A novel gene arrangement among the Stylommatophora by the complete mitochondrial genome of the terrestrial slug Meghimatium bilineatum (Gastropoda, Arionoidea).

Stylommatophora is a main clade of Gastropoda that encompasses approximately 112 gastropod families and may exceed a total of 30,000 species. Twenty-four complete stylommatophoran mitogenomes have been sequenced to date, yet our understanding of mitochondrial evolution in stylommatophorans is still in its infancy. To further expand the set of available mitogenomes, we sequenced the mitogenome of Meghimatium bilineatum (Arionoidea: Philomycidae), a widespread land slug in East Asia. This is the first report on a mitogenome of the superfamily Arionoidea, and indeed on a terrestrial slug. The mitogenome of Meghimatium bilineatum comprises 13,972 bp and exhibits a novel, highly distinctive gene arrangement among the Stylommatophora. Phylogenetic reconstructions based on the sequences of all protein-coding genes consistently recovered Meghimatium bilineatum as sister-group of the Succineidae. A phylogenetic reconstruction based on gene order, however, suggested a highly divergent tree topology, which is less credible when taking into account prior knowledge of stylommatophoran relationships. Our CREx (Common interval Rearrangement Explorer) analysis suggested that three successive events of tandem duplication random loss (TDRL) best explain the evolutionary process of gene order rearrangement in Meghimatium bilineatum from an ancestral stylommatophoran mitogenome. The present example offers new insights into the mechanisms of mitogenome rearrangements in gastropods at large and into the usefulness of mitogenomic gene order as a phylogenetic marker.

Context-Dependent Passive Avoidance Learning in the Terrestrial Slug Limax.

The terrestrial slug Limax has been used as a model animal for studying the neural mechanisms underlying associative olfactory learning. The slug also innately exhibits negative phototactic behavior using its eyes. In the present study, we developed an experimental paradigm for quantification of slug's negative phototaxis behavior, and investigated whether the nature of the negative phototaxis can be modified by learning experience. The experimental set-up consists of light and dark compartments, between which the slug can move freely. During conditioning, the slug was placed in the light compartment, and an aversive stimulus (quinidine sulfate solution) was applied when it reached the dark compartment. After a single conditioning session, the time to reach the dark compartment significantly increased when it was tested following 24 hr or one week. Protein synthesis inhibition immediately following the conditioning impaired the memory retention at one week but not at 24 hr. The retrieval of the memory was context-dependent, as the time to reach the dark compartment did not significantly increase if the slug was placed on a floor with a different texture in the memory retention test. If the aversive stimulus was applied when the slug was in the light compartment, the time to reach the dark compartment did not increase after 24 hr. This is the first report demonstrating the capability of the slug to form context-dependent passive avoidance memory that can be established in a single conditioning session.

Molecular and morphological evidence for nine species in North American Australapatemon (Sudarikov, 1959): a phylogeny expansion with description of the zygocercous Australapatemon mclaughlini n. sp.

Zygocercous (aggregating) cercarial larvae were recently discovered emerging from a physid snail during a molecular survey of cercariae from molluscs in lakes in central Alberta, Canada. This manuscript delves into the characterization of these cercariae through morphological and molecular techniques and provides the first genetic information for a zygocercous larval trematode. Analyses of cytochrome c oxidase I of mitochondrial DNA and two partial regions of nuclear ribosomal DNA sequences revealed the zygocercous cercariae to belong to the genus Australapatemon Sudarikov, 1959. Further analyses of sequences of Australapatemon burti (Miller, 1923), from cercariae and adults collected from across North America, indicate a complex of nine genetically-distinct lineages within this species, a surprising level of diversity. The zygocercous cercariae, along with adult worms collected from ducks in Manitoba, Canada, and from Mexico, represent one of these lineages, and are herein described as Australapatemon mclaughlini n. sp. Seven lineages cannot yet be identified, but one is tentatively identified as Australapatemon burti.

