Reproductive behavior in the squid Sepioteuthis australis from South Australia: interactions on the spawning grounds

Squid behavior is synonymous with distinctive body patterns, postures, and movements that constitute a complex visual communication system. These communications are particularly obvious during reproduction. They are important for sexual selection and have been identified as a potential means of species differentiation. Here we present a detailed account of copulation, mating, and egg deposition behaviors from in situ observations of the squid Sepioteuthis australis from South Australia. We identified four mating types from 85 separate mating attempts: "Male-upturned mating" (64% of mating attempts); "Sneaker mating" (33%); "Male-parallel" (2%); and "Head-to-head" (1%). Intervals between successive egg deposition behaviors were clearly bimodal, with modes at 2.5 s and 70.0 s. Ninety-three percent of egg capsules contained 3 or 4 eggs (mean = 3.54), and each egg cluster contained between 218 and 1922 egg capsules (mean = 893.9). The reproductive behavior of S. australis from South Australia was different from that described for other cephalopod species. More importantly, comparison between these results and those for other populations of S. australis suggests that behavior may differ from one population to another.     

Relationship between benthic insects (Ephemeroptera, Plecoptera, Coleoptera, Trichoptera) and temperature in small and medium-sized streams in Germany: A multivariate study

Benthic insect communities (Ephemeroptera, Plecoptera, Coleoptera, Trichoptera) were studied together with water temperature and environmental parameters in streams between June 2000 and June 2001. The sampling area consisted of 20 sites in small and medium-sized streams located in the lower mountainous area of Central Europe. Temperature was recorded nearly continuously and several physicochemical and environmental variables were assessed. Macroinvertebrates were sampled both in spring and summer. Data-sets of species abundance and occurrence were analysed using multivariate techniques and were correlated to the thermal and environmental conditions of the streams. The temperature preferences of the species were compared to published data-sets on their autecological characteristics. Up to 29% of the variability in the Ephemeroptera, Plecoptera, Trichoptera and Coleoptera community was explained by summer temperature variation in the data-sets for both small and medium-sized streams. A smaller, but significant part of the variability in species distribution was explained by conductivity, substratum type, and the percent coverage of local riparian forest. Compared to small streams, temperature was less important for the macroinvertebrate composition in medium-sized streams. This result is likely due to the more tolerant, eurythermic species composition in larger streams. A total of 33 Ephemeroptera, Plecoptera, Coleoptera and Trichoptera taxa were positively correlated and 28 taxa were negatively correlated to summer temperature patterns. The temperature preferences of taxa considered in this study were related to species traits, such as egg dormancies and life cycle plasticity.     

Ultrastructural and histochemical studies of the egg capsules of Perla marginata (Panzer, 1799) and Dinocras cephalotes (Curtis, 1827) (Plecoptera : Perlidae)

Eggshells of stone flies P. marginata and D. cephalotes (Plecoptera : Perlidae), inhabiting mountain streams, were examined using scanning and transmission electron microscopes, a phase-contrast light microscope and histochemical methods to detect proteins, lipids and polysaccharides.The eggshells of the species investigated consist of a vitelline envelope, chorion and gelatinous sheet decorated on its outer surface with mushroom-like structures. An anchoring structure (attachment disc) is situated on the posterior pole of the egg. The structure and function of the attachment disc, as well as the possible taxonomic applications, are discussed. The morphology and histochemical composition of all these elements of the shell clearly demonstrate good adaptation to land and aquatic habitats; the chorion consists of 2 layers, the internal layer being finely perforated by numerous aeropyles. The external layer, with fewer, regularly placed aeropyles, protects the egg interior against dehydration in the land habitat. The gelatinous sheet seems to provide additional protection. Mushroom-like structures, situated on its surface, correspond with the positions of aeropylar openings. These and other interrelations between chorion structure and function are discussed.     

A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.     

Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses

Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga “Oophila amblystomatis” (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the ‘Oophila’ clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae.     

