Defenses of whirligig beetles against native and invasive frogs.

Many native insects have evolved defenses against native predators. However, their defenses may not protect them from non-native predators due to a limited shared history. The American bullfrog, Aquarana catesbeiana (Anura: Ranidae), which has been intentionally introduced to many countries, is believed to impact native aquatic animals through direct predation. Adults of whirligig beetles (Coleoptera: Gyrinidae), known for swimming and foraging on the water surface of ponds and streams, reportedly possess chemical defenses against aquatic predators, such as fish. Although whirligig beetles potentially encounter both bullfrogs and other frogs in ponds and lakes, the effectiveness of their defenses against frogs has been rarely studied. To assess whether whirligig beetles can defend against native and non-native frogs, we observed the behavioral responses of the native pond frog, Pelophylax nigromaculatus (Anura: Ranidae), and the invasive non-native bullfrog, A. catesbeiana, to native whirligig beetles, Gyrinus japonicus and Dineutus orientalis, in Japan. Adults of whirligig beetles were provided to frogs under laboratory conditions. Forty percent of G. japonicus and D.orientalis were rejected by P. nigromaculatus, while all whirligig beetles were easily consumed by A. catesbeiana. Chemical and other secondary defenses of G. japonicus and D. orientalis were effective for some individuals of P. nigromaculatus but not for any individuals of A. catesbeiana. These results suggest that native whirligig beetles suffer predation by invasive non-native bullfrogs in local ponds and lakes in Japan.

Description of the larva of Cybisterlewisianus Sharp, 1873 (Coleoptera, Dytiscidae, Cybistrinae).

We describe for the first time, the larvae of Cybister (Cybister) lewisianus Sharp, 1873, an endangered species of diving beetle in Japan, emphasizing the chaetotaxy of the cephalic capsule, head appendages, legs, last abdominal segment, and urogomphi. Cybisterlewisianus larvae are characterized by a longer third article of antenna 3 than the sum of the first and second articles; rounded apex of parietal setae 1-3; labium seta 8 absent; elongated trochanter seta 4, not multi-branched; rounded apex of abdominal setae 1, 12, and 13 (instar I); narrow lateral projections of the frontoclypeus; pronotum without two dark-brown longitudinal stripes dorsally (instar III); and the base of the thick row of small setae on the inner edge of the mandible angulate and projecting medially (all instars).

Bombardiers and assassins: mimetic interactions between unequally defended insects.

In defensive mimicry, resemblance between unequally defended species can be parasitic; this phenomenon has been termed quasi-Batesian mimicry. Few studies have used real co-mimics and their predators to test whether the mimetic interactions were parasitic. Here, we investigated the mimetic interaction between two well-defended insect species, the bombardier beetle Pheropsophus occipitalis jessoensis (Coleoptera: Carabidae) and the assassin bug Sirthenea flavipes (Hemiptera: Reduviidae), using their potential predator, the pond frog Pelophylax nigromaculatus (Anura: Ranidae), which coexists with these insect species in the same habitat in Japan. We observed behavioural responses of this frog species (adults and juveniles) to adult Ph. occipitalis jessoensis and adult S. flavipes under laboratory conditions. Among the frogs, 100% and 75% rejected Ph. occipitalis jessoensis and S. flavipes, respectively, suggesting that, compared with the assassin bug S. flavipes, the bombardier beetle Ph. occipitalis jessoensis is more well-defended against frogs. An assassin bug or a bombardier beetle was provided to a frog that had encountered the other insect species. Frogs with a history of assassin bug encounter demonstrated a lower rate of attack toward bombardier beetles. Similarly, frogs with a history of bombardier beetle encounter demonstrated a lower rate of attack toward assassin bugs. Therefore, both the bombardier beetle Ph. occipitalis jessoensis and the assassin bug S. flavipes benefit from the mimetic interaction.

A new species of Pseudovelia Hoberlandt, 1950 (Hemiptera: Heteroptera: Veliidae) from Honshu, Japan.

The genus Pseudovelia occurs at the margins of freshwater bodies and is widely distributed in the Palearctic and Oriental regions. It is represented by 29 species in the East and Southeast Asian mainland, of which five are known from Japan. Described here is Pseudovelia lasiomma sp. nov. from Lake Biwa, Honshu, Japan. In addition, a key is provided to facilitate the identification of the six species of Pseudovelia now known from Japan.

Improved fertility of frozen-thawed porcine spermatozoa with 3,3-dimethylglutaric anhydride poly-L-lysine as a novel cryoprotectant.

