Morphological and physiological development of anterior thoracic stretch receptors in two isopods, <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> and <Emphasis Type="Italic">Ligia exotica</Emphasis>

Abdominal muscle receptor organs (MROs) monitor the position and movement of abdomen in crustaceans. Thoracic segments of decapods are fused and immovable. It is speculated that MROs had retrograded simple shape, N-cells that lost receptor muscles, a receptor cell and accessory nerves. We focused on the effect of segmental movement in respect to thoracic N-cells and MROs in isopods that have movable thoracic segments. Armadillidium vulgare rolled up its body segments. Ligia exotica swam by quick movement of the posterior thoracic segments. Both isopods possessed N-cells and MROs in the thorax. N-cells were a simple structure, but N-cells from the second and third thoracic segments of A. vulgare had a muscle strand. MROs^T3–T4 (from the third and fourth thoracic segments) of A. vulgare had two receptor muscles. MROs^T3–T4 of L. exotica had one long receptor muscle. N-cells of both species and MROs of A. vulgare showed slowly adapting stretch-activated discharges. MROs of L. exotica showed both slowly and rapidly adapting discharges. The stretch-activated responses of N-cells and MROs inhibited each other. N-cells or MROs in the thorax of isopods are not related to the segmental structure. The morphology and physiology of N-cells and MROs are specialized to species–specific behaviors.     

Calcium translocations during the moulting cycle of the semiterrestrial isopod <Emphasis Type="Italic">Ligia hawaiiensis</Emphasis> (Oniscidea,Crustacea)

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO₃ deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. ⁴⁵Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.     

Two new species of <Emphasis Type="Italic">Ligia</Emphasis> Fabricius, 1798 (Crustacea: Isopoda: Ligiidae) from coasts of the Persian and Aden gulfs

Two new species of Ligia are described, L. persica sp. nov. from the Persian Gulf and L. yemenica sp. nov. from the Gulf of Aden. Ligia persica occurs along the northern coasts of the Persian Gulf and around some Iranian islands such as Qeshm and Kish. A comparison of SEM micrographs shows that the shape and ornamentation of distal parts of the appendix masculina are reliable characters for the identification of morphologically similar Ligia species. They are species-specific and of great importance in the taxonomy of the genus.     

Phylogenetic Analysis and Fluorescence <Emphasis Type="Italic">In Situ</Emphasis> Hybridization Detection of Archaeal and Bacterial Endosymbionts in the Anaerobic Ciliate <Emphasis Type="Italic">Trimyema Compressum</Emphasis>

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 μg ml⁻¹), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host’s fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.     

Gut symbiotic bacteria in the cabbage bugs <Emphasis Type="Italic">Eurydema rugosa</Emphasis> and <Emphasis Type="Italic">Eurydema dominulus</Emphasis> (Heteroptera: Pentatomidae)

The cabbage bugs Eurydema rugosa Motschulsky and Eurydema dominulus (Scopoli) (Heteroptera: Pentatomidae: Strachiini) possess a number of crypts in a posterior region of the midgut, which are filled with bacterial symbiont cells. Here we characterized the gut symbionts of Eurydema stinkbugs using molecular phylogenetic and histological techniques. Specific gammaproteobacteria were consistently identified from the posterior midgut of E. rugosa representing nine populations and E. dominulus representing six populations, respectively. The bacterial 16S rRNA gene sequences were identical within the species but slightly different (98.2% sequence identity) between the species. Molecular phylogenetic analysis revealed that the Eurydema symbionts formed a well-defined monophyletic group in the Gammaproteobacteria. The symbionts were phylogenetically distinct from the gut symbionts of the stinkbug families Acanthosomatidae, Plataspidae, Parastrachiidae, Scutelleridae, and other pentatomid species, suggesting multiple evolutionary origins of the gut symbiotic bacteria among diverse stinkbugs. In situ hybridization confirmed that the symbiont is located in the cavity of the midgut crypts. Aposymbiotic insects of E. rugosa, which were produced by egg surface sterilization, were viable but suffered retarded growth, reduced body weight, and abnormal body color, suggesting the biological importance of the symbiont for the host.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Replication and Host Cell Division in <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachias replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.     

Evolutionary history of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background  

Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods. Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and among host species are not well understood. We sequenced a portion of the wsp gene from the Wolbachia genome infecting 138 individuals from eleven geographically distributed native populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals to infer the transmission patterns of Wolbachia in S. invicta.     

