Edible insects are important sources of essential nutrients and have the potential to contribute to malnutrition reduction and food security in the Republic of Benin. However, their consumption is always restricted to a limited number of sociocultural groups. To determine how the consumption of insects could be promoted as an alternative food source, this study documents the endogenous knowledge associated with edible insects and, the main factors that govern their perception and frequency consumption.A survey was conducted towards 479 rural households consuming edible insects through 91 villages of Atacora, Alibori, Zou, and Plateau departments using individual interviews with a semi-structured questionnaire. The survey was focused on the inventory of edible insects and the documentation of consumers’ acceptance, frequencies and motive reason of consumption, local uses, and accessibility to edible insects. Samples of edible insects were collected and preserved in 70% alcohol for taxonomic identification.Brachytrupes membranaceus Drury being the most widespread and consumed. Six factors affecting edible insect availability were identified with the chemical pollution as the most important. Besides their food use (63.2%), edible insects in the study area were used for several purposes. We find that ethnicity, religion, age, education level, and monthly frequency of insect consumption are the main factors influencing the local perception of edible insects. Indeed ethnic group, religion ethnicity, and market accessibility have a positive influence on edible insect consumption frequency. The Hierarchical Clustering of Principal Components has allowed us to classify the interviewees into 3 groups with different perceptions of entomophagy and their characteristics will make it possible to better orient the strategies for promoting entomophagy in the Republic of Benin.The majority of surveyed people (79.1%) were consumers of edible insects since many years ago (29.1 ± 17.2 years). Insect species belonging to 17 genera of 7 families and 3 orders of insects were used as food, with Religion and tradition are among the main factors that influence entomophagy in Benin Republic. The development of a national strategy to promote entomophagy should take into account the recorded insect consumption motivations, and their different uses by each ethnic group, and mainly target young people. Consumed for centuries, edible insects were first promoted as an alternative sustainable source of nutrients for humans and livestock by Meyer-Rochow  who recoIn the Republic of Benin, consumption of edible insects is restricted to a limited number of sociocultural groups, such as the Waama, Dendi, Ottamari, Bariba and Nagot. Although some studies have been conducted on edible insects in Benin, they are mainly limited to their diversity, gathering method, collection place, seasonal availability, consumption patterns, and consumer perception , 12–16. The development of strategies allowing the acceptance of entomophagy by Beninese populations requires a better understanding of the factors influencing the perception and consumption of edible insects. Indeed, several factors such as consumers’ socio-demographic characteristics, consumers’ external environment, and factors related to the insects consumed are known as influencing the acceptance or rejection of entomophagy . UnfortuThe aim of this study was to (1) document the consumption frequency, and local uses of edible insects; (2) examine the local perception of consumers about entomophagy; (3) investigate factors influencing consumers’ perception and consumption of edible insects.The survey was carried out in 91 villages located across the departments of Atacora (Latitudes/ Longitude: 10° 45′ 0" N/1° 40′ 0" E), Alibori (Latitudes/ Longitude: 11°19′ 0" N/2°55′ 0" E), Zou (Latitudes/ Longitude: 7° 15′ 0" N/2° 10′ 0" E) and Plateau (Latitudes/ Longitude: 7° 10′ 0" N/2° 34′ 60" E) in the Republic of Benin  by two trained investigators. The oral consent of the participants was taken after the presentation of the objectives of the survey. Translators were recruited locally and impregnated with the questionnaire to facilitate exchanges with the interviewees. The number of surveyed households varied from one department to another, depending on the edible insect’s consumers at the level of each surveyed village Table . The col2) or Fisher test was performed to assess the relationship between insect consumption practices and ethnic groups. Principal component analysis (PCA) using the Facto Mine R package was performed [The descriptive analysis was carried out to summarize the sociodemographic characteristics of the surveyed households. Chi-square  of each of the edible insect species inventoried in the study area was calculated following Alves et al.  using thThe fidelity level (FL), the percentage of respondents claiming the use of a certain species of edible insects for the same major purpose, was calculated for the most frequently reported diseases or ailments as: disease .Hierarchical Clustering on Principal Components (HCPC) was performed to assess the influence of social, cultural, and economic characteristics of respondents on the perception of insect consumption. The HCPC consisted of a Multiple Correspondence Analysis (MCA) realised on the perception measures and on the social characteristics of the respondents, followed