In this study, we determined the efficacy of 3,3-dimethylglutaric anhydride poly-L-lysine (DMGA-PLL) as a cryoprotectant for porcine spermatozoa. Porcine spermatozoa were cryopreserved in a freezing extender containing 3% (v/v) glycerol and various concentrations of DMGA-PLL. At 12 h after thawing, the motility index of spermatozoa cryopreserved with 0.25% (v/v) DMGA-PLL (25.9) was significantly (P < 0.01) higher than that of spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (10.0-16.3). In addition, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% DMGA-PLL (22.8%) was significantly (P < 0.01) higher than that of embryos derived from spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (7.9%-10.9%). The mean number of total piglets born to sows inseminated with spermatozoa cryopreserved without DMGA-PLL (9.0) was significantly (P < 0.05) lower than that of total piglets born to sows inseminated with spermatozoa stored at 17°C (13.8). However, when spermatozoa cryopreserved with 0.25% DMGA-PLL were used for artificial insemination, the mean number of total piglets (11.7) was not significantly different from that obtained following artificial insemination using spermatozoa stored at 17°C. The results showed the usefulness of DMGA-PLL as a cryoprotectant in the cryopreservation of porcine spermatozoa.

Revision of the family Haliplidae (Insecta, Coleoptera) in Japan.

The Japanese members of Haliplidae were reviewed and 13 species in two genera are recognized. A new species, Haliplusmoriisp. nov. is described from Honshu; it is similar to Haliplusjaponicus Sharp, 1873, but belongs to a different subgenus. Haliplusdiruptus J. Balfour-Browne, 1946, syn. nov. is treated as a junior synonym of Halipluskotoshonis Kano & Kamiya, 1931. The records of Haliplusdavidi Vondel, 1991 from Japan are regarded as misidentifications of H.kotoshonis. Haliplusbasinotatuslatiusculus Nakane, 1985, syn. nov. is treated as a junior synonym of H.basinotatus. Haliplusangustifrons Régimbart, 1892 known from south and southeast Asia, is newly recorded from Japan.

Nagisavelia hikarui, a new genus and species of Mesoveliinae (Hemiptera: Heteroptera: Mesoveliidae) inhabiting shingle beaches in Japan.

A new genus and species of Mesoveliinae, Nagisavelia hikarui n.gen. and n.sp., is described from rocky intertidal beaches in Japan. The new taxon is compared to the other 10 genera of Mesoveliinae, and a character matrix for the identification of eight genera is provided. A key to facilitate the identification of the seven species of Mesoveliidae now reported from Japan is also provided.

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant.

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.

Active behaviour of terrestrial caterpillars on the water surface.

Most butterfly and moth larvae (Lepidoptera) are terrestrial. When terrestrial caterpillars accidentally fall into water, they may drown or be preyed upon by aquatic predators before they can safely reach land. However, how terrestrial caterpillars escape aquatic environments and predators remains unclear. In July 2018, we observed a terrestrial caterpillar actively moving forward on the surface of a pond in Japan until it successfully reached the shore. To further investigate this behaviour in terrestrial caterpillars, we experimentally placed larvae of 13 moth species (four families) on a water surface under laboratory and field conditions. All caterpillars floated. Larvae of seven species moved forward on the water surface, whereas those of six species did not. A total of two types of behaviour were observed; in Dinumma deponens, Hypopyra vespertilio, Spirama retorta, Laelia coenosa, Lymantria dispar (all Erebidae), and Naranga aenescens (Noctuidae), larvae swung their bodies rapidly from side to side to propel themselves along the water surface (i.e., undulatory behaviour); in contrast, larvae of Acosmetia biguttula (Noctuidae) rapidly moved the abdomen (posterior segments) up and down for propulsion along the water surface (i.e., flick behaviour). Although thoracic legs were not used for undulatory and flick behaviour, rapid movements of the abdomen were used to propel caterpillars on the water surface. We also observed that undulatory and flick behaviour on the water surface aided caterpillars in escaping aquatic predators under field conditions. In addition, we investigated the relationship between body size and undulatory behaviour on the water surface in the erebid S. retorta under laboratory conditions. The frequency and speed of forward movement on the water surface increased with body length. Together, these results show that the rapid movement of elongated bodies results in forward propulsion on the water surface, allowing some terrestrial caterpillars to avoid drowning or aquatic predators. We further suggested potential factors related to morphology, host plant habitat, and defensive behaviour that may have led to the acquisition of aquatic behaviour in terrestrial caterpillars.

Shell-breaking predation on gastropods by Badister pictus (Coleoptera, Carabidae) with strikingly asymmetric mandibles.