Development and ultrastructure of the thickened serosa and serosal cuticle formed beneath the embryo in the stonefly Scopura montana Maruyama, 1987 (Insecta,Plecoptera, Scopuridae)

We aimed to describe the development and ultrastructure of the thickened serosa and serosal cuticle formed beneath the embryo of Plecoptera, using Scopura montana of Scopuridae as a euholognathan representative. Using transmission electron microscopy, we found that the egg membranes were composed of a thick exochorion, a thicker endochorion consisting of two sublayers, and an extremely thin vitelline membrane. The egg membrane construction represents a groundplan feature of the euholognathan egg membranes. The serosa converges beneath the embryo to form a thickened serosa, comprising cells in a radial arrangement, in association with the formation of the amnioserosal fold. The thickened serosa then deposits the thickened serosal cuticle, consisting of four layers differing in fine structure and electron density. After achieving its secretory function, the thickened serosa then disintegrates, and the liberated serosal cells float for a short period in the peripheral region of the egg inside. Collectively, our findings should provide the basis for further characterization of the serosal structures concerned, but we were unable to corroborate previous studies assigning the thickened serosa and serosal cuticle in Plecoptera to the water absorption function.     

Chemical defense in the egg masses of benthic invertebrates: an assessment of antibacterial activity in 39 mollusks and 4 polychaetes.

Many marine invertebrates deposit benthic egg masses that are potentially vulnerable to microbial infection. To help counter this threat these species may have evolved some form of chemical protection for their encapsulated embryos. In this study the egg masses from 7 marine mollusks were tested for antibacterial activity against 4 marine pathogens: Enterococcus sericolicida, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio harveyi. Extracts from all of these egg masses were found to inhibit the growth of at least 1 marine bacterium at concentrations that approximate the natural concentration of extract in the egg masses. The egg masses of 39 mollusks and 4 polychaetes were then tested for antibacterial activity against 3 human pathogenic bacteria; Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Activity was detected in the egg masses from 34 species, including 2 polychaetes and mollusks from two classes and 18 families. Antibacterial activity in molluskan egg masses was found to extend across the marine, estuarine, freshwater, and terrestrial environments. Both gelatinous egg masses and tough egg capsules were found to inhibit microbial growth, suggesting that physical protection alone may not be sufficient to protect the eggs. Antimicrobial activity was observed in the fresh egg masses but not in the well-developed egg masses of a subset of species. The results of this study indicate that a wide range of invertebrates use chemical defense to protect their early stage embryos against bacterial infection.     

Perlesta ephelida, a new Nearctic stonefly species (Plecoptera, Perlidae)

A new Nearctic species of Perlidae (Insecta, Plecoptera), Perlesta ephelidasp. n., is described from the male, female, and egg stages. This species has been previously reported as, or confused with, Perlesta shubuta Stark from several central and eastern U.S. states. Perlesta ephelida is distinctive from Perlesta shubuta and other regional Nearctic congeners mainly according to male genitalic and egg characteristics. Perlesta ephelida is a widely-distributed eastern Nearctic species, whereas Perlesta shubuta appears to be restricted to a narrow latitudinal belt in the Gulf Coast region from Louisiana east conservatively to the Florida panhandle. The egg of Perlesta shubuta is depicted with scanning electron microscopy for the first time.     

Fine structure of reproductive glands in two primitive marine pulmonates (Basommatophora: Siphonariidae)

Within the clade Euthyneura the marine basommatophorans are particularly neglected. More morphological and molecular studies are needed because their phylogenetic relationships with other pulmonates remain unresolved. The present study examines the most conspicuous reproductive gland, the glandular complex in two marine limpets, Siphonaria capensis and S. serrata (Pulmonata: Basommatophora) at both gross and fine structural levels. These two sympatric species with different developmental modes were selected to compare the structure and function of this enormous glandular structure. In both S. capensis and S. serrata, the glandular complex shows an undifferentiated state composed of an acidophilic albumen gland and a basophilic mucous gland. The glands contain secretory cells and supporting cells (= ciliated cells) that are highly ciliated. When the histochemical properties of the glandular complex were compared with those of siphonariid egg masses (of each species) it could be established that the albumen gland was responsible for the production of perivitelline fluid whereas the mucous gland secreted substances that help in the assembly of mucous layers surrounding the egg capsules. We suggest that the presence of a single glandular complex comprised of two glands is the most primitive organization of reproductive glands in pulmonates. Furthermore, the histology, fine structure and histochemistry of these glands are very similar to those of the reproductive glands of opisthobranchs.     

