Lateralised courtship behaviour and its impact on mating success in Ostrinia furnacalis (Lepidoptera: Crambidae).

Lateralisation is a well-established phenomenon observed in an increasing number of insect species. This study aims to obtain basic details on lateralisation in courtship and mating behaviour in Ostrinia furnacalis, the Asian corn borer. We conducted laboratory investigations to observe lateralisation in courtship and mating behaviours in adult O. furnacalis. Our goal was also to detect lateralised mating behaviour variations during sexual interactions and to elucidate how these variances might influence the mating success of males. Our findings reveal two distinct lateralised traits: male approaches from the right or left side of the female and the direction of male turning displays. Specifically, males approaching females from their right side predominantly exhibited left-biased 180° turning displays, while males approaching females from the left-side primarily displayed right-biased 180° turning displays. Notably, left-biased males, executing a 180° turn for end-to-end genital contact, initiated copulation with fewer attempts and began copulation earlier than their right-biased approaches with left-biased 180° turning displays. Furthermore, mating success was higher when males subsequently approached the right side of females during sexual encounters. Left-biased 180° turning males exhibited a higher number of successful mating interactions. These observations provide the first report on lateralisation in the reproductive behaviour of O. furnacalis under controlled laboratory conditions and hold promise for establishing reliable benchmarks for assessing and monitoring the quality of mass-produced individuals in pest control efforts.

Fine mapping of the Cepaea nemoralis shell colour and mid-banded loci using a high-density linkage map.

Molluscs are a highly speciose phylum that exhibits an astonishing array of colours and patterns, yet relatively little progress has been made in identifying the underlying genes that determine phenotypic variation. One prominent example is the land snail Cepaea nemoralis for which classical genetic studies have shown that around nine loci, several physically linked and inherited together as a 'supergene', control the shell colour and banding polymorphism. As a first step towards identifying the genes involved, we used whole-genome resequencing of individuals from a laboratory cross to construct a high-density linkage map, and then trait mapping to identify 95% confidence intervals for the chromosomal region that contains the supergene, specifically the colour locus (C), and the unlinked mid-banded locus (U). The linkage map is made up of 215,593 markers, ordered into 22 linkage groups, with one large group making up ~27% of the genome. The C locus was mapped to a ~1.3 cM region on linkage group 11, and the U locus was mapped to a ~0.7 cM region on linkage group 15. The linkage map will serve as an important resource for further evolutionary and population genomic studies of C. nemoralis and related species, as well as the identification of candidate genes within the supergene and for the mid-banding phenotype.

Environmental DNA metabarcoding reveals comparable responses to agricultural stressors on different trophic levels of a freshwater community.

Freshwater habitats are under stress from agricultural land use, most notably the influx of neonicotinoid pesticides and increased nutrient pressure from fertilizer. Traditional studies investigating the effects of stressors on freshwater systems are often limited to a narrow range of taxa, depending heavily on morphological expertise. Additionally, disentanglement of multiple simultaneous stressors can be difficult in field studies, whereas controlled laboratory conditions do not accurately reflect natural conditions and food webs. To overcome these drawbacks, we investigated the impacts of two agricultural stressors (the neonicotinoid insecticide thiacloprid and fertilizer) in full-factorial design in a semi-natural research site, using environmental DNA sampling to study three different taxonomic groups representing three trophic levels: bacteria (decomposers), phytoplankton (primary producers), and chironomids (consumers). The results show considerable impact of both stressors across trophic levels, with an additive effect of fertilizer and thiacloprid on community composition at all levels. These findings suggest that agricultural stressors affect the entire food web, either directly or through cascade reactions. They are also consistent with morphological assessments that were performed in the same study site, even at a lower number of replicates. The study presented shows that the use of multimarker environmental DNA provides a more comprehensive assessment of stressor impacts across multiple trophic levels, at a higher taxonomic resolution than traditional surveys. Additionally, many putative novel bioindicators for both agricultural stressors were discovered. We encourage further investigations into stressors impacts at different trophic levels, which will lead to more effective monitoring and management of freshwater systems.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

Changes in richness and community composition of ectomycorrhizal fungi among altitudinal vegetation types on Mount Kinabalu in Borneo.