Snails in the desert: Assessing the mitochondrial and morphological diversity and the influence of aestivation behavior on lineage differentiation in the Australian endemic Granulomelon Iredale, 1933 (Stylommatophora: Camaenidae).

Progressive aridification since the mid-Miocene has had a significant influence on the evolution of the biota in the arid zone of central Australia. Especially moisture sensitive groups, such as snails, are often restricted to topographically complex areas, which have acted as refugia in an otherwise inhospitable environment. This historical fragmentation is deemed to be a potent agent of allopatric lineage diversification. Camaenid land snails are amongst only a few terrestrial gastropods that have managed to survive in the arid zone probably due to their ability to escape desiccation through aestivation. Here, we present the first study of the mitochondrial lineage differentiation in an endemic land snail genus from the Australian 'Red Centre', Granulomelon Iredale, 1933. Exposing significant incongruence between mtDNA phylogeny and morphology-based taxonomy, we completely revise the species and genus level taxonomy of this camaenid group. We demonstrate that this genus contains three species, G. grandituberculatum, G. adcockianum and G. squamulosum, which have so far been assigned to different genera: Granulomelon Iredale, 1933 (junior synonym: Baccalena Iredale, 1937), Basedowena Iredale, 1937 and Pleuroxia Ancey, 1887. Two of these species are widespread comprising multiple divergent mitochondrial lineages. Based on a molecular clock estimate, these lineages diverged approximately during the mid-Pleistocene, a period of particularly severe aridification. The phylogeographic patterns are consistent with an isolation-by-distance model in one species but not the other. We suggest that these differences can be attributed to their distinctive aestivation behavior.

Olfactory Memory Storage and/or Retrieval Requires the Presence of the Exact Tentacle Used During Memory Acquisition in the Terrestrial Slug Limax.

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.

A molecular phylogeny of camaenid land snails from north-western Australia unravels widespread homoplasy in morphological characters (Gastropoda, Helicoidea).

North-western Australia harbours more than 300 species of camaenid land snail in 41 genera exhibiting considerable and well documented morphological diversity. We performed Bayesian and Maximum Likelihood analyses of concatenated nuclear (28S) and mitochondrial (COI, 16S) DNA sequences from 140 species of 37 of these genera plus an additional 27 extralimital species in order to resolve their phylogenetic relationships and to address the significance of morphological characters for the delineation of monophyletic taxa. While north-western Australian Camaenidae in their entirety are not monophyletic with respect to extralimital groups, they underwent extensive in situ-diversification in several independent phylogenetic radiations. A Maximum Likelihood-based character history reconstruction revealed extensive convergence in all studied shell characters and in three out of four genital features across this group. However, in some genera close morphological similarity is best explained by the retention of ancestral characters. We hypothesize that both morphological stasis in some groups and convergent character evolution in others can to a large extent be attributed to adaptive transformations in response to historically increasing aridity throughout north-western Australia in concert with structural constraints.

Thermal Impact and the Relevance of Body Size and Activity on the Oxygen Consumption of a Terrestrial Snail, Theba pisana (Helicidae) at High Ambient Temperatures.

Metabolism, mainly driven by oxygen consumption, plays a key role in life, as it is one of the main ways to respond to extreme temperatures through internal processes. Theba pisana, a widespread Mediterranean land snail, is exposed to a wide range of ambient temperature. In this species the oxygen consumption was tested as a response variable by multiple regression modelling on the "explanatory" variables shell-free mass, temperature, and relative humidity. Our results show that the oxygen consumption of T. pisana can be well described (73.1%) by these three parameters. In the temperature range from 23 °C to 35 °C the oxygen consumption decreased with increasing temperature. Relative humidity, in the range of 67% to 100%, had the opposite effect: if it increases, oxygen consumption will increase as well. Metabolism is proportional to an individual's mass to the power of the allometric scaling exponent α, which is between 0.62 and 0.77 in the mentioned temperature range. CT scans of shells and gravimetry revealed the shell-free mass to be calculated by multiplying the shell diameter to the third power by 0.2105. Data were compared to metabolic scaling exponents for other snails reported in the literature.