A revision of the Oriental stonefly genus Phanoperla (Plecoptera: Perlidae)

The genus Phanoperla (=Dyaperla Banks, 1939) (Plecoptera: Perlidae: Perlini) is revised and generic diagnoses are provided for adults and larvae. Diagnostic and constitutive characters of the tribe Neoperlini are discussed, and Chinoperla Zwick, 1980, is shown to be the closest relative of Phanoperla. Many past misidentifications of Phanoperla species have been corrected by the use of characters recently recognized as important, namely the structure of the internal genitalia of male and female specimens and particularly the complex pattern of spines on the male penial sac made visible by eversion of this structure, and details of sculpturing of the egg chorion.
The following species of Phanoperla are recognized: amorpha sp.n.; anomala (Banks, 1939); bakeri (Banks, 1924); ceylonica Kawai, 1975; comuta sp.n.; flaveola (Klapálek, 1921), comb.n. ( =clarissa (Banks, 1913), syn.n.), (=Neoperla hageni Banks, 1920, syn.n.), (= N.consimilis Banks, 1924, syn.n.); guttata sp.n.; himalayana Zwick, 1977 (= siwalika Harper, 1977); limosa (Hagen, 1858); maculata sp.n.; maindroni (Navas, 1926), comb.n.; malayana sp.n.; minutissima (Enderlein, 1909); nana sp.n.; nervosa Banks, 1939; nuwara Kawai, 1975; omega sp.n.; pallipennis (Banks, 1938); parva sp.n.; pumilio (Klapálek, 1921), comb.n.; peniculus Kawai, 1969a; schmidi sp.n.; sertispina Jewett, 1975; srilanka sp.n.; sumatrae sp.n.; testacea (Hagen, 1858); wedda sp.n. P.claggi (Banks, 1938) is a nomen nudum.
All species are (re-)described and figured; all primary types have been examined. A key to species is provided. Most species can be assigned to one of seven species groups which are defined.
Phanoperla is endemic to the Oriental Region. Species groups are generally widespread, but individual species are in most cases known only from restricted areas.     

Comparative spermatology in Plecoptera (Insecta): an ultrastructural investigation on four species

The ultrastructure of spermatozoa of four Plecoptera species, Leuctra fusca Linnaeus, Brachyptera risi (Morton), Taeniopteryx stankovitchi Ikonomov, T. kuehtreiberi (Aubert), was investigated by light microscopy, scanning and transmission electron microscopy, and immunofluorescence microscopy. The spermatozoa of all the species have a complex acrosome, are filiform and flagellate, with a '9+9+2' axoneme flanked by two mitochondrial derivatives. However, the structure shows a certain interspecific heterogeneity. L. fusca has a short conical nucleus in the apical position and an axoneme flanked by two accessory bodies. In the sperm of the Taeniopterygidae, the nucleus is a long cylindrical body which lies between the two mitochondrial derivatives flanking the axoneme for most of its length. Our results suggest a close phylogenetic affinity between Plecoptera and the other orders of Polyneoptera, although the proposed sister-group relationship between Plecoptera and Embioptera cannot be confirmed.     

