A new open-access journal, Zoological Letters, was launched as a sister journal to Zoological Science, in January 2015. The new journal aims at publishing topical papers of high quality from a wide range of basic zoological research fields. This review highlights the notable reviews and research articles that have been published in the first year of Zoological Letters, providing an overview on the current achievements and future directions of the journal.

In the present study, we performed behavioral analyses of the habituation of backward escape swimming in the marbled crayfish, Procambarus fallax. Application of rapid mechanical stimulation to the rostrum elicited backward swimming following rapid abdominal flexion of crayfish. Response latency was very short—tens of msec—suggesting that backward swimming is mediated by MG neurons. When stimulation was repeated with 10 sec interstimulus intervals the MG-like tailflip did not occur, as the animals showed habituation. Retention of habituation was rather short, with most animals recovering from habituation within 10 min. Previous experience of habituation was remembered and animals habituated faster during a second series of experiments with similar repetitive stimuli. About half the number of stimulus trials was necessary to habituate in the second test compared to the first test. This promotion of habituation was observed in animals with delay periods of rest within 60 min following the first habituation. After 90 min of rest from the first habitation, animals showed a similar time course for the second habituation. With five stimuli at 15 min interval during 90 min of the rest, trained animals showed rapid habituation, indicating reinforcement of the memory of previous experiments. Crayfish also showed dishabituation when mechanical stimulation was applied to the tail following habituation.

It is often desirable but difficult to retrieve information on the mature phenotype of an immature tissue sample that has been subjected to gene expression analysis. This problem cannot be ignored when individual variation within a species is large. To circumvent this problem in the butterfly wing system, we developed a new surgical method for removing a single forewing from a pupa using Junonia orithya; the operated pupa was left to develop to an adult without eclosion. The removed right forewing was subjected to gene expression analysis, whereas the non-removed left forewing was examined for color patterns. As a test case, we focused on Distal-less (Dll), which likely plays an active role in inducing elemental patterns, including eyespots. The Dll expression level in forewings was paired with eyespot size data from the same individual. One third of the operated pupae survived and developed wing color patterns. Dll expression levels were significantly higher in males than in females, although male eyespots were smaller in size than female eyespots. Eyespot size data showed weak but significant correlations with the Dll expression level in females. These results demonstrate that a single-wing removal method was successfully applied to the butterfly wing system and suggest the weak and non-exclusive contribution of Dll to eyespot size determination in this butterfly. Our novel methodology for establishing correspondence between gene expression and phenotype can be applied to other candidate genes for color pattern development in butterflies. Conceptually similar methods may also be applicable in other developmental systems.

The adult newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown. Here, to approach this issue we focused on Pax6, a transcription factor known to be expressed in RPESCs. We first identified four classes (v1, v2, v3 and v4) of Pax6 variants in the eye of adult newt, Cynops pyrrhogaster. These variants were expressed in most tissues of the intact eye in different combinations but not in the RPE, choroid or sclera. On the basis of this information, we investigated the expression of Pax6 in RPE cells after the NR was removed from the eye by surgery (retinectomy), and found that two classes (v1 and v2) of Pax6 variants were newly expressed in RPE cells 10 days after retinectomy, both in vivo and in vitro (RLEC system). In the RLEC system, we found that Pax6 expression is mediated through a pathway separate from the MEK-ERK pathway, which is required for cell cycle re-entry of RPE cells. These results predict the existence of a pathway that may be of fundamental importance to a better understanding of the reprogramming of RPE cells in vivo.

The freshwater crustacean genus Daphnia has been used extensively in ecological, developmental and ecotoxicological studies. Daphnids produce only female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable conditions and external stimuli, they produce male offspring. Although we reported that exogenous exposure to juvenile hormones and their analogs can induce male offspring even under female-producing conditions, we recently established a male induction system in the Daphnia pulex WTN6 strain simply by changing day-length. This male and female induction system is suitable for understanding the innate mechanisms of sexual dimorphic development in daphnids. Embryogenesis has been described as a normal plate (developmental staging) in various daphnid species; however, all studies have mainly focused on female development. Here, we describe the developmental staging of both sexes during embryogenesis in two representative daphnids, D. pulex and D. magna, based on microscopic time-course observations. Our findings provide the first detailed insights into male embryogenesis in both species, and contribute to the elucidation of the mechanisms underlying sexual differentiation in daphnids.