The distribution patterns of tropical ectomycorrhizal (ECM) fungi along altitudinal gradients remain largely unknown. Furthermore, despite being an iconic site for biodiversity research, virtually nothing is known about the diversity and spatial patterns of fungi on Mt Kinabalu and neighbouring mountain ranges. We carried out deep DNA sequencing of soil samples collected between 425 and 4000 m above sea level to compare richness and community composition of ECM fungi among altitudinal forest types in Borneo. In addition, we tested whether the observed patterns are driven by habitat or by geometric effect of overlapping ranges of species (mid-domain effect). Community composition of ECM fungi was strongly correlated with elevation. In most genera, richness peaked in the mid-elevation montane forest zone, with the exception of tomentelloid fungi, which showed monotonal decrease in richness with increasing altitude. Richness in lower-mid- and mid-elevations was significantly greater than predicted under the mid-domain effect model. We provide the first insight into the composition of ECM fungal communities and their strong altitudinal turnover in Borneo. The high richness and restricted distribution of many ECM fungi in the montane forests suggest that mid-elevation peak richness is primarily driven by environmental characteristics of this habitat and not by the mid-domain effect.

Drosophila pachea asymmetric lobes are part of a grasping device and stabilize one-sided mating.

BACKGROUND: Multiple animal species exhibit morphological asymmetries in male genitalia. In insects, left-right genital asymmetries evolved many times independently and have been proposed to appear in response to changes in mating position. However, little is known about the relationship between mating position and the interaction of male and female genitalia during mating, and functional analyses of asymmetric morphologies in genitalia are virtually non-existent. We investigated the relationship between mating position, asymmetric genital morphology and genital coupling in the fruit fly Drosophila pachea, in which males possess an asymmetric pair of external genital lobes and mate in an unusual right-sided position on top of the female. RESULTS: We examined D. pachea copulation by video recording and by scanning electron microscopy of genital complexes. We observed that the interlocking of male and female genital organs in D. pachea is remarkably different from genital coupling in the well-studied D. melanogaster. In D. pachea, the female oviscapt valves are asymmetrically twisted during copulation. The male's asymmetric lobes tightly grasp the female's abdomen in an asymmetric 'locking' position, with the left and right lobes contacting different female structures. The male anal plates, which grasp the female genitalia in D. melanogaster, do not contact the female in D. pachea. Experimental lobe amputation by micro-surgery and laser-ablation of lobe bristles led to aberrant coupling of genitalia and variable mating positions, in which the male was tilted towards the right side of the female. CONCLUSION: We describe, for the first time, how the mating position depends on coupling of male and female genitalia in a species with asymmetric genitalia and one-sided mating position. Our results show that D. pachea asymmetric epandrial lobes do not act as a compensatory mechanism for the change from symmetric to one-sided mating position that occurred during evolution of D. pachea's ancestors, but as holding devices with distinct specialized functions on the left and right sides.

Fitness benefits of the fruit fly Rhagoletis alternata on a non-native rose host.

Many species have been introduced worldwide into areas outside their natural range. Often these non-native species are introduced without their natural enemies, which sometimes leads to uncontrolled population growth. It is rarely reported that an introduced species provides a new resource for a native species. The rose hips of the Japanese rose, Rosa rugosa, which has been introduced in large parts of Europe, are infested by the native monophagous tephritid fruit fly Rhagoletis alternata. We studied differences in fitness benefits between R. alternata larvae using R. rugosa as well as native Rosa species in the Netherlands. R. alternata pupae were larger and heavier when the larvae fed on rose hips of R. rugosa. Larvae feeding on R. rugosa were parasitized less frequently by parasitic wasps than were larvae feeding on native roses. The differences in parasitization are probably due to morphological differences between the native and non-native rose hips: the hypanthium of a R. rugosa hip is thicker and provides the larvae with the possibility to feed deeper into the hip, meaning that the parasitoids cannot reach them with their ovipositor and the larvae escape parasitization. Our study shows that native species switching to a novel non-native host can experience fitness benefits compared to the original native host.

