring in the target taxon but also the morpho-functional significance of its adaptive collagenic anatomical traits. In addition, data may give insights on both marine collagen sustainable applied researches along with evolutionary and phylogenetic analyses, thus highlighting sponges as a key renewable source for inspired biomaterials. Therefore, we also promote bioresources sustainable exploitation with the aim to provide new donors of marine collagen, thereby supporting conservation of wild populations/species.

Hygroreceptors in the larva of Libellula depressa (Odonata: Libellulidae).

Ultrastructural and electrophysiological (single-cell recordings) investigations were carried out on the coeloconic sensilla borne by the apical antenna of the larvae of Libellula depressa (Odonata: Libellulidae). These sensilla appear as pegs located in pits. One of them is a compound sensillum constituted of two fused pegs in a common pit and the other two are single pegs located in separated pits close to each other. Coeloconic sensilla show position and ultrastructural details very similar to those described in insect hygroreceptors. The electrophysiological recordings on the apical antennae of the last larval instar of L. depressa clearly show the presence of moist and dry cells responding antagonistically to humidity changes. This study gives the first evidence of hygroreceptors in dragonfly larvae and represents the first electrophysiological approach to larval sensilla of aquatic insects. The presence of hygroreceptors in L. depressa larvae is in agreement with the hygropositive response shown by these insects in laboratory and field behavioural experiments.

Development of long-term primary cell aggregates from Mediterranean octocorals.

In lower metazoans, the aggregative properties of dissociated cells leading to in vitro stable multicellular aggregates have furnished a remarkable experimental material to carry out investigations in various research fields. One of the main expectations is to find good models for the study in vitro of the first steps of biomineralization processes. In this study, we examined five common Mediterranean gorgonians (Paramuricea clavata, Corallium rubrum, Eunicella singularis, Eunicella cavolinii, and Eunicella verrucosa) using mechanical cell aggregate production techniques. In particular, we investigated the conditions of aggregate formation, their number and survival in experimental conditions, the DNA synthesizing activity using 5'-bromo-2'-deoxyuridine (BrdU) tests, and the response to calcein addition and observed the secretion of newly formed sclerites. The BrdU tests showed that cell proliferation depends on the size of aggregates and on the presence/absence of symbiotic zooxanthellae. With epifluorescent and confocal imaging from calcein addition assays, we observed the presence of calcium ions within cells, a possible clue for prediction of sclerite formation or calcium deposition. The species-specific efficiency in production of cell aggregates is correlated to the size of polyps, showing that the higher density of polyps and their diameter correspond to higher production of cell aggregates. Regarding the long-term maintenance, we obtained the best results from E. singularis, which formed multicellular aggregates of 0.245 mm ± 0.086 mm in size and maintained symbiotic association with zooxanthellae throughout the experimental run. Formation of sclerites within aggregates opens a wide field of investigation on biomineralization, since de novo sclerites were observed around 30 d after the beginning of the experiment.

Invertebrate diversity in relation to chemical pollution in an Umbrian stream system (Italy).

We used self-organizing maps (SOM, neural network) to bring out patterns of benthic macroinvertebrate diversity in relation to river pollution. Fourteen stations were sampled over various seasons in the Nestore drainage basin (Central Italy) and characterized for macroinvertebrate communities, nutrient and heavy metal concentrations. Physicochemical variables were introduced into a SOM previously trained with macroinvertebrate data. Patterns of communities matched spatial and seasonal changes in environmental conditions, including water chemistry related to economic activities in the catchment. Although our analyses did not allow us to establish the specific effect of any given environmental parameter upon macroinvertebrate community composition based on the field study, they enabled us to map the ecological health of river ecosystems in a readily interpretable manner.

The caddisfly Ceraclea fulva and the freshwater sponge Ephydatia fluviatilis: a successful relationship.

The association between the aquatic phases of the caddisfly Ceraclea fulva (Trichoptera, Leptoceridae) and the freshwater sponge Ephydatia fluviatilis (Porifera, Spongillidae) has been investigated by means of scanning and transmission electron microscopy (SEM, TEM). Ceraclea fulva habitually feeds on sponges and builds its case by using the siliceous spicules of the sponge, which are arranged side by side, inter-crossed, cemented with silk, and organised in layers. In the newly hatched larva, the case is strengthened exclusively by cemented siliceous spicules, while during growth, the insect adds sponge fragments to it. The fine organisation of the sponge tissues growing on the case proves that the sponge is functional. Inter-spaced, small protrusions, derived from the outermost compact silk layer, form a series of "bridges" enhancing case/sponge adhesion. Tube-case shape varies according to the aquatic developmental phase of the insect: in the mature larva and pupa, this shelter carries larger sponge fragments dorsally. The caddisfly acts as carrier of the sponge, thus facilitating its dispersal and the colonisation of new habitats. This justifies regarding this association as a successful mutualistic relationship, and not as a unilateral parasitic behaviour on the part of the insect.