by a hierarchical clustering analysis (HCA) on the principal components from the MCA. To select the variables for each perception group, we measured the difference between the class values and the overall values. These statistics were converted into a criterion called the value test (V-test) to perform a selection on the variables, and thus determine the most characteristic variables , 29. TheA multivariable logistic regression of the outcome of edible insect consumption (No/Yes) on the independent variables  was conducted to determine their association. The conditional association of the different independent variables with the outcome was quantified using odds ratios.All the analyses were performed in R version 4.3  and a sip = 0.795) and level of education (p = 0.402) in the frequency of consumption of edible insects. Throughout the study area, insect consumption is usually family-based (79.3%). However, in the plateau department, entomophagy is much more practiced by parents (Table p < 0.001), age (p =  < 0.001), and religion (p =  < 0.001). More illiterate (44.9%), Christian (37.9%), and adult (57.4%) respondents continue to practice entomophagy compared to others  were still consumers of edible insects and since several years ago (29.1 ± 17.2 years). The remaining surveyed people (20.9%) have consumed edible insects at least once in the past but no longer consume them. Most of the surveyed men (49.7%) consume edible insects against 29.4% of the surveyed women. Contrary to the other departments where the consumption of insects by the surveyed people is more recent, those surveyed in the Atacora department have consumed them since their childhood (39.3 ± 14.3 years of consumption on average). Among the nutrient’s sources of the surveyed households, edible insects represent 10 and 30% of annual intake , houses (37.3%), savannas (15.2%), forests 3.4%), public places (2.9%), and mountains (2.9%). Six factors affecting the accessibility to edible insects were identified in the study area Fig. . The mos%, publicCybister sp.) to 1 (B. membranaceus) (Table A. ruficornis citrina) and tobacco crickets (B. membranaceus) are the only ones listed as sold in markets mainly in the Alibori department , edible insects in the study area were used in traditional medicine (19%), spiritual uses (8.8%), poultry feed (6.1%), animal trap (1.3%), sold in markets (1%), and for fishing (0.6%). The use values of edible insects in the study area varied from 0.14  Table . These ient Fig. . These iThe results of the principal component analysis showed that the factorial design formed by axis 1 and 2 explains 88.6% of the initial total variability of the different traditional uses of edible insects by ethnic groups  showed that 79.52% of the initial variability of the perception measures and social characteristics was retained on the first 60 axes of the Multiple Correspondence Analysis (MCA). The hierarchical clustering analysis (HCA) identified three groups of perceptions related to sociocultural and economic characteristics Fig. .Fig. 10Pp < 0.001) and Cotimba , animists  with a high level of education  and whose frequency of insect consumption is average . The second cluster included 59.40% of the respondents mainly Yorouba  and Ottamari  consumers with a positive perception of entomophagy and its promotion (Table p = 0.003) and older persons  who practice the religion of Christianity  and who have a high frequency of insect consumption . However, these respondents are unaware of the health benefits of eating edible insects and do not adhere to their sales. The third group including 26.29% of the respondents belonging to the Dendi  and Fon  ethnic groups, practicing Islam  who are very favourable to entomophagy and its promotion (Table p < 0.001) and a low frequency of insect consumption . They have knowledge of the health benefits of edible insects and are willing to buy them despite their low monthly consumption.The first cluster, which included 14.31% of respondents was characterized by consumers with a very negative perception of entomophagy and are not ready to promote it Table . These rp < 0.001) that the proportion of participants who eat the insect is associated with the ethnic group, and religion and also linked to their accessibility of market (Table p = 0.92), age group (p = 0.21), education (p = 0.54), and source of income (p = 0.98). The results of the full model taking into account all the factors indicated that the covariates considered in this model, with the exception of the ethnic group, religion, and market accessibility, were no evidence (p > 0.05) on edible insect consumption. Thus, this model leads to the same results as the univariate tests performed on each of the factors. Therefore, edible insect consumption is associated with an ethnic group, religion, and market accessibility. Indeed, more Ottamari respondents (p = 0.004) consume insects than other ethnic group participants (Table p = 0.03) consume weakly insects than Christians and animists. As access to the market becomes increasingly difficult (p < 0.001), people consume more insects  (Coleoptera: Buprestidae) to our knowledge has never been mentioned as edible insect in Benin. This could suggest that entomophagy is experiencing a positive development in Benin with new species of insects consumed. The dominance of Orthoptera, Blattaria, and Coleoptera among the edible