Quantitative analysis of amygdalin and prunasin in Prunus serotina Ehrh. using (1) H-NMR spectroscopy.

INTRODUCTION: Prunus serotina is native to North America but has been invasively introduced in Europe since the seventeenth century. This plant contains cyanogenic glycosides that are believed to be related to its success as an invasive plant. For these compounds, chromatographic- or spectrometric-based (targeting on HCN hydrolysis) methods of analysis have been employed so far. However, the conventional methods require tedious preparation steps and a long measuring time. OBJECTIVE: To develop a fast and simple method to quantify the cyanogenic glycosides, amygdalin and prunasin in dried Prunus serotina leaves without any pre-purification steps using (1) H-NMR spectroscopy. METHODS: Extracts of Prunus serotina leaves using CH3 OH-d4 and KH2 PO4 buffer in D2 O (1:1) were quantitatively analysed for amygdalin and prunasin using (1) H-NMR spectroscopy. Different internal standards were evaluated for accuracy and stability. The purity of quantitated (1) H-NMR signals was evaluated using several two-dimensional NMR experiments. RESULTS: Trimethylsilylpropionic acid sodium salt-d4 proved most suitable as the internal standard for quantitative (1) H-NMR analysis. Two-dimensional J-resolved NMR was shown to be a useful tool to confirm the structures and to check for possible signal overlapping with the target signals for the quantitation. Twenty-two samples of P. serotina were subsequently quantitatively analysed for the cyanogenic glycosides prunasin and amygdalin. CONCLUSION: The NMR method offers a fast, high-throughput analysis of cyanogenic glycosides in dried leaves permitting simultaneous quantification and identification of prunasin and amygdalin in Prunus serotina.

A new, unusually large, Clavicornaltica Scherer, 1974 flea beetle from Borneo, described and sequenced in the field by citizen scientists (Coleoptera, Chrysomelidae, Galerucinae).

Background: The genus Clavicornaltica Scherer 1974 consists of very small, soil-dwelling flea beetles in South, Southeast and East Asia. Due to their diminutive size and morphological similarities, very little is known about their ecology and taxonomical diversity. It is likely that further studies will reveal this genus to be much more speciose than the 30 species currently recognised. New information: A new species of Clavicornaltica from Brunei Darussalam is described, C.mataikanensis Otani et al., sp. nov. This is the second species of this genus recorded from Ulu Temburong National Park.

New records of moth flies (Diptera, Psychodidae) for the Dutch Fauna.

Background: Prior to this study, the moth flies in The Netherlands were represented by 61 species. Our findings derive from a citizen-science expedition in the Vondelpark in Amsterdam, one of the oldest public parks and best known parks in The Netherlands. The combination of citizen science and the exploration of a well-known urban park has allowed us to contribute to the knowledge of moth fly species present in The Netherlands. The findings from this study provide valuable insights into the distribution, taxonomy and genetic resources of Psychoda and Panimerus species, enhancing our understanding of insect biodiversity and promoting future research in this field. New information: Our study provides two new geographical records of the moth flies in The Netherlands, namely, Psychodauniformata Haseman, 1907 and Panimerusmaynei (Tonnoir, 1920) elevating the total number of species to 63. Furthermore, we provide re-descriptions of the females of Panimerusnotabilis (Eaton, 1893) and P.goetghebueri (Tonnoir, 1919). Additionally, we make available for the first time, the sequence of the 5'-end of the cytochrome c oxidase subunit I (COI) gene or COI Barcodes for Panimerusnotabilis, P.goetghebueri and P.maynei. These COI Barcodes serve as valuable tools for future species identification within the genus.