The epipharyngeal sensilla of the damselfly Ischnura elegans (Odonata, Coenagrionidae).

The knowledge on Odonata adult mouthparts sensilla is scanty and, notwithstanding the epipharynx in the labrum is considered an organ of taste, no ultrastructural investigation has been performed so far on this structure in Odonata. The labrum of the adult of the damselfly Ischnura elegans (Odonata, Coenagrionidae) shows on its ventral side the epipharynx with sensilla represented by articulated hairs and by small pegs located at the apex of slightly raised domes. Under scanning and transmission electron microscope, the articulated hairs, with a well developed socket and tubular body, have the typical structure of bristles, the most common type of insect mechanoreceptors, usually responding to direct touch; the pegs, showing an apical pore together with a variable number of sensory neurons (from two to five), the outer dendritic segments of which show a dendrite sheath stopping along their length, have features typical of contact chemoreceptors.

First evidence of the use of olfaction in Odonata behaviour.

Dragonflies and damselflies are among the most ancient winged insects. Adults belonging to this order are visually oriented and are considered anosmic on the basis of neuroanatomical investigations. As a consequence, the chemical ecology of these predatory insects has long been neglected. Morphological and electrophysiological data demonstrated that dragonfly antennae possess olfactory sensilla. Additionally, a neuroanatomical study revealed the presence of spherical knots in the aglomerular antennal lobe that could allow for the perception of odour. However, the biological role of the antennal olfactory sensilla remains unknown, and no bioassay showing the use of olfaction in Odonata has been performed thus far. Here, we demonstrate through behavioural assays that adults of Ischnura elegans are attracted by olfactory cues emitted by prey; furthermore, using electrophysiological single-cell recordings, we prove that the antennal olfactory sensilla of I. elegans respond to prey odour. Our results clearly demonstrate the involvement of antennal olfactory sensilla in Odonata predation, thus showing, for the first time, the use of olfaction in Odonata biology. This finding indicates that the nervous system of Odonata is able to receive and process olfactory information, suggesting that the simple organisation of the antennal lobe does not prevent the use of olfaction in insects.

The mechanoreceptors on the endophytic ovipositor of the dragonfly Aeshna cyanea (Odonata, Aeshnidae).

This study investigates the mechanoreceptors located on the cutting valvulae of the ovipositor of the dragonfly Aeshna cyanea (Aeshnidae), using both SEM and TEM, with the aim of providing an overview of the sensory equipment of an odonatan endophytic ovipositor. Four kinds of sensilla have been described. Notwithstanding their different external and internal morphology, they show features typical of mechanoreceptors. Three of them are evident along the external surface of the two cutting valvulae in the form of sub-spherical pegs, pit organs type 1 (holes) and pit organs type 2 (depressions), these last are similar to amphinematic scolopidia, while the fourth type is represented by subintegumental mononematic scolopidia having no direct relationship with the cuticle. In spite of their structural differences, the morphology of the described mechanoreceptors is consistent with performing a main role in allowing the perception of compression/stretching of the thick cuticle of the valvulae and their bending due to the pressure acting on the distal end of the ovipositor during substrate penetration. Such an organization is coherent with the need of endophytic Odonata to be able to evaluate the stiffness of the plant where to lay eggs.

The gustatory sensilla on the endophytic ovipositor of Odonata.

The present paper aims at describing the fine structure of coeloconic sensilla located on the cutting valves of the endophytic ovipositor of two Odonata species, the anisopteran Aeshna cyanea (Aeshnidae) and the zygopteran Ischnura elegans (Coenagrionidae), by carrying out parallel investigations under SEM and TEM. In both species these coeloconic sensilla are innervated by four unbranched neurons forming four outer dendritic segments enveloped by the dendrite sheath. One dendrite terminates at the base of the peg forming a well developed tubular body, while the other three enter the peg after interruption of the dendrite sheath. The cuticle of the peg shows an apical pore and a joint membrane. This last feature, together with the tubular body and the suspension fibers, represent the mechanosensory components of the sensillum while the pore and the dendrites entering the peg allow chemoreception. The ultrastructural organization of these coeloconic sensilla is in agreement with the one reported for insect gustatory sensilla. Our investigation describes for the first time typical insect gustatory sensilla in Odonata. Electrophysiological and behavioral studies are needed to verify the role that these structures can perform in sensing the egg-laying substrata.