insects in Benin is not surprising. Indeed, these insects’ orders are in the top five of most commonly consumed in Sub-Saharan Africa [B. membranaceus is among the most preferred edible insects.Our results showed that 17 species of insects are consumed in the four surveyed regions. This number of edible insect species is low comparatively to other studies conducted in Benin , 15, andn Africa , and acrn Africa , 41. As n Africa , in NigeThe use of chemical pesticides to protect farmlands was the main constraint hindering the accessibility of edible insects to populations. Indeed, edible insects are mainly caught in cropping areas, where they are also considered by some farmers as crop pests and killed with pesticides. In fact, some studies showed that edible insects collected in crop fields have a high quantity of pesticide residues on and recommended their farm rearing according to appropriate regulations to ensure safety , 44. It A. ruficornis citrina and B. membranaceus) mentioned as sold in the markets of Alibori department were also cited as having a commercial value in Nigeria [Diverse uses of edible insects were recorded in the study area. As in other countries, edible insects are used for medicinal purposes –47, cult Nigeria , and Cam Nigeria . It is tThe perception of entomophagy and the frequency of consumption of edible insects depends on ethnic groups. In fact, Dendi, Fon, Yorouba and Ottamari ethnic groups showed a good perception of entomophagy and clear desire to promote it. However, some respondents of Yorouba and Ottamari ethnic groups are unaware of the health benefits of eating edible insects. This ignorance of the nutritional benefits of edible insects despite their consumption could be explained by the fact that they consume the insects only by food habit or just for flavour. It is, therefore, necessary to organize awareness-raising on the nutritional benefits linked to the consumption of edible insects towards this type of consumer to reinforce their inclusion as a regular source of nutrients in their diet.Our results showed that respondents from the Cotimba and Ottamari ethnic groups, who still practice endogenous religions have a negative perception of entomophagy compared to those who are Christians or Muslims. Indeed, it is known that religions prohibiting the consumption of animals and their derivatives significantly influence the practice of entomophagy , 53. It Our results showed that the consumption of edible insects represents a small part of the total food intake, and is however fully rooted in the eating habits of the surveyed people. Several reasons motivating the consumption of insects have been recorded and must be exploited in the definition of policy to promote entomophagy in the study area. Various synthetic chemical pesticides are used in edible insect collection fields suggesting the possibility of consuming insects containing residues harmful to the health of populations. The development and popularization of edible insect farming methods accessible to low-income populations is therefore recommended. In addition, awareness sessions on the nutritional benefits associated with the consumption of edible insects must be organized for their regular inclusion in the diet of populations. A differential perception of entomophagy across ethnic groups was noted with a negative perception among the Cotimba ethnic group. Ethnicity and religion are the main factors taken into account in the definition of policies to promote entomophagy in Benin.


Pinus sylvestris, the late phases of somatic embryogenesis were investigated on seven cell lines in a set of two trials . During the germination phase, a shortened and simplified in vitro period, a cold storage option and basal media were explored. Viable plants were obtained from all treatments. However, there is still the need to improve germination and related protocols together with growing regimes for Pinus sylvestris. The improvements to protocols presented here, particularly for Pinus radiata, result in greater survival and quality of somatic emblings, leading to reduced costs and increased confidence in the technology. Simplified protocols using a cold storage option show great promise and, with some further research, could lead to reductions in the cost of the technology.Somatic embryogenesis (SE) is an advanced vegetative propagation technology that, when used in combination with breeding and cryopreservation, offers the forest industry a powerful tool for the deployment of elite genotypes. Germination and acclimatization are critical and cost-intensive phases in the production of somatic plants. The efficient conversion of somatic embryos into robust plants is a necessity if a propagation protocol is to be successfully adopted by the industry. In this work, these late phases of the SE protocol of two pine species were investigated. A shortened germination protocol and more controlled acclimatization were investigated for Pinus radiata, testing embryos from 18 embryogenic cell lines. A more simplified protocol, including a cold storage phase, was also compared among 10 of these cell lines. A shortened germination period and more controlled protocols significantly improved the acclimatization of somatic embryos directly from the lab to the glasshouse. When results for all cell lines were pooled, there were significant improvements in all growth