A new semi-slug of the genus Microparmarion from Brunei, discovered, described and DNA-barcoded on citizen-science 'taxon expeditions' (Gastropoda, Stylommatophora, Ariophantidae).

Background: During citizen-science expeditions to the Ulu Temburong National Park, Brunei, several individuals were collected of a semi-slug species of the genus Microparmarion that, based on morphology and in-the-field DNA-barcoding, was found to be an undescribed species. New information: In this paper, we describe Microparmarionsallehi Wu, Ezzwan & Hamdani, n. sp., after field centre supervisor Md Salleh Abdullah Bat. We provide details on the external and internal reproductive morphology, the shell and the ecology of the type locality, as well as a diagnosis comparing it with related species. DNA barcodes were generated for five individuals and used for a phylogenetic reconstruction. Microparmarionsallehi sp. n. and M.exquadratus Schilthuizen et al., 2019 so far are the only Bornean species of the genus that live in lowland forest; other species are found in montane forests.

The complete mitogenome of Cylindrus obtusus (Helicidae, Ariantinae) using Illumina next generation sequencing.

BACKGROUND: This study describes how the complete mitogenome of a terrestrial snail, Cylindrus obtusus (Draparnaud, 1805) was sequenced without PCRs from a collection specimen that had been in 70% ethanol for 8 years. The mitogenome was obtained with Illumina GAIIx shot gun sequencing. Although the used specimen was collected relatively recently and kept in a DNA-friendly preservative (not formalin as frequently used with old museum specimens), we believe that the exclusion of PCRs as facilitated by NGS (Next Generation Sequencing) removes a great obstacle in DNA sequencing of collection specimens. A brief comparison is made between our Illumina GAIIx approach and a similar study that made use of the Roche 454-FLX platform. RESULTS: The mtDNA sequence of C. obtusus is 14,610 bases in length (about 0.5 kb larger than other stylommatophoran mitogenomes reported hitherto) and contains the 37 genes (13 protein coding genes, two rRNAs and 22 tRNAs) typical for metazoans. Except for a swap between the position of tRNA-Pro and tRNA-Ala, the gene arrangement of C. obtusus is identical to that reported for Cepaea nemoralis. The 'aberrant' rearrangement of tRNA-Thr and COIII compared to that of other Sigmurethra (and the majority of gastropods), is not unique for C. nemoralis (subfamily Helicinae), but is also shown to occur in C. obtusus (subfamily Ariantinae) and might be a synapomorphy for the family Helicidae. CONCLUSIONS: Natural history collections potentially harbor a wealth of information for the field of evolutionary genetics, but it can be difficult to amplify DNA from such specimens (due to DNA degradation for instance). Because NGS techniques do not rely on primer-directed amplification (PCR) and allow DNA to be fragmented (DNA gets sheared during library preparation), NGS could be a valuable tool for retrieving DNA sequence data from such specimens. A comparison between Illumina GAIIx and the Roche 454 platform suggests that the former might be more suited for de novo sequencing of mitogenomes.

Phylogenetic reconstruction and shell evolution of the Diplommatinidae (Gastropoda: Caenogastropoda).

The fascinating and often unlikely shell shapes in the terrestrial micromollusc family Diplommatinidae (Gastropoda: Caenogastropoda) provide a particularly attractive set of multiple morphological traits to investigate evolutionary patterns of shape variation. Here, a molecular phylogenetic reconstruction, based on five genes and 2700 bp, was undertaken for this family, integrated with ancestral state reconstruction and phylogenetic PCA of discrete and quantitative traits, respectively. We found strong support for the Diplommatininae as a monophyletic group, separating the Cochlostomatidae into a separate family. Five main clades appear within the Diplommatininae, corresponding with both coiling direction and biogeographic patterns. A Belau clade (A) with highly diverse (but always sinistral) morphology comprised Hungerfordia, Palaina, and some Diplommatina. Arinia (dextral) and Opisthostoma (sinistroid) are sister groups in clade B. Clade C and D solely contain sinistral Diplommatina that are robust and little ornamented (clade C) or slender and sculptured (clade D). Clade E is dextral but biogeographically diverse with species from all sampled regions save the Caroline Islands. Adelopoma, Diplommatina, Palaina, and Hungerfordia require revision to allow taxonomy to reflect phylogeny, whereas Opisthostoma is clearly monophyletic. Ancestral state reconstruction suggests a sinistral origin for the Diplommatinidae, with three reversals to dextrality.