The adults and larvae of some groups in the coleopteran family Carabidae are known to prey on snails (Mollusca: Gastropoda). Most species of the carabid tribe Licinini are believed to feed on live snails. However, the snail-eating behavior of only a few species has been studied. Whether adults of the licinine genus Badister can prey on live snails was tested by providing 155 live snails of 20 species (eleven terrestrial and nine aquatic species) to adults of Badister pictus Bates, 1873, and observing their behavior under laboratory conditions. Six of the 20 snail species have an operculum that can close the aperture of the shell. Each B. pictus adult attacked all of the snails provided. Badister pictus successfully preyed on ten terrestrial and six aquatic snail species. These beetles used their strikingly asymmetrical mandibles to break the dextral shells along the dorsal part of the whorls from the outer lip of the aperture towards the apex, allowing subsequent consumption of the soft bodies. However, 41.9% of snails could not be eaten by B. pictus adults. The rate of predation success by B. pictus decreased with increasing shell size and thickness of snails. In addition, the presence of an operculum decreased the rate of predation success by B. pictus. The results show that the shell size, thickness, and operculum of some snail species could play important roles in preventing B. pictus mandibles from breaking the shells. Therefore, Badister beetles may exert selective pressure on the evolution of defensive shell structures in small-sized snails.

Description of larvae of Japanese Macronychini (Coleoptera: Elmidae: Elminae).

Larvae of 15 species of Macronychini, subfamily Elminae, belonging to the genera Sinonychus Jäch Boukal, Paramacronychus Nomura, Zaitzeviaria Nomura, Zaitzevia Champion, and Urumaelmis Satô were described based on scanning electron microscope (SEM) observation. Larvae of eleven of these species, S. tsujunensis Yoshitomi Nakajima, Zaitzeviaria gotoi (Nomura), Zaitzeviaria brevis (Nomura), Zaitzeviaria kuriharai Kamite, Ogata Satô, Zaitzevia elongata Nomura, Zaitzevia aritai Satô, Zaitzevia yaeyamana Satô, Zaitzevia awana (Kôno), Zaitzevia nitida Nomura, Zaitzevia tsushimana Nomura, and U. uenoi (Nomura) are described for the first time.

Unexpected discovery of a new Podonychus species in Kyushu, Japan (Coleoptera, Elmidae, Elminae, Macronychini).

Podonychus gyobu sp. nov., a second species of the genus Podonychus Jäch & Kodada, 1997, hitherto known only from Indonesia, is described from Kyushu, Japan. This new species is similar to P. sagittarius Jäch & Kodada, 1997, but differs from it in the straight penis, arcuate 2nd labial palpomere, and in the 3rd antennomere being longer than wide. The endophallic structures and the larva of P. gyobu sp. nov. are described. A character matrix of the Macronychini genera and a key to the Japanese genera are provided.

Description of adults and larvae of Orientelmis parvula (Nomura amp; Baba, 1961) (Coleoptera: Elmidae) with their molecular phylogenetic analysis.

The elmid beetle Orientelmis parvula (Nomura Baba, 1961) is a rare and endangered species in Japan. Mouth parts and genitalia in adults of both sexes and larval morphology are described based on scanning electron microscope observations. The larva of the genus Orientelmis Shepard, 1998 is described for the first time. The systematic position of the genus is discussed based on the morphology and a phylogenetic tree inferred from the mitochondrial COI, and nuclear ArgK and 18S gene sequences. A new key for larvae of known species and genera of Japanese Elmidae is given.

A new species of the genus Ochthebius (Coleoptera, Hydraenidae) from the Ogasawara Islands, Japan, with a description of the larva.

A new species of the genus Ochthebius, O. (O.) sasakiisp. nov., is described from the Ogasawara Islands, Japan, with a description of the larva. This record is the first of the family Hydraenidae from the Ogasawara Islands. This species belongs to the punctatus species group and is similar to two Japanese species, O. (O.) inermis Sharp, 1884 and O. (O.) danjo Nakane, 1990, but it differs from them in both adult and larval characters. The larva of O. (O.) inermis is also described for comparison.

Description of larvae of genera Stenelmis, Ordobrevia and Nomuraelmis (Coleoptera: Elmidae: Elminae) of Japan with their molecular taxonomy.

Larvae of 10 elmid species of the genera Stenelmis, Ordobrevia and Nomuraelmis were described based on SEM observation. Of these, S. hisamatsui, S. ishiharai, S. hayashii, O. amamiensis were described for the first time, and N. amamiensis was redescribed. The morphological types of the larvae corresponded to the monophyletic groups defined by the mitochondrial COI gene tree. A new key for larvae of all species from Japan with known larva is given. The larvae of the type species of Stenelmis and Ordobrevia are also described.

Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices.