Life history and population ecology of Radix swinhoei (Lymnaeidae) in nearshore regions of a hypereutrophic plateau lake.

Accurate assessment of life history and population ecology of widespread species in ultra-eutrophic freshwater lakes is a prerequisite for understanding the mechanisms by which widespread species respond to eutrophication. Freshwater pulmonate (Radix swinhoei) is widespread and abundant in many eutrophic water bodies in Asia. Despite its key roles in eutrophic lake systems, the information on life history and population ecology of R. swinhoei is lacking, especially in ultra-eutrophic freshwater plateau lakes. Here, we conducted a 1-year survey of R. swinhoei with monthly collections to measure the life history traits (life span and growth), annual secondary production, and population size structure of R. swinhoei in nearshore regions with a high seasonally variation of nutrients in Lake Dianchi, a typic hypereutrophic plateau lake in Southwest China. Our results showed that R. swinhoei had the highest biomass in autumn and had the lowest in winter. Its maximum potential life span was 2.5 years, with three recruitment periods (November, March, and July) within a year. Its annual secondary production and P/B ratio were 137.19 g WW/m2 and 16.05, respectively. Redundancy analysis showed that eutrophication-related environmental factors had weak correlations with population size structure of R. swinhoei. Our results suggested that R. swinhoei is a typical r-strategist with high secondary production and thrive in eutrophic environment. Our study can help better understand the mechanisms for widespread species to survive eutrophication and could also be relevant for biodiversity conservation and management of eutrophic ecosystems.

First record of an agriolimacid slug in Southeast Asia - Deroceras laeve (O. F. Müller, 1774) (Gastropoda: Pulmonata) recently introduced to the Socialist Republic of Vietnam.

Several individuals of the terrestrial slug Deroceras laeve were collected in 2018 in the Hoàng Liên Son mountain range of northern Vietnam. The three specimens which were investigated anatomically were aphallic or hemiphallic. A partial COI sequence verified the species identity. This is the first discovery of D. laeve and also of the slug family Agriolimacidae on the Indochinese Peninsula. The collecting site is situated near a cable-car station and below a tourist complex on Fansipan mountain, both of which had just been built by a Swiss-Austrian company between 2013 and 2016. This and the fact that the species had not been found elsewhere in the surrounding area, although searched for thoroughly, indicate that D. laeve is most probably a recent introduction, potentially with building material from Austria or Switzerland.

Complete mitochondrial genome of the Endangered species Ellobium chinense (Pulmonata, Ellobiidae) from Korea.

The complete mitochondrial genome of Ellobium chinense (Ellobioidea, Ellobiidae), an Endangered species in South Korea, is reported here for the first time. The mitogenome of E. chinense is 13,979 base pairs in total length and includes 13 PCGs, small and large rRNAs, and 21 tRNAs. Twelve genes are encoded on the light-strand and 24 genes on the heavy-strand. Compared to four other ellobiid species, the PCGs of E. chinense have a conserved gene order except for the positions of ND4L and ND4. These data provide useful molecular information for phylogenetic studies concerning ellobiids and related species.

A new terrestrial snail species (Gastropoda: Bulimulidae) from the Región de Antofagasta, northern Chile.

A new species of Scutalus Albers, 1850 (Gastropoda: Bulimulidae), Scutalus chango sp. n., is described from a coastal area of northern Chile. Empty shells of this new species were found buried in sand and under boulders and rocks in the foothills of the Chilean Coastal Range at Paposo, Región de Antofagasta. This new species is distinguished from all other Chilean terrestrial snails by its slender shell with a flared and reflected aperture, and by the presence of a columellar fold. This is the first record of Scutalus in Chile, and the southernmost record for this endemic South American bulimulid genus. The presence of this species in Paposo highlights the need for further research and for conservation guidelines in coastal areas of northern Chile, which have comparatively high levels of biodiversity and endemism.