No evidence for asymmetric sperm deposition in a species with asymmetric male genitalia.

Background: Asymmetric genitalia have repeatedly evolved in animals, yet the underlying causes for their evolution are mostly unknown. The fruit fly Drosophila pachea has asymmetric external genitalia and an asymmetric phallus with a right-sided phallotrema (opening for sperm release). The complex of female and male genitalia is asymmetrically twisted during copulation and males adopt a right-sided copulation posture on top of the female. We wished to investigate if asymmetric male genital morphology and a twisted gentitalia complex may be associated with differential allocation of sperm into female sperm storage organs. Methods: We examined the internal complex of female and male reproductive organs by micro-computed tomography and synchrotron X-ray tomography before, during and after copulation. In addition, we monitored sperm aggregation states and timing of sperm transfer during copulation by premature interruption of copulation at different time-points. Results: The asymmetric phallus is located at the most caudal end of the female abdomen during copulation. The female reproductive tract, in particular the oviduct, re-arranges during copulation. It is narrow in virgin females and forms a broad vesicle at 20 min after the start of copulation. Sperm transfer into female sperm storage organs (spermathecae) was only in a minority of examined copulation trials (13/64). Also, we found that sperm was mainly transferred early, at 2-4 min after the start of copulation. We did not detect a particular pattern of sperm allocation in the left or right spermathecae. Sperm adopted a granular or filamentous aggregation state in the female uterus and spermathecae, respectively. Discussion: No evidence for asymmetric sperm deposition was identified that could be associated with asymmetric genital morphology or twisted complexing of genitalia. Male genital asymmetry may potentially have evolved as a consequence of a complex internal alignment of reproductive organs during copulation in order to optimize low sperm transfer rates.

Species identification skills predict in-depth knowledge about species.

To raise biodiversity awareness effectively, communicators should be aware of knowledge levels in their audiences. Species identification skills have been used in the past as a measure of what people know about species, yet it is not known whether they serve as good indicators. To study the link between species identification and in-depth species knowledge, we presented an animal knowledge test to an online audience of over 7,000 Dutch adults, and used correlation and regression analyses to determine the extent to which species identification predicts in-depth knowledge about species' origin, habitat, diet, and behavior. We found that in-depth knowledge was higher in those who correctly identified species as compared with those who did not correctly identify species, for all four types of in-depth knowledge. Moreover, as compared to alternative variables (work, age, gender, and educational level), species identification was by far the best predictor for in-depth knowledge about species. However, species identification levels were generally higher than levels of in-depth knowledge, and knowledge gaps and misconceptions were uncovered. The results confirm the value of species identification tests, but also highlight limitations and challenges that should be taken into account when establishing knowledge levels and communicating biodiversity.

Animal biodiversity and specificity in children's picture books.

While animal biodiversity is declining globally, cultural representations of animals are highly prevalent in society and play an increasing part in shaping children's perceptions of animal diversity. We studied animal portrayals in children's picture books in the Netherlands, and coded over 2,200 animals from 217 award-winning books. We found a strong bias toward vertebrates, mammals in particular. Mammals were featured more often than other animals, played more prominent roles in the story, and were visually and textually specified more strongly. Furthermore, exotic and domestic species outnumbered native species. Picture books currently are likely to reinforce children's perceptions toward only a small part of animal biodiversity. While we realize that picture books have other primary aims, picture book makers could be inspired and encouraged to diversify and specify their portrayals of the natural world. This would broaden children's perceptions of the animal kingdom and could help foster lasting connections to biodiversity.

