Philippa Kaur

Project 1: mesocosm field experimentsMesocosm experiments took place at Lockwood Farm located in Hamden, Connecticut. Individual mesocosms were composed of black 20 L cylindrical plastic containers filled with 12 L tap water and seeded with 10 mg of a 3:2 ratio liver powder/brewer’s yeast mixture and 1 g of grass hay. Drain-holes were drilled into the sides of each container 5 mm from the 12 L surface to allow flooding for Aedes spp. egg emergence and to allow overflow beyond this level due to precipitation. Four experimental mesocosm clusters were dispersed throughout the Lockwood Farm in microhabitats previously sampled in Eastwood et al.22. Clusters contained 4 mesocosms spaced 3 m apart in a 2 × 2 grid. We utilized four L. sphaericus treatment levels in each cluster: no L. sphaericus, the LC50 (0.053 ITU/ml) and LC95 (1.0 ITU/ml) for Culex pipiens derived from Burtis et al.3, and the label rate of L. sphaericus (~ 1.2 ITU/ml). All treatments were derived from VectoLex WDG. Prior to insecticide application, we prepared 1 L of a 1000 ITU/ml stock solution. To inoculate each mesocosm, we measured the depth of the container’s water column, calculated water volume, and applied the appropriate amount of stock to achieve the target LC value. Replicate insecticide treatments were randomized within each cluster, and insecticides were applied 30-days post mesocosm seeding with nutrients. All mesocosms in each cluster were rotated within the 2 × 2 grid each week. Two clusters were then randomly chosen for a second application of L. sphaericus 30-days post initial insecticide application.To sample the larval habitat of each mesocosm, we performed a figure-8 sweep with an aquarium fish net (4 × 3-in. opening, Penn-Plax) each Monday and Thursday of the week for each week of the experiment. Sweep contents were washed from the net into a white photo development pan, and pupae were removed for in-lab identification after eclosion following a dichotomous key23. All larvae were then returned to the mesocosm. This sampling protocol minimized destruction of larval habitats and influence of interspecific interactions due to removal sampling.In addition to sampling containers for pupae, we collected water samples from each container for an in-lab bioassay to determine the realized mortality of the larval environment. Due to time constraints of the field crew, a 50% randomized sample of containers were sampled on Monday with the remaining 50% sampled on Thursday of each sampling week. Bioassay procedures followed McMillan et al.24 for Cx. pipiens with the addition of screening mortality in CAES’ Ae. albopictus colonies. We finally performed in-lab susceptibility trials to L. sphaericus with larvae from CAES’ Cx. pipiens and Ae. albopictus colonies to confirm each species’ colony varied in their sensitivity to the product. Briefly, 15 3rd to 4th instar larvae of each species per replicate dose were exposed to a wide range of L. sphaericus concentrations and mortality was recorded 24-h post-exposure. Lethal concentrations were then estimated from a generalized linear model with mortality (corrected for mortality in untreated control replicates) as the response term and the log10-dose as the predictor term.Primary endpoints from the field experiment included the number and species identity of pupae collected from each mesocosm. We compared total weekly pupal collections per mesocosm using a generalized linear mixed model (GLMM) framework with treatment level and cluster ID as fixed effects, species ID and week of collection as a random effect, and a Poisson-error distribution. We repeated this analysis excluding all collected Culex spp. to examine how the L. sphaericus treatments impacted the more tolerant Aedes spp. The primary endpoint for the mortality assays was the corrected larval mortality. We initially compared mortality using a species-specific GLMM with L. sphaericus treatment concentration and treatment period as fixed effects, week of collection as a random effect, and a binomial-error distribution. Preliminary analyses revealed negligible variance attributed to week of collection, so all subsequent models were a GLM. All analyses were performed in R V4.1.325 using the following packages: tidyverse26, gridExtra27, ggplot228, ggeffects29, and glmmTMB30.Project 2: laboratory competition assaysCompetition assays took place at CAES’ main facility in New Haven, CT. This facility contains an Ae. albopictus colony (founded circa 2014 from Stratford, CT) and a Cx. pipiens colony (founded circa 2018 from New Haven, CT;). Colony maintenance for each species was similar: larval rearing pans consisted of approx. 