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Inoda T.,Conservation Laboratory of Rare Water Insects | Inoda T.,Toho University | Hardling R.,Lund University | Kubota S.,Toho University
Zoological Science | Year: 2012

Many species of Dytiscus diving beetles exhibit intrasexual dimorphism, e.g., the elytra is smooth in some females and grooved in others. However, the expression of the grooves and whether they are a product of heredity or the environment remain unknown. One Japanese species, Dytiscus sharpi sharpi Wehncke, 1875, also shows female dimorphism, with grooved and smooth morphs, while D. sharpi validus Rgimbart, 1899, only has a single morph (the grooved type). A hybrid of the two species should therefore provide a means of sorting out how the grooves are inherited. We found two independent wetlands of D. sharpi sharpi in Chiba Prefecture, Japan. One was a place where a high proportion of grooved females lived, and the others had high proportions of smooth females. After five to eight generations of beetles from two populations with different proportions of grooved females were reared under aquarium conditions constituting a common garden design, i.e., water temperature, water depth, and presence of a plant for oviposition, the differences remained. We mated smooth virgin females of D. sharpi sharpi with males of D. sharpi validus to obtain hybrid offspring. The elytral traits of the hybrid females produced only grooved forms. These results suggested that the female dimorphism is determined by genetics, and that the grooved morph was dominant over the smooth one, independent of environmental factors. In addition, the hybrid insects did not differ from the two subspecies insects in larval survivorship, pupation success, or sex ratio. They also showed neither morphological abnormality nor reduced survival. © 2012 Zoological Society of Japan.


Suzuki G.,Toho University | Inoda T.,Toho University | Inoda T.,Conservation Laboratory of Rare Water Insects | Kubota S.,Toho University
Entomological Science | Year: 2012

Nonlethal DNA sampling is highly desirable in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from endangered diving beetles (Dytiscus sharpi sharpi Wehncke, Cybister lewisianus Sharp and Cybister brevis Aubé), we amputated the antennae of these endangered diving beetles and investigated the impact of the amputation on reproductive behaviors, egg-laying and lifespan. Diving beetles with either one or no antennae copulated without delay and laid eggs, comparable to the pairs of intact beetles under breeding conditions. The lifespan of antennae-amputated D. sharpi sharpi was the same as that of the intact beetles. A single antenna was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome-c oxidase subunitI (COI), and the sequence of the COI gene could be determined directly. The PCR-ready genomic DNA was available both in fresh antennae isolated from living beetles and in old antennae from whole beetles preserved for at least 5-6years in pure ethanol. These results suggest that an antenna is a good sampling site for isolating genomic DNA from endangered diving beetles without sacrificing and disturbing reproductive behaviors such as mating and egg-laying, or lifespan. © 2012 The Entomological Society of Japan.


Inoda T.,Conservation Laboratory of Rare Water Insects | Inoda T.,Toho University | Miyazaki Y.,Conservation Laboratory of Rare Water Insects | Kitano T.,Tokai University | Kubota S.,Toho University
Entomological Science | Year: 2015

Nonlethal DNA sampling is a highly recommendable method in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from larvae of endangered diving beetles (Cybister brevis, C.lewisianus, C.limbatus, C.rugosus, Dytiscus sharpi sharpi and D.sharpi validus), we obtained the larval exuvia (molted skin) of these endangered diving beetles under laboratory conditions. A single exuvia 24h after molting was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome-c oxidase subunitI (COI), and the sequence of the COI gene could be determined directly. Sequences obtained from the exuvial samples were used to further find similarities within DDBJ/EMBL/GenBank. Genomic DNA from the samples was successfully isolated, and we identified the species. This process suggests that exuvia provides a good sample for extracting DNA from endangered diving beetle larvae without killing them. © 2015 The Entomological Society of Japan.


Inoda T.,Conservation Laboratory of Rare Water Insects | Inoda T.,Toho University | Suzuki G.,Toho University | Ohta M.,Conservation Laboratory of Rare Water Insects | Kubota S.,Toho University
Entomological Science | Year: 2012

Three species of the Japanese diving beetle Dytiscus have been identified: D. dauricus Gebler, 1832; D. marginalis czerskii Zeitzev, 1953; and D. sharpi. At present, the latter consists of the subspecies D. sharpi sharpi Wehncke, 1875 and D. sharpi validus Régimbart, 1899 based on the comparative data of mitochondrial DNA cytochrome-c oxidase I (COI) sequences. Many Dytiscus species have smooth and grooved elytra, which are female dimorphic traits. For many years it has been thought that Japanese D. marginalis czerskii has a single morph, that is, only grooved females, although there were some collecting reports of smooth females occurring at the foot of Mt. Chokaisan in Yamagata and Akita Prefectures. However, the population of smooth females (smooth population) has not yet been identified by DNA markers. To understand the species status of the smooth population, we sequenced 769bp of COI of a male derived from a smooth mother insect and compared it with the sequence from a known grooved female. The sequences of 769bp of the COI gene in the smooth population were identical to that in the grooved female, indicating that Japanese D. marginalis czerskii has female dimorphic traits. © 2012 The Entomological Society of Japan.