Complete mitochondrial genome of Platevindex sp. (Gastropoda: Pulmonata: Systellommatophora: Onchidiidae).

The complete mitochondrial genome sequence of Platevindex sp. is firstly described in the article. The mitogenome (13,908 bp) contains 22 tRNA genes, 2 ribosomal RNA genes and 13 protein-coding genes, and 1 putative control region (CR). CR is not well characterized due to lack of discrete conserved sequence blocks. This characteristic is similar with CRs of other invertebrate mitochondrial genomes. The characteristic is the typical bivalvia mitochondrial gene composition.

Identification and qualification of 500 nuclear, single-copy, orthologous genes for the Eupulmonata (Gastropoda) using transcriptome sequencing and exon capture.

The qualification of orthology is a significant challenge when developing large, multiloci phylogenetic data sets from assembled transcripts. Transcriptome assemblies have various attributes, such as fragmentation, frameshifts and mis-indexing, which pose problems to automated methods of orthology assessment. Here, we identify a set of orthologous single-copy genes from transcriptome assemblies for the land snails and slugs (Eupulmonata) using a thorough approach to orthology determination involving manual alignment curation, gene tree assessment and sequencing from genomic DNA. We qualified the orthology of 500 nuclear, protein-coding genes from the transcriptome assemblies of 21 eupulmonate species to produce the most complete phylogenetic data matrix for a major molluscan lineage to date, both in terms of taxon and character completeness. Exon capture targeting 490 of the 500 genes (those with at least one exon >120 bp) from 22 species of Australian Camaenidae successfully captured sequences of 2825 exons (representing all targeted genes), with only a 3.7% reduction in the data matrix due to the presence of putative paralogs or pseudogenes. The automated pipeline Agalma retrieved the majority of the manually qualified 500 single-copy gene set and identified a further 375 putative single-copy genes, although it failed to account for fragmented transcripts resulting in lower data matrix completeness when considering the original 500 genes. This could potentially explain the minor inconsistencies we observed in the supported topologies for the 21 eupulmonate species between the manually curated and 'Agalma-equivalent' data set (sharing 458 genes). Overall, our study confirms the utility of the 500 gene set to resolve phylogenetic relationships at a range of evolutionary depths and highlights the importance of addressing fragmentation at the homolog alignment stage for probe design.

Complete mitochondrial genome of Vaginulus alte and Homoiodoris japonica.

The complete mitochondrial genome sequence of Vaginulus alte and Homoiodoris japonica are described first in the article. The mitogenomes (14 772 bp and 14 601 bp) contain 22 tRNA genes, two ribosomal RNA genes, and 13 protein-coding genes, and one putative control region (CR). CR is not well characterized due to the lack of discrete conserved sequence blocks. This characteristic is similar with CRs of other invertebrate mitochondrial genomes, which is the typical bivalvia mitochondrial gene composition.

Complete mitochondrial genome of the giant African snail, Achatina fulica (Mollusca: Achatinidae): a novel location of putative control regions (CR) in the mitogenome within Pulmonate species.

The whole sequence (15,057 bp) of the mitochondrial DNA (mtDNA) of the terrestrial snail Achatina fulica (order Stylommatophora) was determined. The mitogenome, as the typical metazoan mtDNA, contains 13 protein-coding genes (PCG), 2 ribosomal RNA genes (rRNA) and 22 transfer RNA genes (tRNA). The tRNA genes include two trnS without standard secondary structure. Interestingly, among the known mitogenomes of Pulmonata species, we firstly characterized an unassigned lengthy sequence (551 bp) between the cox1 and the trnV which may be the CR for the sake of its AT bases usage bias (65.70%) and potential hairpin structure.