200 eggs (on papers, Ae. albopictus, or as egg rafts, Cx. pipiens) in ~ 2 L RO water and initiated with ~ 20 ml of a 1% 3:2 liver powder/brewer’s yeast slurry. Pans were held at 25.5 °C and 80% humidity and fed ~ 20 ml of the 1% slurry every other day. Pupae were removed to an eclosion chamber and adults were allowed access to 10% sucrose solution ad libitum. Aedes albopictus females were given access to defibrinated sheep’s blood (HemoStat©) through a Hemotek membrane feeder for 1 h every 2–3 weeks and moistened, fluted filter paper was provided to collect eggs. Culex pipiens females were given access to a live, restrained buttonquail overnight once per week and a small cup seeded with 5 ml 1% slurry and 15 RO ml water was provided to collect egg rafts. The use of buttonquail was reviewed and approved in accordance with CAES Institutional Animal Care and Use Committee.We performed two experiments. All experiments consisted of the following treatments: variable ratios of Ae:Cx larvae and two L. sphaericus treatments (no treatment and 0.01 ITU/ml). Larval density (40 per container) remained constant across all replicate treatments, but Ae:Cx ratios varied from 40/0, 30/10, 20/20, 10/30, and 0/40. Nutrients supplied were a low concentration (3 mg larva−1) of a 3:2 liver powder/brewer’s yeast mix applied at the beginning of the experiment. Temperature was held constant at the colony maintenance level. Assays took place in 300 ml disposable plastic cups filled with 100 ml of RO water. The first experiments consisted of the addition of the 40 larvae as newly hatched individuals (+/− 1 day between species’ hatch) at the appropriate ratios, the larval diet, and the 0.01 ITU/ml concentration (diluted from a lab stock of 1000 ITU/ml). Assays were monitored daily until all larvae were dead and/or all larvae pupated. Experiment 2 consisted of the addition of only the Cx. pipiens larvae and the larval diet. After all Cx. pipiens had pupated, containers were treated with L. sphaericus and then the Ae. Albopictus larvae were added.Primary endpoints included species-specific pupation success. Preliminary analyses in a GLMM framework revealed negligible variance attributed to a replicate ID random effect; replicate as a random term also interfered with model convergence. Preliminary analyses further revealed there was neither a significant interaction nor an improvement in the Akaike Information Criterion between the L. sphaericus treatment and initial starting condition terms. Thus, we adopted a GLM rather than a GLMM framework in all further analyses, and species-specific mortality was analyzed as a binomial response term with treatment and initial starting conditions included as fixed effects All analyses were performed in R V4.1.325 using the following packages: tidyverse26, gridExtra27, and ggplot228. More

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Study areaWe conducted the study in the best-preserved stands of the Białowieża Forest, strictly protected within the Białowieża National Park (hereafter BNP; coordinates of Białowieża village: 52°42′N, 23°52′E). The extensive Białowieża Forest (c. 1500 km2) straddles the Polish-Belarusian border, where the climate is subcontinental with annual mean temperatures during May–July of 13–18 °C, and mean annual precipitation of 426–940 mm66,67.The forest provides a unique opportunity to observe animals under conditions that likely prevailed across European lowlands before widespread deforestation and forest exploitation by humans66,68,69. The stands have retained a primeval character distinguished by a multi-layered structure, frequent fallen and standing dead trees, and a high species richness66,70. The stands are composed of about a dozen tree species of various ages, up to several hundred years old. The interspecific interactions and natural processes have been little affected by direct human activity.We conducted observations mostly within the three permanent study plots (MS, N, W), totalling c. 130 ha, and in other nearby fragments of primeval oak-lime-hornbeam Tilio-Carpinetum or mixed deciduous-coniferous Pino-Quercetum stands. However, a small number of observations from adjacent managed deciduous forest stands were also included. For details