Inoda T.,Conservation Laboratory of Rare Water Insects | Ohba S.-Y.,Nagasaki University | Rullan J.K.,University of the Philippines at Manila
Aquatic Insects | Year: 2013

Gonad maturation in wild Cybister brevis Aubé, 1838 from Japan was investigated to determine the physiological mating season. Breeding experiments showed that hatching occurred from May to September, and the peak was observed in July. The gonadosomatic index (GSI) in collected females (ovary development) showed the highest value in May. The GSIs were low in other seasons. On the other hand, the GSIs in collected males (testes and accessory glands) remained at constant values during the experimental period. Interestingly, high sperm motility was exhibited in May and September, whereas it was low in December and March. This indicates the maturation difference between females and males. Females mature only at the beginning of the breeding season (May), but males do so from May to September. This result suggests that males have a wide-ranging maturation period and earlier sexual maturation than females, ensuring a successful fertilization. © 2014, © 2014 Taylor & Francis.


Inoda T.,Conservation Laboratory of Rare Water Insects | Ladion T.M.A.,Independent Scholar--Avrin, William F.
Aquatic Insects | Year: 2016

A box trap was developed for effective collection of large predaceous diving beetles. The floating trap, which was fabricated from a plastic box with two funnel mouths equipped with mesh lids, can be opened only when beetles enter the trap. Considerable attention was paid to the trap's performance as it was quantitatively evaluated in laboratory conditions in detail using Cybister and Dytiscus diving beetles. Without the mesh lid on the trap mouth (negative control test), the number of beetles in the trap was the highest at 2–3 hours. However, most beetles escaped from the trap without a mesh lid within six hours of starting the experiment. When eight beetles were put into a trap without a mesh lid, all of them had escaped from it after eight hours. On the contrary, beetles did not escape from a trap with a mesh lid. This result and testing in the field suggest that trap lids have a significant role in averting the escape of beetles from the trap. © 2016 Informa UK Limited, trading as Taylor & Francis Group


Inoda T.,Conservation Laboratory of Rare Water Insects | Kitano T.,Tokai University
Applied Entomology and Zoology | Year: 2013

For conservation purposes, and to supply critically endangered insects for laboratory use, a system for artificial breeding is crucial. However, in the case of carnivorous insects such as diving beetles, the larvae must be isolated because they are cannibalistic. We developed a method for mass breeding the larvae of two diving beetles, Dytiscus sharpi sharpi (Wehncke) and Dytiscus sharpi validus (Régimbart) (Coleoptera: Dytiscidae), which are designated critically endangered species in Japan. Ten to twenty larvae were raised in a small tank (35 cm × 25 cm × 10 cm; water depth 7 cm) with Rana ornativentris (Werner) tadpoles as prey. At low prey density, ~80 % of the larvae were cannibalized. At moderate prey density, 50-60 % were cannibalized. However, at high prey density, <3 % were cannibalized. Well-fed mass-bred adults were larger than individually bred and field-collected adults. This mass breeding method can be used for the conservation and breeding of these rare diving beetles in a manageable number of aquaria. © 2013 The Japanese Society of Applied Entomology and Zoology.


Inoda T.,Conservation Laboratory of Rare Water Insects | Balke M.,Zoologische Staatssammlung | Balke M.,Ludwig Maximilians University of Munich
Entomological Science | Year: 2012

We sequenced 628 base pairs of the mitochondrial cytochrome-c oxidase subunit1 (cox1) gene to investigate the genetic differentiation among Japanese Dytiscus diving beetles (Coleoptera: Dytiscidae). Dytiscus beetles are a charismatic part of the fauna of lakes, ponds and swamps. Three species are known from Japan: D.dauricus Gebler, 1832; D.marginalis czerskii Zeitzev*, 1953; and D.sharpi Wehncke, 1875. Adults of D.sharpi collected in Chiba and Ishikawa Prefectures were morphologically highly similar, but here we found that they differed by 20 cox1 base pairs (3.18%). Our results imply that conservation strategies especially for Japanese D.sharpi sharpi and D.sharpi validus Régimbart, 1899 might need to be adjusted to address the presence of two evolutionarily significant units. © 2011 The Entomological Society of Japan.


Inoda T.,Conservation Laboratory of Rare Water Insects | Kamimura S.,Chuo University
Journal of Insect Behavior | Year: 2014

Diving beetle larvae use their mandibles in two ways: capturing prey and sucking their body fluid. Catching and consuming the prey’s most nutritious body part leads to the highest feeding efficiency. To test this, Dytiscus sharpi sharpi larvae were given tadpoles (Rana ornativentris) as food and their feeding behaviors were observed. Dytiscus larvae preferred to catch tadpoles by the abdomens rather than by other parts. Tadpoles soon became immobilized and in most cases the beetle larvae started eating abdomens first. Beetle larvae tried to change biting site to tadpole’s abdomen when the tadpole was initially caught by the head or tail. More food was absorbed from the abdomen than the head or tail suggesting that the feeding behavior of beetle larva is optimized to obtain nutrition efficiently from the tadpole abdomen. © 2014, Springer Science+Business Media New York.

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