The antennal lobe of Libellula depressa (Odonata, Libellulidae).

Here we describe the antennal lobe of Libellula depressa (Odonata, Libellulidae), identified on the basis of the projections of the afferent sensory neurons stemming from the antennal flagellum sensilla. Immunohistochemical neuropil staining as well as antennal backfills revealed sensory neuron terminal arborizations covering a large portion of the antennal lobe. No clear glomerular structure was identified, thus suggesting an aglomerular antennal lobe condition as previously reported in Palaeoptera. The terminal arbors of backfilled sensory neurons do, however, form spherical knots, probably representing the connections between the few afferent neurons and the antennal lobe interneurons. The reconstruction revealed that the proximal part of the antennal nerve is divided into two branches that innervate two spatially separated areas of the antennal lobe, an anterioventral lobe and a larger posteriodorsal lobe. Our data are consistent with the hypothesis that one tract of the antennal nerve of L. depressa contains olfactory sensory neurons projecting into one of the sublobes, while the other tract contains thermo-hygroreceptive neurons projecting into the other sublobe.

Life-cycle traits of Paraleucilla magna, a calcareous sponge invasive in a coastal Mediterranean Basin.

The calcareous sponge Paraleucilla magna, originally observed along the Brazilian coast (Atlantic Ocean), is the only allochthonous invasive species of Porifera reported in the Mediterranean Sea. A 1-year investigation of the population dynamics and life-cycle of this exotic species in the Mar Piccolo di Taranto (southern Italy, central Mediterranean Sea) has provided a good opportunity to test how environmental variations can influence its life-cycle and to ascertain what strategy can be adopted to successfully colonize a new environment. In the Mar Piccolo di Taranto, P. magna exhibits marked temporal changes in biomass. The studied specimens reproduce almost all year round, showing a seasonal pattern that peaks during warm months. This prolonged sexual activity allows P. magna to continuously produce young specimens, with repeated recruitment events taking place throughout the year, thus offsetting the seasonal mortality of adult specimens. This r-strategy enables the non-indigenous sponge to achieve a high degree of maintenance over relatively long periods (ten years at least).

Olfaction in dragonflies: electrophysiological evidence.

The problem of olfaction in Paleoptera (Odonata, Ephemeroptera) cannot be considered fully elucidated until now. These insects have been traditionally considered anosmic, because their brain lacks glomerular antennal lobes, typically involved in Neoptera odor perception. In order to understand if the presumed coeloconic olfactory receptors described on the antennal flagellum of adult Odonata are really functioning, we performed an electrophysiological investigation with electroantennogram (EAG) and single cell recordings (SCR), using Libellula depressa L. (Odonata, Libellulidae) as a model species. Odors representing different chemical classes such as (Z)-3-hexenyl acetate (acetate ester), (E)-2-hexenal, octanal (aldehydes), (Z)-3-hexen-1-ol (alcohol), propionic acid, butyric acid (carboxylic acids), and 1,4-diaminobutane (amine) were tested. Most of the tested chemicals elicited depolarizing EAG responses in both male and female antennae; SCR show unambiguously for the first time the presence of olfactory neurons in the antennae of L. depressa and strongly support the olfactory function of the coeloconic sensilla located on the antennal flagellum of this species. Electrophysiological activity may not necessarily indicate behavioral activity, and the biological role of olfactory responses in Odonata must be determined in behavioral bioassays. This study represents a starting point for further behavioral, electrophysiological, neuroanatomical and molecular investigation on Odonata olfaction, a research field particularly interesting owing to the basal position of Paleoptera, also for tracing evolutionary trends in insect olfaction.

The spermatodesm of Cloeon dipterum (L.): fine structure and sperm movement.

Sperm bundles of the ephemeropteran Cloeon dipterum (L.) were found in the seminal vesicle. They are apically protected by cyst cells and sperm anterior regions are embedded in a secretion produced by these cells. Cyst cells further degenerate making sperm cells free. The sperm bundles are able to perform a forward progression only when sperm are stick together with their tail posterior tips. On the contrary, when sperm have their tail ends free, they beat but are unable to progress. The functional significance of the sperm bundles is discussed.