of the study area see71,72,73.Study speciesOur study system focused on ground-nesting Wood Warblers Phylloscopus sibilatrix, blowflies Protocalliphora azurea, and Myrmica or Lasius ants, which occurred in the birds’ nests.The Wood Warbler is a small (c. 10 g) insectivorous songbird that winters in equatorial Africa and breeds in temperate European forests, typically rearing one or two broods each year74. Wood Warblers build dome-shaped nests for each breeding attempt, composed of woven grass, leaves and moss, and lined with animal hair73. The nests are situated on the ground among moderately sparse vegetation, often under a tussock of vegetation or near a fallen tree-branch or log (see examples in Supplementary Fig. S2)53,75. The breeding season of Wood Warblers begins in late April–early May and ends in July–August, when nestlings from replacement clutches (after initial loss) or second broods leave the nest. The typical clutch size in BNP is 5–7 eggs, and the nestling stage lasts 12–13 days74,76.Wood Warbler nests are inhabited by various arthropods, including Myrmica ruginodis or M. rubra ants, and less often Lasius platythorax, L. niger or L. brunneus. The ants foraged and/or raised their own broods within the Wood Warbler nests52. The Myrmica and Lasius ant species are common in Europe77,78. Their colonies contain from tens to thousands of workers, and can be found on the forest floor, e.g. in soil, within or under fallen dead wood, in patches of moss, or among fallen tree-leaves53,77,78. All of the ant species found in the Wood Warbler nests are predators of other arthropods77,79,80.Blowflies, Protocalliphora spp., are obligatory blood-sucking (hematophagous) ectoparasites that reproduce within bird nests. The occurrence, abundance, and impact of blowflies on Wood Warbler offspring is largely unknown, similar to many other European songbirds that build dome-shaped nests. Adult blowflies emerge in late spring and summer to lay eggs on the birds’ nesting material or directly onto the skin of typically newly hatched nestlings14,26. The blowfly larvae hatch within two–three days, and develop in the structure of warm bird nests for another 6–15 days, during which they emerge intermittently to feed on host blood, before finally pupating within the nests14,25,26,27.Data collectionNest monitoring and measurements of nestlingsWe searched for Wood Warbler nests daily from late April until mid-July in 2018–2020, by following birds mainly during nest-building. Nests were assigned to a deciduous or mixed deciduous-coniferous habitat type, depending on the tree stand where they were found. We inspected nests systematically, according to the protocol described in Wesołowski and Maziarz76. The number of observer visits was kept to a minimum to reduce disruptions for birds or potential risks of nest predation.We aimed to establish the dates of hatching (day 0 ± 1 day), nestlings vacating the nest (fledging; ± 1 day) or nest failure (± 1–2 days). When nestlings hatched asynchronously, the hatching date corresponded to the earliest record of nestling hatching. The dates of fledging or nest failure were the mid-dates between the last visit when the nestlings were present in the nest, and the following visit, when the nest was found empty. Nest failure was primarily due to predation, which is the main cause of the Wood Warbler nest losses in BNP76,81 and elsewhere in Europe82,83.To assess fitness consequences for birds of variable weather conditions, blowfly abundance and/or ant presence, we measured nestling growth and determined brood reduction (i.e. the mortality of chicks in the nest) from hatching until fledging. To define brood reduction, we assessed the number of hatchlings (nestlings up to 4 days old) and the number of fledglings leaving the nests. To ensure accurate counting and avoid premature fledging of nestlings, we established the number of fledglings on the day of measurement, when all nestlings were temporarily extracted from the nest.We measured nestling growth on a single occasion when they were 6–9 days old (median 8 days), almost fully developed but too young to leave the nest. The measurements lasted for less than 10–15 min at each nest to minimise any potential risk of attracting predators. For each nestling we measured (using a ruler) the emerged length of the longest (3rd) primary feather vane (± 0.5 mm) on the left wing84,85, and body mass to the nearest 0.1 g using an electronic