Complete mitochondrial genome of Peronia verruculata (Gastropoda: Pulmonata: Systellommatophora: Onchidiidae).

The complete mitochondrial genome sequence of Peronia verruculata is firstly described in the article. The mitogenome (13,861 bp) contains 22 tRNA genes, 2 ribosomal RNA genes and 13 protein-coding genes. The characteristic is the typical bivalvia mitochondrial gene composition.

Proximate factors influencing egg cannibalism in the land snail Arianta arbustorum (Pulmonata, Helicidae).

Factors affecting oophagy among siblings in the land snail Arianta arbustorum were studied in 3 populations from different altitudes in Switzerland. The degree of egg cannibalism in A. arbustorum is a function of hatching asynchrony since the earliest hatched snails will devour the unhatched eggs in the same clutch. Clutch size, egg density and amount of vegetable food available to newly hatched snails did not affect the degree of cannibalism. Snails from 3 populations were similar in terms of incubation time and intrinsic hatching asynchrony of the clutches. However, they differed in degree of cannibalism when the hatching asynchrony had been experimentally increased. Snails from a lowland forest showed a higher degree of cannibalism than did those from an alpine mountain slope. The parent snails differed in terms of incubation time and hatching synchrony in their clutches. Under natural conditions, the length of the hatching spread and, as a result, the degree of cannibalism will depend additionally on the mode of oviposition (batches or single; clumped or dispersed), on the spatial heterogeneity of egg-laying places and on climatic conditions (e.g. drought).

Gastrocopta (Mollusca, Gastropoda, Pupillidae) in the Pilbara region of Western Australia.

Six species of Gastrocopta have been identified from the Pilbara region, Western Australia, by means of comparative analyses of shell and mtDNA variation. Three of these species, Gastrocopta hedleyi, Gastrocopta larapinta and Gastrocopta servilis, have been recorded in the Pilbara for the first time. Gastrocopta sp. CW1 is probably new to science and might be endemic to the region. By contrast, Gastrocopta hedleyi, Gastrocopta larapinta and Gastrocopta mussoni are shown to be widespread.

Darwininitium - a new fully pseudosigmurethrous orthurethran genus from Nepal (Gastropoda, Pulmonata, Cerastidae).

A new genus and species of pseudosigmurethrous orthurethran pulmonate of the family Cerastidae, Darwininitium shiwalikianumgen. n. and sp. n. is described from the Lesser Himalaya of Nepal. It represents the first record of an orthurethran with a fully developed pseudosigmurethrous pallial system, having a completely closed secondary ureteric system. Biogeographically this new taxon provides a significant range extension for the family north of the previously known distribution range.

Terrestrial molluscs of Pemba Island, Zanzibar, Tanzania, and its status as an "oceanic" island.

Pemba is thought to have had a longer and/or stronger history of isolation than its better-known counterpart, Unguja. The extent to which the biota support this hypothesis of greater oceanicity have been debated. Here, Pemba's terrestrial mollusc ("land-snail") fauna is surveyed and reviewed for the first time. We find at best equivocal evidence for the following hallmarks of greater oceanicity: impoverishment, imbalance, and a high rate of endemism. At least 49 species are present, families are represented in typical proportions, and there are only between two and four island-endemic species - i.e. a 4% to 8% rate of endemism. For land-snails, isolation thus seems to have been short (Pleistocene) or, if longer, weak. Nevertheless, Pemba does host endemic and globally rare species. Forty-five percent of the species found, including most of these, is restricted to forest reserves, with Ngezi Forest Reserve particularly rich. A further 45% are able to tolerate the island's woody cultivated habitats. One new snail species (Cyclophoridae: Cyathopoma) and one new slug species (Urocyclidae: Dendrolimax pro tem.) are described. New data and illustrations are provided for other taxa.