Control of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> and <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> in cucumber by <Emphasis Type="Italic">Amblyseius swirskii</Emphasis>

Bemisia tabaci Genn. (Hemiptera: Aleyrodidae) and Frankliniella occidentalis (Thysanoptera: Thripidae) are major pests in greenhouse grown cucumber crops. Recently, Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) was shown an effective biological control agent of both pests. Hence, perhaps both pests can be controlled simultaneously by this predator. However, with simultaneous infestation of both pests, synergistic effects, or interference could affect biological control and perhaps require changes in release rates of the predator. Thus, the aim of the present study was to evaluate different release rates of A. swirskii to control both pests under a worst case scenario of rapid immigration into a cucumber greenhouse. Two experiments were conducted, one simulating the influx of whiteflies alone (whitefly experiment) and the other immigration of whiteflies and thrips together (whitefly plus thrips experiment). Three treatments were compared in the whitefly experiment: (1) B. tabaci alone, (2) B. tabaci + 25 A. swirskii m⁻² and (3) B. tabaci + 75 A. swirskii m⁻². The high release rate was more effective than the low rate in controlling B. tabaci alone. The high rate was subsequently tested against B. tabaci and F. occidentalis for the whitefly and thrips experiment in which five treatments were compared: (1) B. tabaci alone, (2) F. occidentalis alone, (3) B. tabaci + 75 A. swirskii m⁻², (4) F. occidentalis + 75 A. swirskii m⁻² and (5) B. tabaci + F. occidentalis + 75 A. swirskii m⁻². This rate of A. swirskii controlled whiteflies and thrips either alone or together. Therefore, 75 A. swirskii m⁻² should be an adequate rate for controlling both pests either alone or simultaneously in cucumber greenhouses.     

Detection of <Emphasis Type="Italic">Rickettsia</Emphasis> and a Novel <Emphasis Type="Italic">Haemaphysalis shimoga</Emphasis> Symbiont Bacterium in Ticks in Thailand

In this study, we identified two Haemaphysalis species present at the Khao Yai National Park in Thailand and investigated the presence of rickettsia in these ticks. A total of 166 Haemaphysalis specimens were collected randomly under leaves along visitor paths at five locations in the park. Male and female adults of two different Haemaphysalis species, H. shimoga and H. lagrangei, were identified. Polymerase chain reaction (PCR) analysis revealed Rickettsia bacteria in these two Haemaphysalis species; this study represents the first time such presence has been reported in Thailand. The infection rates of Rickettsia were in both H. shimoga (7.41%) and H. lagrangei (10.17%) at these locations in addition to two pools of Haemahysalis nymphs (28.57%). Furthermore, 25.93% of H. shimoga showed positive results that matched Haemaphysalis longicornis symbionts (92% sequence identity) and the Coxeilla burnetti 16S ribosomal RNA gene (90% sequence identity). We propose that this is a novel H. shimoga symbiont bacterium in Thailand and might be a novel Coxeilla-like agent or Coxeilla sp. found in H. shimoga. In contrast, we did not observe any Wolbachia bacteria, which also belong to the order Rickettsiales, in the same group of Haemaphysalis ticks. Furthermore, PCR was used to detect three other genera of bacteria, Anaplasma, Ehrlichia and Borrelia, none of which were identified in the Haemaphysalis ticks studied.     

Bacterial Uptake by the Marine Sponge <Emphasis Type="Italic">Aplysina aerophoba</Emphasis>

Sponges (Porifera) are filter feeders that take up microorganisms from seawater and digest them by phagocytosis. At the same time, many sponges are known to harbor massive consortia of symbiotic microorganisms, which are phylogenetically distinct from those in seawater, within the mesohyl matrix. In the present study, feeding experiments were performed to investigate whether phylogenetically different bacterial isolates, hereafter termed “food bacteria,” microbial seawater consortia, and sponge symbiont consortia are taken up and processed differently by the host sponge. Aplysina aerophoba retained high numbers of bacterial isolates and microbial seawater consortia with rates of up to 2.76 × 10⁶ bacteria (g sponge wet weight)^–1 h^–1, whereas the retention of sponge symbionts was lower by nearly two orders of magnitude [5.37 × 10⁴ bacteria (g sponge wet weight)⁻¹ h^–1]. In order to visualize the processing of a food bacterium within sponge tissues, the green fluorescent protein-labeled Vibrio strain MMW1, which had originally been isolated from A. aerophoba, was constructed. Incubation of this strain with A. aerophoba and subsequent visualization in tissue cryosections showed its presence in the choanocytes and/or endopinacocytes lining the canals but, unlike latex beads, not in deeper regions of the mesohyl, which suggests digestion of the bacteria upon contact with the host. Denaturing gradient gel electrophoresis (DGGE) was performed on the incubation seawater to monitor the changes in phylogenetic composition after incubation of the sponge with either seawater or sponge symbiont consortia. However, the DGGE experiment provided no evidence for selective processing of individual lineages by the host sponge. In conclusion, this study extends early studies by Wilkinson et al. (Proc R Soc London B 220:519–528, 1984) that sponges, here A. aerophoba, are able to differentiate between food bacteria and their own bacterial symbionts.     