balance. The length of the feather vane is closely linked to feather growth86 and is one of the characteristics of nestling growth85,87. We treated the length of the primary feather vane and body mass as indices of nestling growth rate under varying conditions of weather, blood-sucking ectoparasites, or ant presence.Extraction of arthropods from bird nestsTo assess the number of blowflies and to establish the presence of ants, we checked the contents of 129 nests (including 11 nests from the managed forest stands) at which Wood Warbler nestlings had been measured. The sample included 86 successful breeding attempts (where a minimum of one nestling successfully left the nest), 27 failed (predated) nests (remnants of nestlings were found, but the nest structure remained intact), and 16 nests with an unknown fate (nestlings were large, so were capable of leaving the nest, but no family were located or other signs indicating fledging).Due to ethical reasons, we were unable to collect the Wood Warbler nests and extract the ectoparasites and ants from them while they were in use by the birds. Removing the nests and replacing them with dummy nests would cause unacceptable nest desertion by adults. Therefore, we assessed the occurrence and number of blowflies or ant presence after Wood Warbler nestlings fledged or the breeding attempts failed naturally. We retrospectively explored the changes in blowfly infestation14, including the effect of ant presence53 in the same nests.We collected nests from the field as soon as a breeding attempt ended, within approximately five days (median 1 day) following fledging or nest failure (nest structure remained intact). The delay of nest collection would not bias the ectoparasite infestation, as blowfly larvae pupate within bird nests and stay there after the hosts abandon their nests; puparia can be still found in nests collected in autumn or winter14. As the likelihood of finding ant broods (larvae or pupae associated with workers) was rather stable with the delay of nest collection53, the method seemed reliable also for assessing the presence of ant broods (35 of all 71 Wood Warbler nests containing ants). Only the number of nests with lone foraging ant workers could be underestimated, potentially inflating the uncertainty of tested relationships. However, as ants usually re-use rich food resources88, foraging Myrmica or Lasius ant workers might regularly exploit warbler nests, increasing the chances of finding the insects in the collected nests.Wood Warbler nests were collected in one piece, with each placed into a separate sealed and labelled plastic bag. We carefully inspected the leaf litter around the nests, and the soil surface under them, to make sure that all blowfly larvae or pupae were collected. We transported the collected nests to a laboratory, where we stored them in a fridge for up to 5–6 days before the arthropod extraction.To establish the number of blowflies and the presence of ants, in 2018, we carefully pulled apart the nesting material and searched for the arthropods amongst it 52. We gathered all blowfly pupae or larvae and a sample of ant specimens into separate tubes, labelled and filled with 70–80% alcohol, for later species identification. For nests collected in 2019–2020, we extracted the arthropods with a Berlese-Tullgren funnel. During the extraction, which usually lasted for 72 h, each nest was covered with fine metal mesh and placed c. 15 cm under the heat of a 40 W electric lamp. The arthropods were caught in 100 ml plastic bottles containing 30 ml of 70–80% ethanol, installed under each funnel. After the arthropod extraction, we carefully inspected the nesting material in the same way as in 2018, to collect any blowflies that remained within the nests. The quality of information collected on the number of ectoparasites and ant presence should be comparable each year.Weather dataWe obtained the mean daily temperatures and rainfall sums from a meteorological station, operated by the Meteorology and Water Management National Research Institute in the Białowieża village, 1–7 km from the study areas.Data analysesWeather conditions affecting blowfly ectoparasitesTo explore the impact of weather on blowfly ectoparasites, for each Wood Warbler nest we calculated average temperatures from daily means, and total sums of rainfall from daily sums, for the two time-windows in which we assumed the impact of weather would be of greatest importance:

i.