A new giant keelback slug of the genus Limax from the Balkans, described by citizen scientists.

Background: Despite their large size, striking colouration and genital extravagance, the taxonomy of the European giant keelback slugs of the genus Limax is still poorly understood. Preliminary morphological and molecular data suggest that many unnamed or unrecognised species exist, especially in the Alps, the Mediterranean and the Balkans. New information: We organised a citizen science expedition to Durmitor National Park in Montenegro and discovered a new species, genetically distinct, but morphologically similar to the sympatric L.cinereoniger Wolf 1803 and describe it as L.pseudocinereoniger.

Evo-devo of shell colour in gastropods and bivalves.

Recent technical innovations are revealing surprising patterns in mollusc shell pigmentation, such as an unexpectedly modest role for melanins and rapid divergences in the mix of pigments used to achieve similar colour patterns. The elucidation of the molecular genetic basis of shell pigmentation has been slow, probably because of the high genome complexity of gastropods and bivalves. Recent work within the old field of evolutionary ecology of shell pigmentation allows a greater role for the analysis of large-geographic-scale patterns (sometimes employing citizen-science data), as well as experimental field studies. However, the field remains dominated by land snails as model organisms, while colour pattern evolution in marine gastropods and bivalves, particularly those not exposed to visual predators, remains mysterious.

The draft genome sequence of the grove snail Cepaea nemoralis.

Studies on the shell color and banding polymorphism of the grove snail Cepaea nemoralis and the sister taxon Cepaea hortensis have provided compelling evidence for the fundamental role of natural selection in promoting and maintaining intraspecific variation. More recently, Cepaea has been the focus of citizen science projects on shell color evolution in relation to climate change and urbanization. C. nemoralis is particularly useful for studies on the genetics of shell polymorphism and the evolution of "supergenes," as well as evo-devo studies of shell biomineralization, because it is relatively easily maintained in captivity. However, an absence of genomic resources for C. nemoralis has generally hindered detailed genetic and molecular investigations. We therefore generated ∼23× coverage long-read data for the ∼3.5 Gb genome, and produced a draft assembly composed of 28,537 contigs with the N50 length of 333 kb. Genome completeness, estimated by BUSCO using the metazoa dataset, was 91%. Repetitive regions cover over 77% of the genome. A total of 43,519 protein-coding genes were predicted in the assembled genome, and 97.3% of these were functionally annotated from either sequence homology or protein signature searches. This first assembled and annotated genome sequence for a helicoid snail, a large group that includes edible species, agricultural pests, and parasite hosts, will be a core resource for identifying the loci that determine the shell polymorphism, as well as in a wide range of analyses in evolutionary and developmental biology, and snail biology in general.

Microbiome and environment explain the absence of correlations between consumers and their diet in Bornean microsnails.

Classical ecological theory posits that species partition resources such that each species occupies a unique resource niche. In general, the availability of more resources allows more species to co-occur. Thus, a strong relationship between communities of consumers and their resources is expected. However, correlations may be influenced by other layers in the food web, or by the environment. Here we show, by studying the relationship between communities of consumers (land snails) and individual diets (from seed plants), that there is in fact no direct, or at most a weak but negative, relationship. However, we found that the diversity of the individual microbiome positively correlates with both consumer community diversity and individual diet diversity in three target species. Moreover, these correlations were affected by various environmental variables, such as anthropogenic activity, habitat island size, and a possibly important nutrient source, guano runoff from nearby caves. Our results suggest that the microbiome and the environment explain the absence of correlations between diet and consumer community diversity. Hence, we advocate that microbiome inventories are routinely added to any community dietary analysis, which our study shows can be done with relatively little extra effort. Our approach presents the tools to quickly obtain an overview of the relationships between consumers and their resources. We anticipate our approach to be useful for ecologists and environmentalists studying different communities in a local food web.