The aquatic insects of a standard small plain river in NE France,with emphasis on remarkable species

A five-year macroinvertebrate study was conducted on a 55 km river (le Rupt-de-Mad, Lorraine region, north-eastern France), a standard for the region. A list of 300 species was drawn up, and remarkable species were listed for some better known orders: Ephemeroptera, Plecoptera, Trichoptera and Odonata. Some faunistic results are emphasised: about 42% of the identified species were more or less ubiquitous, 26% were meso- to polysaprobic species of potamon, present only in the main course of the river, while 31% were rather stenoecious species restricted to certain tributaries. Fifty-one remarkable species were listed, taking into account their regional status, according to IUCN categories: more than three quarters were hosted in the small tributaries, and 55% found exclusively in these latter (versus 23.5% only present in the main course of the river). Calcareous lotic tributaries were hosting particularly original communities with many remarkable species. Ephemeroptera, Plecoptera and Trichoptera were pertinent groups to assess the global faunistic interest of lotic habitats, but lentic habitats are probably better evaluated using other groups, e.g. Odonata and Coleoptera; the latter unfortunately poorly known from an ecological point of view.     

Long-term effects of prey-availability, partnering and temperature on overall egg capsule output of 'New Zealand flatworms', Arthurdendyus triangulates

Arthurdendyus triangulatus is an invasive terrestrial flatworm that preys on earthworms. To assess A. triangulatus egg capsule production, flatworms were maintained in ventilated polypropylene tubs (7.5 L) kept in controlled environment (CE) chambers or outdoors in the ground. Controlled environment chambers were maintained at 8°C or 14°C, flatworms kept singly or paired within tubs and offered Eisenia fetida according to a weight equalling one‐eighth or one‐half of the mean flatworm weight, or left unfed. The tubs were a successful method for keeping flatworms, with some surviving for over one year. The greatest number of egg capsules produced by an individual A. triangulatus was nine over a 16 week period for a flatworm kept at 14°C and fed at the one‐half regime (0.56 egg capsules flatworm^‐1 week^‐1). Although the effects of treatments varied with CE chambers, there was some evidence from flatworms kept outdoors, that feeding affected egg capsule output, with those flatworms fed at the one‐half regime tending to produce more egg capsules (P= 0.057). Flatworms at the one‐eighth regime or that were unfed produced progressively lighter egg capsules and substantially declined in weight themselves. Nevertheless, even unfed flatworms continued to produce egg capsules for 18 weeks. The lightest egg capsule weighed 8 mg, whilst the heaviest was 180 mg. In the CE chambers at 14°C, there was evidence for two different reproductive/survival strategies. Some flatworms produced cumulatively more egg capsules the longer they survived, whereas others lived longer but produced fewer egg capsules. Flatworms kept without a partner still produced egg capsules up to 35 weeks later. Egg capsules contained a mean of 4.14 (CE chambers) or 4.62 (outdoors) juveniles, with a maximum of 11. Overall, juveniles were 45% of the weight of egg capsules, although larger egg capsules had more juveniles, which comprised a greater proportion of the egg capsule. The conversion of earthworm prey to egg capsule production was estimated at 13%.     

Free fatty acids and sterols in the benthic spawn of aquatic molluscs, and their associated antimicrobial properties

The free lipid content of extracts from the spawn of 17 molluscs were analysed by gas chromatography/mass spectrometry. These extracts encompass the encapsulated embryos and extraembryonic structures from benthic gelatinous egg masses and leathery egg capsules covering five taxonomic groups. Palmitic and stearic acids were the dominant saturated fatty acids and oleic acid was the principal unsaturated acid found in the spawn. Cholesterol was the dominant sterol and the only sterol found in the spawn from every species. Extracts from gelatinous egg masses were found to contain proportionally more fatty acids compared to leathery egg capsules. No unsaturated fatty acids were found in any of the leathery egg capsules, including five neogastropods and one littorinimorph. Unsaturated fatty acids were present in all of the gelatinous egg masses, including two other littorinimorphs. This is the first study to demonstrate that unsaturated fatty acids possess significant bacteriolytic activity against four aquatic pathogens. Encapsulated Anaspidea egg masses contain relatively high concentrations of these unsaturated fatty acids and a lipid mixture modeled on these extracts was strongly bacteriolytic at concentrations down to 0.0001 mg/ml. By comparison, lipid mixtures modeled on extracts from the spawn of four other molluscan taxa with higher proportions of saturated fatty acid and cholesterol, were only partially active against some of the bacteria at 0.1 mg/ml. Thus, unsaturated fatty acids could explain the antimicrobial activity previously reported in lipid extracts of some, but not most, molluscan spawn. MDS ordination and ANOSIM revealed significant taxonomic differences in the composition of free lipids from molluscan spawn, suggesting that lipid analyses may be useful in future systematic studies of the Mollusca.     