the early nestling stage, when Wood Warbler nestlings were 1–4 days old. During this stage, female blowflies require a minimum temperature of c. 16 °C to become active and oviposit in bird nests27. Thus, cool and wet weather in the early nestling stage should reduce the activity of ovipositing blowflies, leading to less frequent ectoparasite infestation of Wood Warbler nests.

ii.

The late nestling stage, when the warbler nestlings were aged between over four days old and until fledging or nest failure. During this stage, blowfly larvae grow and develop in bird nests after hatching a few days after oviposition14,25,26,27. As the temperature of bird nests strongly depends on ambient temperatures21, mortality of blowfly larvae should increase in cool weather, resulting in fewer ectoparasites in nests collected shortly after the fledging of birds29.

Weather conditions affecting Wood Warbler nestling growthTo explore the impact of weather on nestling growth, for each nest we calculated the average temperatures and total sums of rainfall for the period when nestlings were over four days old and until their measurement, usually on day 8 from hatching (see above). During this stage, nestlings are no longer brooded by a parent74, so must balance their energetic expenditure between growth (feather length and body mass) or thermoregulation89. Thus, we expected that the gain in body mass and the growth of flight feathers would be reduced in nestlings during cool and wet weather, when maintaining a stable body temperature would be costly90.Statistical analysesAll statistical tests were two-tailed and performed in R version 4.1.091.The changes in blowfly infestation of the Wood Warbler nestsTo test the changes in blowfly infestation of warbler nests, we used zero-augmented negative binomial models (package pscl in R;92,93), which deal with the problem of overdispersion and excess of zeros92. In this study, hurdle and zero-inflated models fitted with the same covariates had an almost identical Akaike Information Criterion (AIC). Therefore, we presented only the results of hurdle models, which are easier to interpret than zero-inflated models. Hurdle models consisted of two parts: a left-truncated count with a negative binomial distribution representing the number of blowflies in infested nests, and a zero hurdle binomial estimating the probability of blowfly presence. We used models with a negative binomial distribution, which had a much lower AIC than with a Poisson distribution on a count part.We designed the most complex (global) model that contained a response variable of the number of blowflies in each of the 129 Wood Warbler nests. The covariates were: mean ambient temperature, total sum of rainfall, presence (or absence) of ants in the same nests, habitat type (deciduous vs mixed deciduous-coniferous forest), study year (2018–2020), the number of nestlings hatched (brood size), and nest phenology (the relative hatching date of Wood Warbler nestlings, as days from the median hatching date in a season: 23 May in 2018, 25 May in 2019 and 29 May in 2020). The initial global model also contained the two-way interaction terms that we suspected to be important: between temperature and rainfall, temperature and presence of ants, and rainfall and presence of ants.To explore all potentially meaningful subsets of models, we used the same covariates on both parts (count and binomial) of the global model. We performed automated model selection with the MuMIn package94, starting from the most complex (global) model and using all possible simpler models (i.e. all subsets)95. To attain the minimum sample size of c. 20 data points for each parameter96, we limited the maximum number of parameters to six in each part (count or binomial) of the candidate models.As some of the interaction terms appeared insignificant in the initial model selection, to minimise the risk of over-parametrisation, we included only the significant interaction term on a count part of the final global model. As described above, we performed model selection again. We tested linear relationships, as the quadratic effects of weather variables (presuming temperature or rainfall optima) appeared insignificant.To test whether blowfly infestation changed with weather in the early or late nestling stages, we twice repeated the procedure described above. The first global model included the mean ambient temperature and the total sum of rainfall for the early nestling stage, and the second global model contained weather variables for the late nestling stage. The remaining covariates were the same.A practice of including the same sets of covariates on count and binomial parts has been previously questioned97. However, our approach allowed us to comply with these objections97, as we presented only the most parsimonious models (with ΔAICc  More

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A roaring trans-European herring trade that began in the Viking Age might have depleted stocks1.