Electrophysiological identification of thermo- and hygro-sensitive receptor neurons on the antennae of the dragonfly Libellula depressa.

Recent ultrastructural investigations on Odonata antennal flagellum describe two types of sensilla styloconica, T1 and T2. The styloconic sensilla are located in pits, at the bottom of deep cavities, and share common features typical of thermo-hygroreceptors. In order to ascertain if the Odonata antennae are involved in hygroreception and thermoreception, we carried out electrophysiological recordings (single cell recordings, SCR) from adult males and females of Libellula depressa L., 1758. After contact was established, the antenna was stimulated by rapid changes in temperature and humidity. The present research shows the occurrence of a dry (DC), a moist (MC) and a cold (CC) receptor neurons on the antennal flagellum of L. depressa. These data demonstrate for the first time the presence of functional thermo-hygroreceptors on the antennal flagellum of dragonflies. The present results extend our knowledge of the not visual sensory modalities of Odonata, a field of research unexplored so far.

The antennal sensilla of adult mayflies: Rhithrogena semicolorata as a case study.

The present paper reports on an ultrastructural investigation of the sensilla on the antennal flagellum of the imago (SEM and TEM) and subimago (SEM) of the mayfly Rhithrogena semicolorata. Sensilla coeloconica of two different sizes, named big and small pegs, are distinguishable in both stages. The big pegs are lobe-shaped with a porous cuticle. The small pegs are more or less conical with a smooth cuticle. TEM seriate sections revealed that the big pegs are innervated by three neurons, which enter the peg. The dendrite sheath opens at the entrance to the peg and leaves the dendrites in contact with the outside through pores and pore tubules. This structure is in agreement with that reported for single-walled olfactory receptors. The small pegs may be innervated by two or four neurons. In both cases, two unbranched dendrites enter the peg and are in close association with the peg wall. These show features typical of hygroreceptors. In the small pegs innervated by four neurons, the other two neurons stop short of entering the peg, and one of them branches. This last could constitute the thermoreceptive unit. The function of the fourth neuron is unknown.The presence of olfactory sensilla in adult mayflies, such as Paleoptera, is relevant to the study of evolutionary trends in insect odour perception. Hygro-thermoreceptors are important in such fragile aquatic insects, which need a moist environment for moulting and reproduction.

The antennal sensilla of the adult of Libellula depressa (Odonata: Libellulidae).

An ultrastructural investigation (SEM, TEM) on the antennal flagellum of the adult of the dragonfly Libellula depressa (Odonata:Libellulidae) revealed sensilla located in pits on the lateral-ventral side of the antenna. These sensilla are represented by sensilla coeloconica and by deeply sunken sensilla. The sensilla coeloconica are innervated by three unbranched dendrites, which enter the peg and show a dendrite sheath ending at the base of the peg. The peg has no socket and its cuticle is irregular with wide pore-like structures at the base of which actual pores are visible. The structure of these coeloconic sensilla is in agreement with that reported for single-walled insect chemoreceptors. The deeply sunken sensilla are represented by two kinds of sensilla styloconica, named type-1 and type-2, located at the bottom of deep cavities appearing as simple openings on the antennal surface. These sensilla are no-pore sensilla with inflexible socket and unbranched dendrites and, notwithstanding their structural differences, share common features typical of thermo/hygroreceptors. The presence of chemoreceptors in adult dragonflies sheds light on evolutionary trends in insect perception; the previously unknown occurrence of thermo/hygroreceptors in dragonflies is very important in view of the reported ability of Odonata to thermoregulate heliothermically.

The vacuolar cells of Oscarella lobularis (Porifera, Demospongiae): Ulatrastructural organization, origin, and function.

This work is concerned with the ultrastructural organization and some histochemical features of the vacuolar cells of the demosponge Oscarella lobularis. Vacuolar cells are characterized by large clear vacuoles containing an aqueous fluid. They are commonly found in the mesohyl of this sponge and tend to constitute a sort of parenchyma in the choanosome. Mobile cells of the mesohyl appear to differetiate into vacuolar cells through the progressive formation of wide cytoplasmic lacunae. We have identified four types of cells showing progressive transformation toward the vacuolar cell type. Precursors (types 1-4) of the vacuolar cells probably derive from endopinacocytes, since they share several histochemical and ultrastructural characteristics with them. Our data support the notion that vacuolar cells are involved in the synthesis of collagen, act as a mechanical support of the sponge body, and are eventually extruded from the sponge through the canals of the aquiferous system.