The temperature of two welsh lakes and its effect on the distribution of two freshwater insects

Temperature data are presented from the littoral zones of two lakes in North Wales, Llyn Coron and Llyn Dinas. Recording, mainly on a continuous basis, was over a two year period. Despite their different situations, the lakes had similar temperature regimes. Regional weather factors were of greater importance than local variations. However, some differences were present, especially during the summer months. Laboratory experiments were conducted to determine the temperature relationships of Nemoura avicularis Morton (Plecoptera) and Leptophlebia vespertina (L.) (Ephemeroptera), common species in Llyn Dinas but absent from Llyn Coron. Aspects considered included nymphal temperature tolerance and the effect of temperature on egg development and emergence. From the background of the results, it was concluded that the differences in temperature regime between the two lakes were insufficient to explain the absence of the two species from Llyn Coron.     

A trait-based approach to assess the vulnerability of European aquatic insects to climate change

Aquatic insects are the dominant taxon group in most freshwater ecosystems. As temperature is the main driver of their life cycle development, metabolic activity, and geographic distribution, these macroinvertebrates are particularly suitable for large scale and comparative studies of freshwater community responses to climate change. A dataset of bio-ecological traits of 1,942 Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa was used to analyze (1) the relationships among traits, (2) the potential vulnerability of EPT species to climate change, and (3) the geographical occurrence patterns of these potentially endangered species at the scale of European ecoregions. By means of a fuzzy correspondence analysis (FCA), two gradients emerged: (1) a longitudinal gradient, describing successive upstream–downstream features, and (2) a biogeographical gradient, separating endemic and micro-endemic species from widely distributed taxa. Moreover, aquatic insects of southern European ecoregions emerged as those most endangered in terms of potential vulnerability to climate change. Comparative multi-taxon studies provide important new insights into freshwater ecosystem functioning and responses to climate change, and could be the first step toward developing integrative monitoring or assessment tools (e.g., trait-based indicators at the species level) by means of non-arbitrary statistical methods.     

The invertebrate fauna of the streams in Øvre Heimdalen

The lotic communities in Øvre Heimdalen were completely dominated by insects. The species distribution of stoneflies (Plecoptera), mayflies (Ephemeroptera) and caddisflies (Trichoptera) is given together with autecological notes on the Plecoptera and certain other major species. The species distribution of these groups, especially the Plecoptera, was clearly related to the deciduous vegetation along the streams. There was a reduction in species parallel to a reduction in Salix vegetation.
The main inlet stream to the lake, Øvre Heimdalsvatn, the outlet and the outlet stream had different faunal compositions. The inlet fauna consisted to a large extent of winter growing species, mainly detritus feeders, while the outlet and outlet stream had a fauna dominated by summer growing species, several of which were filter feeders.     

Transmission of nephridial bacteria of the earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

First records of advanced embryos and egg capsules of Bathyraja skates from the deep north-eastern Atlantic

Five variously developed embryos [142–279 mm total length (L_T)] from egg capsules from the Porcupine Seabight, north-eastern Atlantic (1541 m depth) are used to establish the ontogeny and early life history of Bathyraja richardsoni. The capsules of this species and two half-formed ones from the shell glands of a large (1620 mm L_T) female Bathyraja pallida taken off Ireland (c. 1900 m depth) are described and illustrated. The varying degree of yolk sac absorption found in the B. richardsoni embryos is discussed in relation to hatching size and its seeming size independence over c. 20–50 mm within the embryos' total length range. The conservative variation in external morphology with development from advanced embryos to adults among such deep stenobathic Bathyraja skates is commented upon, as is the bathymetric segregation of adults from levels in which egg capsules are deposited and young develop.