Japanese wild boar (Sus scrofa leucomystax) populations have expanded drastically throughout the Japanese Archipelago in recent decades. To elucidate the dispersal patterns of Japanese wild boar in Toyama Prefecture in central Japan, we used a multi-locus microsatellite DNA analysis to determine its population structure and the degree of admixture. The deviation from Hardy-Weinberg equilibrium was detected in either total or separate regional wild boar samples from Toyama Prefecture. This result could be explained by the Wahlund effect resulting from the mixture of samples from different sources. Bayesian structure analysis, assignment test, and factorial correspondence analysis suggested that wild boars around Toyama Prefecture derive from at least two ancestral sources. The migration and possible mating of each individual may have occurred recently and continued in each geographically neighboring region. The present genetic results may be useful for prediction of future dispersal patterns of Japanese wild boar, as well as other animals in expansion.

The mitochondrial DNA (mtDNA) control region (198- to 598-bp) of four ancient Canis specimens (two Canis mandibles, a cranium, and a first phalanx) was examined, and each specimen was genetically identified as Japanese wolf. Two unique nucleotide substitutions, the 78-C insertion and the 482-G deletion, both of which are specific for Japanese wolf, were observed in each sample. Based on the mtDNA sequences analyzed, these four specimens and 10 additional Japanese wolf samples could be classified into two groups— Group A (10 samples) and Group B (4 samples)—which contain or lack an 8-bp insertion/deletion (indel), respectively. Interestingly, three dogs (Akita-b, Kishu 25, and S-husky 102) that each contained Japanese wolf-specific features were also classified into Group A or B based on the 8-bp indel. To determine the origin or ancestor of the Japanese wolf, mtDNA control regions of ancient continental Canis specimens were examined; 84 specimens were from Russia, and 29 were from China. However, none of these 113 specimens contained Japanese wolf-specific sequences. Moreover, none of 426 Japanese modern hunting dogs examined contained these Japanese wolf-specific mtDNA sequences. The mtDNA control region sequences of Groups A and B appeared to be unique to grey wolf and dog populations.

Dietary items of the Japanese eel Anguilla japonica inhabiting estuaries were examined by analyses of the gut (stomach and intestine) contents in two areas in Kyushu, western Japan. In a small estuary in Kagoshima Bay, where seven eel guts were examined, almost all of the dietary organisms consisted of Hemigrapsus crabs and Hediste polychaetes, both of which commonly occurred on tidal flats of this site during our survey on the macrobenthic fauna. In another large estuary in the innermost part of the Ariake Sea, where 14 eel guts were examined, 11 macrobenthic species (nine crustaceans, a polychaete, and a gastropod) were found in the gut contents, mostly consisting of mudflat-specific species. The dietary items are almost completely different not only between the two estuaries, but also among three neighboring sites within the large estuary in the Ariake Sea. These results show that Japanese eels feed on various macrobenthic invertebrates inhabiting estuarine tidal flats at each site. The variety of the prey species occupying different habitats indicates that their foraging areas extend to a wide range of estuarine tidal flats from the upper to lower littoral zones. The physalopterid nematode Heliconema anguillae was found parasitic in eel stomachs in both estuaries. The prevalence of the nematode was higher in the estuary in Kagoshima Bay (100%) than that in the Ariake Sea (43%), although the intensity in the former (4-94 nematodes per infected stomach) was comparable to that of the latter (2–96). The relationship between the nematode infection and the dietary items of Japanese eels is discussed.