characteristics . When the more simplified protocol involving cold storage was tested, improvements were seen in the root architecture. For  Pinus radiata D. Don), a native of North America, is the most widely planted pine in the world. It is extensively cultivated as a plantation timber in many temperate parts of the world, including Australia, Spain, Argentina, Chile, Uruguay, Kenya, South Africa, and New Zealand (NZ), where it is the main commercial forestry species, occupying approximately 87% of plantation forests. In NZ, most tree stocks produced (64%) originate from control pollinated seed, including seedlings, cuttings, and clones. The remaining (36%) originate from OP seed [Radiata pine  is the most common species of the genus Pinus and an economically important species in the Northern Hemisphere. As wood, it is used for building, construction, and furniture, with further processing as veneer, fiberboards, or chipboards, and for pulp and paper production. Its natural range is broad, covering large areas with different climatic conditions in Eurasia, from the West Coast of Europe to the far east of Russia. Within its range, Scots pine is often the dominant species and can occupy up to 65% of the forest area. Within the EU, nearly all the forest reproductive material comes from approved basic materials and is either improved seed orchard seed or seed from selected seed stands. For forest regeneration, both planting and direct seeding are used [Scots pine , germination of somatic embryos, and finally, acclimatization to ex vitro conditions in preparation for field planting. A large amount of work has been done over the last few decades to improve SE protocols in conifers, with most advances occurring in nt to SE . Despitent to SE .When it comes to improving the quality of somatic emblings in conifers, most published studies have focused on the maturation stage of the SE process. The maturation process in culture is initiated with the development of the somatic embryos and terminates with an imposed desiccation prior to germination. For complete maturation to occur, the embryos must achieve both “morphological” and “physiological” maturity ,9,10. ThThe stages of germination and conversion of emblings into plants are extremely important to the overall success of SE on a commercial scale. Loss of plants during these latter stages greatly increases the overall cost of the technology, with the germination step being the most costly due to high labor demands ,15,16. DP. radiata SE process, the resilience of tissue-cultured plants, when transferred to the nursery, is poor. Root systems developed are often a limiting factor for their clonal deployment potential due to scarcity of roots, cessation of their growth and unbalanced architecture [P. sylvestris regenerated following a long, multistep in vitro germination process has been shown to have unbalanced root architecture that may result in bent stems and leaning trees, although suboptimal nursery techniques may have added to these problems [In the current itecture ,30. In Nitecture . Howeverproblems .Picea abies (Norway spruce) SE [Within the last five years, a mounting number of studies in conifers looking at improving germination and acclimatization stages of the SE process have been published. Of particular interest are detailed studies of physical and chemical growing conditions during the germination process in ruce) SE ,24. In tP. radiata and P. sylvestris SE plantlets. A much-simplified protocol was also investigated for both species, where somatic embryos were placed in cold storage prior to short germination on Petri plates and then direct transfer to nursery conditions. The effect of these protocols on the early stages of growth and survival of P. sylvestris and on survival, growth characteristics and root architecture of four-to-five-month-old somatic emblings of P. radiata is reported.The objective of the work presented here was to test a shortened germination protocol developed for Norway spruce  as well At the end of maturation (10–12 weeks), cotyledonary somatic embryos from 18 cell lines were put through two germination/acclimatization treatments: control (a five-week in vitro germination in Petri plates followed by transfer to propagation trays in a glasshouse) and a short germination/micro-plug treatment . At the same time, somatic embryos from 10 of these cell lines were also stored in cold storage, followed by a short germination (two weeks on Petri plates) and planting directly into nursery treatment (Trt 2). All treatments are described by illustration in p < 0.001) of somatic emblings by the repotting stage. When all cell line data were combined, 86% of emblings germinated using this protocol had survived and were able to be repotted, compared to only 64% in the control protocol  and early acclimatization. A combination of a reduced time somatic embryos spent in a germination medium and the inclusion of a micro-plug system had a significantly positive effect on the survival (protocol . Trt 2 (protocol .p < 0.0001). The highest survival was 85% of emblings grown in Trt 1 (short germination/micro-plug protocol), followed by those grown in the control treatment and then those grown in Trt 2 (cold storage/short germination protocol), at 59% and 38%, respectively  and micro-plug environment (Trt 1) and successful conversion and acclimatization in Trt 2 (cold storage/short