Animal cells are generally cryopreserved in cryovials in a cell suspension state containing 5%-10% v/v dimethyl sulfoxide (DMSO) used as a cryoprotective agent. However, cryopreservation of cells in an attached state has not been intensively studied, and the effective freezing solution remains unknown. Here we determined the suitable DMSO concentration for the cryopreservation of human hepatoma HepG2 cells attached to glass and polydimethylsiloxane (PDMS) matrices coated with poly-l-lysine. With the use of the glass matrix, the rate of cell adhesion increased with the DMSO concentration up to 30% v/v in the freezing solution. In contrast, the cell-adhesion rate remained constant in the case of the PDMS matrix irrespective of the DMSO concentration between 10% v/v and 30% v/v. The viability of post-thawed cells attached to glass or PDMS matrix was also investigated. The viability was highest at the DMSO concentration of 20% v/v in the freezing solution. The DMSO concentration of 30% v/v, however, had a cytotoxic effect on the cell viability. Thus, the 20% v/v DMSO concentration was found to be most suitable for the cryopreservation of HepG2 cells in the attached state. This dose is high compared to the DMSO concentration used for the cryopreservation of cells in the suspended state.

The oatmeal nematode Panagrellus redivivus survives moderately low temperatures by freezing tolerance and cryoprotective dehydration.

The cold tolerance abilities of only a few nematode species have been determined. This study shows that the oatmeal nematode, Panagrellus redivivus, has modest cold tolerance with a 50% survival temperature (S (50)) of -2.5°C after cooling at 0.5°C min(-1) and freezing for 1 h. It can survive low temperatures by freezing tolerance and cryoprotective dehydration; although freezing tolerance appears to be the dominant strategy. Freezing survival is enhanced by low temperature acclimation (7 days at 5°C), with the S (50) being lowered by a small but significant amount (0.42°C). There is no cold shock or rapid cold hardening response under the conditions tested. Cryoprotective dehydration enhances the ability to survive freezing (the S (50) is lowered by 0.55°C, compared to the control, after 4 h freezing at -1°C) and this effect is in addition to that produced by acclimation. Breeding from survivors of a freezing stress did not enhance the ability to survive freezing. The cold tolerance abilities of this nematode are modest, but sufficient to enable it to survive in the cold temperate environments it inhabits.

Molecular phylogeny and historical biogeography of the Holarctic wetland leaf beetle of the genus Plateumaris.

Leaf beetles of the genus Plateumaris inhabit wetlands across the temperate zone of the Holarctic region. To explore the phylogeographic relationships among North American, East Asian, and European members of this genus and the origin of the species endemic to Japan, we studied the molecular phylogeny of 20 of the 27 species in this genus using partial sequences of mitochondrial cytochrome oxidase subunit I (COI) and the 16S and nuclear 28S rRNA genes. The molecular phylogeny revealed that three species endemic to Europe are monophyletic and sister to the remaining 11 North American and six Asian species. Within the latter clade, North American and Asian species did not show reciprocal monophyly. Dispersal-vicariance analysis and divergence time estimation revealed that the European and North America-Asian lineages diverged during the Eocene. Moreover, subsequent differentiation occurred repeatedly between North American and Asian species, which was facilitated by three dispersal events from North America to Asia and one in the opposite direction during the late Eocene through the late Miocene. Two Japanese endemics originated from different divergence events; one differentiated from the mainland lineage after differentiation from the North American lineage, whereas the other showed a deep coalescence from the North American lineage with no present-day sister species on the East Asian mainland. This study of extant insects provides molecular phylogenetic evidence for ancient vicariance between Europe and East Asia-North America, and for more recent (but pre-Pleistocene) faunal exchanges between East Asia and North America.

Visualization of uveal perfusion by contrast-enhanced harmonic ultrasonography at a low mechanical index: a pilot animal study.

OBJECTIVE: To evaluate contrast-enhanced harmonic ultrasonography at a low mechanical index for its usefulness in visualizing uveal perfusion. METHODS: The study was performed with 9 rabbits, 6 intact and 3 with focal impaired blood flow in the uvea. Ultrasonography was performed by harmonic imaging (transmit, 5 MHz; receive, 10 MHz) with a contrast agent. The agent was administered at a dose of 50 microL/kg. Transmission power was at a mechanical index of 0.2, which is below the US Food and Drug Administration guideline. The images were compared between the impaired and intact eyes. For uveal measurements, video signal intensity-versus-time plots were generated in all cases. The plots were analyzed to obtain the rate of signal intensity increase and peak signal intensity. RESULTS: A clear increase of signal intensity was observed after contrast agent administration. The signal intensity of the uvea was lower in the impaired eye than in the intact eye. In the impaired eye, the intensity was lower on the side with impaired flow than on the other side. The differences were significant. CONCLUSIONS: Our findings suggest that uveal perfusion can be visualized by contrast-enhanced harmonic ultrasonography in the harmonic imaging mode at a low mechanical index.