Migration through the Eastern Palearctic (EP) flyway by tundra swans (Cygnus columbianus) has not been thoroughly documented. We satellite-tracked the migration of 16 tundra swans that winter in Japan. The objectives of this study were 1) to show the migration pattern of the EP flyway of tundra swans; 2) to compare this pattern with the migration pattern of whooper swans; and 3) to identify stopover sites that are important for these swans' conservation. Tundra swans were captured at Kutcharo Lake, Hokkaido, in 2009–2012 and satellite-tracked. A new method called the “MATCHED (Migratory Analytical Time Change Easy Detection) method” was developed. Based on median, the spring migration began on 18 April and ended on 27 May. Autumn migration began on 9 September and ended on 2 November. The median duration of the spring and autumn migrations were 48 and 50 days, respectively. The mean duration at one stopover site was 5.5 days and 6.8 days for the spring and autumn migrations, respectively. The number of stopover sites was 3.0 and 2.5 for the spring and autumn migrations, respectively. The mean travel distances for the spring and autumn migrations were 6471 and 6331 km, respectively. Seven migration routes passing Sakhalin, the Amur River, and/or Kamchatka were identified. There were 15, 32, and eight wintering, stopover, and breeding sites, respectively. The migration routes and staging areas of tundra swans partially overlap with those of whooper swans, whose migration patterns have been previously documented. The migration patterns of these two swan species that winter in Japan confirm the importance of the Amur River, Udyl' Lake, Shchastya Bay, Aniva Bay, zaliv Chayvo Lake, zal Piltun Lake, zaliv Baykal Lake, Kolyma River, Buyunda River, Sen-kyuyel' Lake, and northern coastal areas of the Sea of Okhotsk.

Chicken is one of the most popular domesticated species worldwide, as it can serve an important role in agricultural as well as biomedical research fields. Because it inhabits almost every continent and presents diverse morphology and traits, the need of genetic markers for distinguishing each breed for various purposes has increased. The whole genome sequencing of three different breeds (White Leghorn, Korean domestic, and Araucana) that show similar coloring patterns, with the exception of the White Leghorn breed, have confirmed previously reported genomic alterations and identified many novel variants. Additionally, the Whole Genome Re-Sequencing (WGRS) approach identified an approximately 4 kb insert within SLCO1B3 responsible for blue egg shell color. Targeted investigation of pigment-related genes corroborated previously reported non-synonymous mutations, and provided deeper insight into chicken coloring, where not a single but a combination of non-synonymous mutations in the MC1R gene is likely to be responsible for altered feather coloring.

Terrestrial pulmonates can form odor-aversion memories once a food odor is presented in combination with an aversive stimulus. Most of the olfactory information ascends via a tentacular ganglion located in the tip of the two pairs of tentacles, and is then transmitted to the higher olfactory center, the procerebrum. The procerebrum is the locus of memory storage and has been shown to be necessary for odor-aversion learning. However, it is unknown whether the procerebrum is the sole locus in which the memory engram resides. By exploiting the regenerative ability of tentacles, here we investigated whether tentacles function merely in transmitting olfactory information to the procerebrum, or constitute a part of the memory engram. We showed that after removal of the tentacles used during memory acquisition, slugs were unable to retrieve the memory, even if these tentacles were regenerated sufficiently to subserve memory function. Our results support the view that tentacles are more than conduits of odor information; they also participate in the formation of the memory engram.

Seasonal changes in physiology of vertebrate animals are triggered by environmental cues including temperature, day-length and oxygen availability. Crucian carp (Carassius carassius) tolerate prolonged anoxia in winter by using several physiological adaptations that are seasonally activated. This study examines which environmental cues are required to trigger physiological adjustments for winter dormancy in crucian carp. To this end, crucian carp were exposed to changing environmental factors under laboratory conditions: effects of declining water temperature, shortening day-length and reduced oxygen availability, separately and in different combinations, were examined on glycogen content and enzyme activities involved in feeding (alkaline phosphatase, AP) and glycogen metabolism (glycogen synthase, GyS; glycogen phosphorylase, GP). Lowering temperature induced a fall in activity of AP and a rise in glycogen content and rate of glycogen synthesis. Relative mass of the liver, and glycogen concentration of liver, muscle and brain increased with lowering temperature. Similarly activity of GyS in muscle and expression of GyS transcripts in brain were up-regulated by lowering temperature. Shortened day-length and oxygen availability had practically no effects on measured variables. We conclude that lowering temperature is the main trigger in preparation for winter anoxia in crucian carp.