germination protocol). The survival of emblings by the end of the experiment germinated and acclimatized under the three treatments differed significantly  of periment .When cell line data were pooled, emblings that were germinated and acclimatized using the short germination/micro-plug protocol (Trt 1) had significantly taller shoots (78.37 mm), longer roots (126.58 mm) and higher root collar diameters (1.28 mm) on average than emblings from both the control  and the cold storage/short germination protocol (Trt 2)  . The other three cell lines  showed no significant differences in average shoot height between the two treatments. When the average shoot heights of the 10 cell lines which went through all 3 treatments were compared, Trt 2 (cold storage/short germination protocol) resulted in shoots that were not significantly taller than those from the control treatment for 7 of these lines. The other three lines resulted in shoots that were not significantly taller than those from Trt 1 (short germination/micro-plug protocol) .For root length, 8 out of the 18 cell lines had significantly longer roots when germinated and acclimatized using Trt 1 compared to those from the control. The other 10 cell lines, although not significantly different, still followed that same trend. Of the 10 cell lines that went through all 3 treatments, 4 showed no significant difference between the three treatments. For the remaining six cell lines, emblings germinated and acclimatized using Trt 2 had significantly shorter roots than those from Trt 1 .Average root collar diameters were significantly larger for 7 out of the 18 cell lines which were germinated and acclimatized using Trt 1 compared to those from the control treatment. The remaining 11 cell lines showed no significant difference between the two treatments, although the same trend was seen. For the 10 cell lines which were also included in Trt 2, 5 showed no significant differences in average root collar diameters in emblings from the 3 treatments, and 4 were significantly lower than emblings from Trt 1 .Root quadrant scores from all surviving emblings from each of the germination/acclimatization protocols were separated into two groups representing either poor root architecture  or good architecture (a score of 3 or 4). When embryos were germinated and acclimatized under Treatment 2 (cold storage/short germination protocol), 72% of emblings fell into the good root architecture category, compared to only 15% of emblings from Treatment 1 (short germination/micro-plug protocol) and 10% of emblings from the control treatment .3 container plugs . All treatments are described in the illustration below in Maturation was carried out for seven cell lines in October and in November 2020. From the maturation carried out in October, 216 cotyledonary embryos from four cell lines (54 embryos per cell line) were germinated in two treatments directly following maturation in December 2020 . The DEC20 trial emblings were transplanted to micro-plugs following germination. All remaining embryos (870 embryos from seven cell lines from both maturation events) were cold stored until June 2021, germinated in three germination treatments and transplanted to nursery conditions in 85 cmp < 0.01) than the non-cold stored cotyledonary embryos , at 2.38 ± 0.02 mm and 2.18 ± 0.04 mm, respectively  were significantly longer (ectively . The sizp < 0.01), respectively (p = 0.81), respectively (p < 0.01), respectively , Scots pine emblings were found to be approximately half of the length of Norway spruce emblings (seen in previous work) after the same duration of in vitro germination. This supported the initial plan to continue the in vitro germination for the second week. For the Dec20 and June21 emblings, when cell line data were pooled, mean shoot lengths after two weeks of in vitro germination were 3.20 ± 0.09 mm and 2.80 ± 0.05 mm (ectively . Mean roectively . The meaectively .p < 0.01), root length (p = 0.014) and total length , the emblings varied in size among cell lines in shoot length . Thus, embryos that could have been interpreted as not germinated were also transplanted to peat to enable tracking of the development of individual embryos. We attempted to apply a threshold in size during in vitro germination to define if the embryo has achieved germination or not by setting the following size limits: Overall length minimum of 4 mm, of which root must be at least 2 mm (they had roughly doubled their size and had visible root growth) .p ≤ 0.01 in all, Post-transplant survival of emblings in the DEC20 trial was 46.8% overall . In the p < 0.03 in all, p ≤ 0.02 in all). When root lengths were grouped (2 mm length groups), the survival was lower when root length was below 2 mm than when root length was 2–6 mm . When the shoot length was between 2–8 mm, the survival was significantly higher than when the shoot length was 1–2 mm , p ≤ 0.02 in all). Additionally, the survival was significantly higher when the shoot length was 6–7 mm (three groups) than when the shoot length was 3–5 mm .In the Dec20 trial, when the emblings were grouped in size classes, survival was significantly lower when the embling length was below 3 mm than in all other groups . When the root length was less than 2 mm, survival was significantly lower than when the root length was 4–6 mm . In shoot length groups, the survival was significantly higher