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doi: https://doi.org/10.1038/d41586-022-03431-y

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The studied viviparous clausiliids developed four types of morphological adaptations that facilitate the delivery of embryos through the shell aperture: (1) reduction of the clausiliar apparatus, (2) decrease of embryonic shell width, (3) widening of the shell canal, and (4) development of a flexible embryonic shell.Reduction of the clausiliar apparatusMembers of the Reinia genus, arboreal species from Japan (Fig. 1), show the most advanced adaptations to live-bearing compared to hypothetical ancestral Phaedusinae. The shell shape in these species is more conical than fusiform, the number of whorls decreases, and the aperture widens. One of the species, R. variegata, features almost full reduction of the clausiliar apparatus that consists of only vestigial folds (Fig. 1F). This species also lacks the clausilium, so the entrance through the aperture is unprotected.Figure 1Different stages of reduction of apertural barriers in members of genus Reinia: R. ashizuriensis (A–C; upper row) and R. variegata (D–F; lower row). (A,D) Adult shells; (B,C,E,F) adult shells with body whorl cut open dorsally in microCT visualisation. cp clausilium plate, il inferior lamella, pr principal plica, sc subcolumellar lamella, sl superior lamella, sp spiral lamella, upp upper palatal plica.Full size imageDecrease of embryonic shell widthAnother adaptation concerns the shape of the embryonic shell (“protoconch”), which becomes very narrow in some viviparous species. This feature is conspicuous because embryonic whorls remain in the adult shell as apical whorls. For instance in S. addisoni (Fig. 2A–D), the apical part being much narrower than the first whorls of the teleoconch is a clear evidence that the growth trajectory has changed abruptly after birth. Other examples include E. cylindrella and E. steetzneri, in which both the protoconch and the teleoconch are very narrow, yet at the borderline between these parts, the shell axis is slightly bent (Fig. 2E–L). We suppose that this feature develops as a result of obstruction during birth.Figure 2Width difference between protoconch and teleoconch in Stereophaedusa addisoni (A–D, upper row), Euphaedusa cylindrella (E–H, middle row), Euphaedusa steetzneri (I–L, lower row). (A,C,E,G,I,K) Adult shells with very narrow apical whorls; (B,F,J) X-rayed adults; (F,J) with retained embryos inside; (D,H,L) X-rays of apical part of adult shell with schematic drawings of a neonate.Full size imageWidening of the shell canalThe third type of adaptation is the widening of the shell canal in the body whorl, allowing for easier passage of the embryo between the lamellae and plicae of the apertural barriers. In this case, the outline of the shell changes only slightly giving the body whorl a more convex appearance. A substantial difference to egg-laying species concerns the apertural barriers: the clausiliar includes a broad clausilium plate and a spirally ascending inferior lamella (Fig. 3A–D). These modifications result in a spacious shell canal in the body whorl, for example in S. addisoni and E. sheridani, that can accommodate the transfer of a large embryo. Table 1 presents neonatal size in these species (shell width ca. 1.2 mm), which is very similar to their clausilium width (ca. 1.1–1.2 mm).Figure 3Two types of clausiliar apparatus occurring in Phaedusinae in microCT visualisation: with spirally ascending inferior lamella and wide clausilium plate (upper row), and with straight ascending inferior lamella and narrow clausilium plate (lower row). (A) T. sheridani adult shell with the body whorl cut open dorsally; (B) clausilium of T. sheridani; (C) clausilium of S. addisoni; (D) clausilium of R. ashizuriensis; (E) Zaptyx ventriosa adult shell with body whorl cut open dorsally; (F) clausilium of Z. ventriosa; (G,H) clausilia of O. miranda. Note, that all depicted species are viviparous.Full size imageTable 1 Shell size of studied Phaedusinae species.Full size tableMost viviparid clausiliids develop one of these three types of modification; some adaptations co-occur within a single species, for example a wide clausilium accompanies