Isolation of New <Emphasis Type="Italic">Symbiodinium</Emphasis> Strains from Tridacnid Giant Clam (<Emphasis Type="Italic">Tridacna crocea</Emphasis>) and Sea Slug (<Emphasis Type="Italic">Pteraeolidia ianthina</Emphasis>) Using Culture Medium Containing Giant Clam Tissue Homogenate

Recent molecular biological studies have revealed that some photosymbiotic invertebrates dwelling in coral reefs host several genetically different dinoflagellates, Symbiodinium species, as symbionts. However, little is known about the difference in physiologic characteristics among these symbionts living in a single host, because some Symbiodinium strains are difficult to culture in vitro. To isolate some of these Symbiodinium strains, we have developed an agar culture medium plate containing antibiotics and a giant clam tissue homogenate. Using-this medium we isolated two new Symbiodinium strains from two molluscan hosts, Tridacna crocea and Pteraeolidia ianthina, each of which hosted two different Symbiodinium strains belonging to Symbiodinium C and D, respectively. The tissue homogenate was essential for the growth of Symbiodinium D. Although it was not essential for the growth of Symbiodinium C, it did stimulate the initial growth. For the isolation of some Symbiodinium strains, isolation medium containing host homogenate is effective.     

Otolith variation in Pacific herring (<Emphasis Type="Italic">Clupea pallasii</Emphasis>) reflects mitogenomic variation rather than the subspecies classification

Pacific herring (Clupea pallasii) is divided into three subspecies: two in northeast Europe and one in the north Pacific Ocean. Genetic studies have indicated that the populations in northeast Europe have derived from the northwest Pacific herring recently, or during the last 10–15 kyr, and that they are distinct from the population in the northeast Pacific. In addition, hybridization between the Pacific herring and the Atlantic herring has been documented. Otolith variation has been considered to be largely affected by environmental variation, but here we evaluate whether the genetic differentiation is reflected in otolith shape differences. A clear difference in otolith shape was observed between the genetically differentiated herring species Clupea harengus from the Atlantic and C. pallasii. The otolith shape of C. p. suworowi in the Barents Sea was different from the shape of C. pallasii in northern Norway and C. p. pallasii from the Pacific. Populations of C. p. pallasii, sampled east and west of the Alaska Peninsula, which belong to two genetically different clades of the C. p. pallasii in the Pacific Ocean, show a clear difference in otolith shape. C. p. suworowi and the local C. pallasii peripheral population in Balsfjord in northern Norway are more similar to the northwest Pacific herring (C. p. pallasii) than to the northeast Pacific herring (C. p. pallasii), both genetically and in otolith shape. The Balsfjord population, known to be influenced by introgression of mtDNA from the Atlantic herring, does not show any sign of admixture in otolith shape between the two species. A revised classification, considering the observed genetic and morphological evidence, should rather group the northwest Pacific herring in the Bering Sea together with the European populations of C. pallasii than with the northeast Pacific herring in the Gulf of Alaska.     

Cytoplasmic incompatibility involving <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Wolbachia</Emphasis> in the white-backed planthopper <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

Cytoplasmic incompatibility (CI) is a reproductive phenotype induced by bacterial endosymbionts in arthropods. Measured as a reduction in egg hatchability resulting from the crossing of uninfected females with bacteria-infected males, CI increases the frequency of bacteria-infected hosts by restricting the fertilization opportunities of uninfected hosts in populations. Wolbachia, a type of alpha-proteobacteria, is well known as a CI inducer in a wide range of arthropod species, while Cardinium, a member of the phylum Bacteroidetes, is known to cause CI in one wasp and three spider mite species. In this study, dual infection with Cardinium and Wolbachia induced strong CI in a single host, Sogatella furcifera (Horváth), a planthopper species that is naturally infected with both bacteria. Specifically, infection with Cardinium alone was found to cause a 76 % reduction in egg development, and dual infection with Cardinium and Wolbachia a 96 % reduction, indicating that Cardinium induces CI and the dual infection raises the CI level. This study was the first to document reproductive alteration by Cardinium in a diploid host species.     