Recently, microRNAs (miRNAs) are focused on the role of biomarker because they are stable in serum and plasma, and some of them express in the specific organs and increase with the organ injury. Thus miRNAs may be very useful as biomarkers for monitoring the health and condition of dolphins and for detecting disorders in aquariums. Here, a small RNA library was made from dolphin lung, liver and spleen, and miRNA expression patterns were then determined for 15 different tissues. We identified 62 conserved miRNA homologs in the dolphin small RNA library and found high expression miRNAs in specific tissues: miR-125b and miR-221 were highly expressed in brain, miR-23b in heart, miR-199a and miR-223 in lung, and miR-122-5p in liver. Some of these tissue-enriched miRNAs may be useful as specific and sensitive diagnostic blood biomarkers for organ injury in dolphins.

Membrane progestin receptor (mPR) α on the cell membrane of the oocyte is involved in the meiotic maturation of vertebrates, including teleosts, but little is known about the role of this membrane-bound follicular receptor. We investigated the ovarian expression of membrane progestin receptor (mPR) mRNA in medaka. In follicles that were destined to ovulate, transcripts of mPRα and mPRγ were expressed in the oocytes as well as the granulosa cells. Transcripts of mPRα and mPRγ were expressed at relatively constant levels in the whole ovary and in the preovulatory follicles throughout the 24-h spawning cycle. In vitro incubation of the preovulatory follicles with recombinant medaka luteinizing hormone caused no significant changes in the expression of mPRα and mPRγ mRNA, suggesting LH-independent follicular expression of these mPR genes. Using HEK293T cells expressing medaka mPRs, forskolin-elevated intracellular cAMP levels were found to be reduced on treatment of the cells with ligand 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP), but only in the cells expressing mPRα. These results indicate that activation of mPRα and mPRγ with DHP may cause differential effects on the granulosa cells. Information obtained from the present study may help to elucidate the role of mPRα and mPRγ in the granulosa cells of the follicles.

Water fleas (Daphnia pulex) normally produce subitaneous eggs that initiate development immediately after oviposition. However, in response to habitat degradation, resting eggs are produced, which are enclosed in a sturdy outer envelope (ephippium) and can survive in harsh environments for an extended time. To understand the molecular mechanism underlying resting egg production in D. pulex, we investigated the genes whose expression patterns played a role in the production and identified the following six candidate genes: Dpfa-1, Dpfa-2, Dpep-1, Dpep-2, Dpep-3, and Dpep-4. These six genes displayed > 40-fold higher expression levels in resting egg-producing animals compared with those in subitaneous egg-producing animals at the period when the ovaries were mature. Dpfa-1 and Dpfa-2 were expressed in the fat cells, and their expression patterns were synchronized with the development of resting egg oocytes in the ovary. In contrast, Dpep-1–4 were expressed in the morphologically altered epidermal cells of the brood chamber with the formation of the ephippium, and their expression patterns were also related to ephippium formation. Our results suggest that the former two genes encode the resting egg-specific components produced by fat cells and that the latter four genes encode the components related to the ephippium formation synthesized by epidermal cells.

The order Scleractinia includes two distinct groups, which are termed “complex” and “robust” as indicated by the molecular phylogeny of mitochondrial 16S ribosomal gene sequences. Since this discovery, coral taxonomists have been seeking morphological characters for grouping this deep division in the order Scleractinia. Recently, morphological characteristics during embryogenesis that facilitate grouping the two clades as “complex” and “robust” were reported, thus clarifying a deep division in the Scleractinia. In the present report, I establish two new suborders, Refertina and Vacatina, on the basis of the embryogenetic morphological characteristics, molecular data, and new observations of Tubastraea coccinea and Cyphastrea serailia embryogenesis. In particular, the embryo of T. coccinea has a possible fertilization membrane that was first observed in the phylum Cnidaria. The new suborder Refertina consists of the families that belong to the “complex” clade and have no or little blastocoel. The new suborder Vacatina is composed of the families that fall into the “robust” clade and have an apparent blastocoel.