a narrow apex. Interestingly, the Reinia genus includes taxa with a gradual escalation of viviparity-related adaptations: R. ashizurensis, with a stout shell shape and a low number of whorls, has fully developed apertural barriers with a broad clausilium plate (Fig. 1A–C), while its congener, R. variegata, has reduced apertural barriers (Fig. 1D–F).Development of a flexible embryonic shellThe fourth type of adaptation found in Phaedusinae concerns the structure of the embryonic shells. We report this adaptation in O. miranda and Z. ventriosa.Oospira miranda is a dextral, often decollated, ground-dwelling species from Vietnam (Fig. 4A). The species is viviparous: during microCT scanning of museum specimens, we found embryos within a parental shell (Fig. 4B); in laboratory culture, we observed neonates immediately after live birth (Fig. 4C,D). Morphological characters recognized in the adult shell, i.e., a wide apex (= wide embryonic shell), straightly ascending inferior lamella, and a narrow clausilium plate (Fig. 3G,H), seemed to exclude the possibility of live-bearing reproduction, as embryos are too large to pass through the shell canal at the narrowest point. The height and width of the neonatal shell (mean values: 5.19 mm, 3.59 mm) evidently exceeds the width of the clausilium plate in this species (1.97 mm) (Table 1). However, under closer examination, we found the shell to be thin and delicate, which we refer to as a ‘soft shell’. In direct examination, the neonatal shell of O. miranda resembles cellophane, which may keep a given shape for a long time but becomes distorted already under slight pressure.Figure 4Viviparous clausiliids and their ‘soft-shelled’ neonates born in laboratory culture. (A–D) O. miranda: adult shell, X-rayed shell with embryo visible inside, neonates; (E–H) Z. ventriosa: adult shell, X-rayed shell with eggs visible inside, neonates.Full size imageA similar adaptation exists in Z. ventriosa, a Taiwanese species with a very wide apex, never decollated, a straight ascending inferior lamella, and a narrow clausilium plate (Figs. 3E,F, 4E,F). This species produces neonates in laboratory culture (Fig. 4G–H). The dimensions of the neonates (mean values: height 3.37 mm, width 2.51 mm) exceed at last twofold the width of the clausilium plate (1.08 mm). The shells of such freshly delivered juveniles, when gently touched with laboratory tweezers, became dented, but not fractured. More intense and stronger pressing can break this dentation.These initial observations, that we made during the maintenance of the laboratory culture, suggested that the neonatal shells of O. miranda and Z. ventriosa have flexible walls. These ‘soft-shells’ seem to be highly malleable during the entire embryonic development period and delivery through apertural barriers, hardening shortly after birth. We further investigated the physical properties of the embryonic shell by means of microcomputed tomography and scanning electron microscopy.Microcomputed tomographyWe scanned ‘soft-shelled’ neonates of O. miranda and Z. ventriosa, together with ‘hard-shelled’ embryos and neonates of S. addisoni and T. sheridani, in order to compare the density and thickness of the shells (Fig. 5).Figure 5Comparison of embryonic shell thickness in clausiliids: ‘soft-shelled’ neonates of Z. ventriosa (A,B,G,H) and O. miranda (C,D,I,J); “hard-shelled” neonate of S. addisoni (E,K) and embryo of T. sheridani (F,L) scanned inside a parental shell. Upper row—microCT visualisation of shell surface; middle row—microCT sections of those specimens; (M–O) X-ray photographs of S. addisoni (embryo from dissected adult) and Z. ventriosa (neonate) enlarged in (N,O), respectively, showing the difference in shell density and thickness; (P) microCT based volume rendering of O. miranda (left) and S. addisoni (right) neonates, showing difference between relative density of their shells.Full size imagePreliminary observations using the two-dimensional X-ray photographs showed a difference in thickness and density between S. addisoni and Z. ventriosa (Fig. 5M, enlarged in N and O, respectively). The 3D visualization of O. miranda and S. addisoni (the same microCT scanning and reconstruction parameters) confirmed the difference between