<Emphasis Type="Italic">Coxiella</Emphasis> Symbionts in the Cayenne Tick <Emphasis Type="Italic">Amblyomma cajennense</Emphasis>

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Colony founding and social parasitism in <Emphasis Type="Italic">Lasius</Emphasis> (<Emphasis Type="Italic">Acanthomyops)</Emphasis>

We investigated colony foundation behavior of three species of the Lasius claviger group, L. latipes, L. interjectus and L. claviger, using field observations and laboratory experiments. Laboratory studies included observing gyne behavior prior to interaction with host colonies (e.g. grouping, overwintering, feeding) and experiments in which gynes were introduced to putative host colonies. Observations of gyne parasitic behavior immediately after mating flights in the field and the discovery of mixed colonies both confirm the parasitic behavior of these species and point toward a wide host species range including two cases of hyperparasitism. L. interjectus and L. latipes enter the host colony immediately after the nuptial flight, while L. claviger may hibernate and enter in the spring. Laboratory experiments showed that L. interjectus gynes prefer to group and suffer lower mortality when in a group, which is consistent with field observations of L. interjectus gynes entering host colonies in large numbers. Details on behavior of L. latipes alpha and beta form gynes are also given.     

Outer membrane nanovesicles of gram-negative bacteria <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> and <Emphasis Type="Italic">Aeromonas salmonicida</Emphasis>

Aeromonas hydrophila and A. salmonicida grown in pure cultures were found to secrete extracellular membrane nanovesicles into the environment. Outer membrane nanovesicle preparations were isolated by differential centrifugation and ultrafiltration and visualized by transmission electron microscopy applying the negative staining technique. Membrane nanovesicle size (10–300 nm) and ultrastructure were determined. The vesicles were especially numerous around bacteria at the edge of small colonies. The process of the vesicle budding off the bacterial cell was observed. On ultrathin sections of rat intestine, outer membrane nanovesicles were revealed among bacterial aggregates of various species of parietal microorganisms. Short chains of such vesicles were also detected inside the glycocalyx between the microvilli of the apical surface of the intestine epitheliocytes. On the basis of the results, together with the literature data, the secretion of the outer membrane nanovesicles by pathogenic gram-negative bacteria, such as A. hydrophila and A. salmonicida, is proposed as a possible mechanism of pathogenesis leading to the host disease, as well as a means for cellular interactions both within the prokaryote population and between the bacteria and the host organism.     

Incidence of <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> endosymbionts in the <Emphasis Type="Italic">Osmia</Emphasis> community in Korea

Sex ratio distorting endosymbionts induce reproductive anomalies in their arthropod hosts. They have recently been paid much attention as firstly texts of evolution of host-symbiont relationships and secondly potential biological control agents to control arthropod pests. Among such organisms, Wolbachia and Cardinium bacteria are well characterized. This study aims at probing such bacteria in the Osmia community to evaluate their potential utilization to control arthropod pests. Among 17 PCR tested species, Osmia cornifrons and a parasitic fly are infected with Wolbachia and a mite species is infected with Cardinium. Phylogenetic tree analyses suggest that horizontal transfer of the bacteria occurred between phylogenetically distant hosts.     

Incidence of the endosymbionts <Emphasis Type="Italic">Wolbachia</Emphasis>, <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Spiroplasma</Emphasis> in phytoseiid mites and associated prey

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in insects and mites and their appeal to researchers’ interest is still increasing. We screened 20 strains of 12 agriculturally relevant herbivorous and predatory mite species for infection with Wolbachia, Cardinium and Spiroplasma by the use of PCR. The majority of specimens originated from Austria and were field collected or mass-reared. Eight out of 20 strains (40%) tested, representing seven of 12 mite species (58%), carried at least one of the three bacteria. We found Wolbachia in the herbivorous spider mites Tetranychus urticae and Bryobia rubrioculus, with the former also carrying Spiroplasma and the latter also carrying Cardinium. Cardinium was furthermore found in two populations of the predatory mite Euseius finlandicus and the spider mite Eotetranychus uncatus. Spiroplasma was detected in the predatory mite Neoseiulus californicus. All bacteria positive PCR products were sequenced, submitted to GenBank and analyzed in BLAST queries. We found high similarities to complete identity with bacteria found in the same and different mite species but also with bacteria found in insect species like ladybirds, butterflies and minute pirate bugs, Orius. We discuss the significance of potential (multiple) infections with the investigated bacteria for biological control.