density and shell thickness of these two species (Fig. 5P).Due to variations in wall thickness within the neonatal shell (e.g., between the first and the second whorls), it is not possible to precisely determine the thickness of the shell wall. The accuracy of the measurement is also limited by the resolution of the microCT scans, especially in the case of the relatively large neonates of O. miranda and Z. ventriosa. When scanning the whole embryonic shell of Z. ventriosa (approximately 3.5 mm in height), the size of the voxel was approximately 1 µm. Thus, we cannot determine the shell thickness down to the nearest micron, but we can estimate it from a few to a dozen microns. A direct comparison between virtual microCT sections of specimens scanned under the same conditions shows a clear difference between the ‘soft-shelled’ and ‘hard-shelled’ taxa (Fig. 5G–L). The ’hard-shelled’ neonates have a shell wall of 30–40 µm thick. We examined the sequence of three ’soft-shelled’ O. miranda specimens that differed in size (the exact time of birth of each of the cultured neonates is unknown, ca. 1–2 days). The larger (older) the neonate was, the thicker the shell. The shell of the largest of the studied O. miranda was up to 20 µm thick. However, the shell wall of this relatively large juvenile (several millimeters in height) still did not reach the thickness of the small ’hard-shelled’ T. sheridani embryo, which was already about 30–40 µm thick, stiff and rigid during the retention in the genital tract. The neonates of O. miranda and Z. ventriosa were much larger than the embryos and neonates of S. addisoni and R. variegata (Table 1), however, the former taxa has much thinner shells.Scanning electron microscopyAfter the non-invasive microCT scan, we scanned embryos and neonates using SEM (Fig. 6). The different properties of the shells of Z. ventriosa and O. miranda vs. S. addisoni and R. variegata were already visible during the preparation of the analysis. Under vacuum conditions, the soft shells of Z. ventriosa and O. miranda shrank and crumpled, creating a cellophane-like surface (Fig. 6A). Embryos and neonates of S. addisoni and R. variegata did not require any special preparation and their shell shape remained unchanged under the vacuum conditions applied during the SEM examination (Fig. 6D,E). To reduce the shell deformations, we freeze-dried the next group of thin-shelled neonates prior to SEM analyses (Fig. 6B,C).Figure 6Neonates of O. miranda (A,B,F,I,L,M,O) and Z. ventriosa (C,G,J,P) in direct comparison with hard-shelled embryos and neonates of R. variegata (D,N,Q) and S. addisoni (E,H,K); SEM microphotographs. The vacuum conditions in SEM led to the shrinkage of the thin O. miranda shell (A); freeze-drying of ‘soft-shelled’ neonates prior to SEM imaging reduced the level of deformity (B,C). Contrastingly, R. variegata and S. addisoni shells do not require special preparation and retain their shape (D,E). (F) The dented surface of O. miranda neonate and SEM-close-up (I) on a cross-section of the shell just a few micrometers thick (arrow in F indicates the region enlarged in I). (G,J) Shell of Z. ventriosa in comparison with similarly ornamented fragment of S. addisoni (H,K); note several times thicker shell in the latter (arrows in G,H indicate the regions enlarged in J,K, respectively). (L,M) Inner surface of intact periostracum which still connects two fragments of broken aragonite shell of O. miranda (the arrow in M indicates the region enlarged in L); note the difference between shell thickness in O. miranda (L,M) and R. variegata (N). All observed specimens have similar crossed-lamellar microstructure (L–Q). However, just as shell thickness, also the number of lamellar layers of alternate orientation within the shell differs (L,M,O,P vs N,Q).Full size imageThe SEM studies allowed for complementary measurements of the shells. In the broken fragments of Z. ventriosa and O. miranda, the thickness of the shell wall ranged from 2–3 µm (Fig. 6F,G,I,J,L,M) to 18 µm in the largest neonate of O. miranda (Fig. 6O). The shells of S. addisoni (Fig. 6H,K) and R. variegata (Fig. 6N) are several times thicker.All analyzed samples have a thin ( More

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