when shoot length was from 6–8 mm or more than 11 mm than in other groups . Additionally, survival was higher when shoot length was 2–4 mm than when shoot length was under 1 mm (p < 0.05 in both comparisons).In the June21 trial, post-transplant survival was low overall 15.3%, c. SurvivLarge-scale uptake of SE into breeding programs of commercially grown conifers is currently being hindered by a number of factors. These factors can be condensed into two overarching areas of concern for the grower; the cost and the quality of the final product ,32. AlthP. radiata in New Zealand is currently being used on a small scale by some of the larger forestry companies. Hesitation around more widespread uptake is mostly based on the cost of the final product. Propagation of P. radiata rooted cuttings from CP material is well established in NZ, and most forestry growers are happy to pay a premium price for them to receive a sturdier plantlet, especially for harsher or more fertile sites [SE of le sites . Incorpole sites . This prle sites . Howeverle sites . While tle sites , it alsole sites . Howeverle sites  and fastP. radiata and P. sylvestris, as well as to improve the acclimatization protocol and the overall quality of somatic emblings of P. radiata. A much-simplified protocol was also attempted for both species, where somatic embryos were placed in cold storage prior to short germination on Petri plates and then directly transferred to nursery conditions. For Scots pine, embryos were measured at the end of maturation (with and without cold storage) as well as after germination and survival, with data recorded after the first growing season. For radiata pine emblings, a morphological assessment was carried out, including some of the standard measurements made to assess the quality of seedlings [The aim of the work presented here was to test a shortened germination protocol developed for Norway spruce  on two peedlings , shoot hhttp://www.vivi.nu, accessed on 23 March 2023), has been successfully applied to both radiata pine and Scots pine. The majority of published SE germination protocols in pines are lengthy. P. taeda somatic embryos are cultured on a germination medium for 7 to 8 weeks in total [P. pinaster [A short in vitro germination on Petri plates, coupled with the use of a micro-plug system designed to assist with the control of humidity, water availability, and gas exchange in the substrate while growing in vitro plantlets  higher following cold storage , suggesting some continued development of embryos. Although the size increment is small, it could be an indicator of changes in storage compounds or even precocious germination [3 containers) than in the DEC20 trial, where they were planted into the controlled micro-plug environment. Although no measurements were made of radiata pine embryos before and after cold storage in this work, it was clear to see that some development of embryos continued during the five to six months in cold storage. Optimum cold storage conditions and timing should be investigated more closely for these species. Varis et al., reported no significant differences in germination rates between somatic embryos that germinated immediately following maturation and those cold stored for three to six months and even up to twelve months  in Norway spruce [In this work, we are unable to separate the effect of cold storage on somatic embryo germination and acclimatization from other factors (short germination/use of micro-plugs) due to the vastly different propagation protocols used in the treatments tested. Despite this, cold storage has been shown to have no adverse effects on germination in some conifers ,41 and mmination . The laty spruce . Castandy spruce .Applying a germination medium refined for Norway spruce (nitrogen content lowered and the source being purely organic) appears to support root growth in Scots pine. However, more studies are required to confirm this. Perhaps similar protocol modifications would be favorable for the media conventionally applied in Scots pine SE. Such a reduction or removal of inorganic nitrogen from germination media has been found to be favorable in several conifer species ,25,27,45p < 0.001) improved survival of somatic emblings compared to the control treatment at the time of repotting, 86 and 64%, respectively  than in the DEC20 trial (3.4 mL). The July conditions in a commercial nursery in Finland are very harsh and have certainly decreased the overall survival. The results from measurements taken following germination on plates for two weeks suggest that when planting directly to larger containers (85 cm3), the emblings should be larger, the roots should be at least 6 mm long, or the total length of emblings should be at least 9 mm to increase the chances of survival. This finding is supported by the result that emblings from the two-step germination survived better in the June21 trial, likely due to their larger size, i.e., longer germination, even though they were the same size after the plate germination. The argument regarding suitable embling size for certain container plug sizes is supported by the survival results from the Dec20 trial, where no additional benefit was achieved by using the two-step in vitro germination. Although the results must be verified with a larger sample size, these initial results could then be applied to improve the germination protocols and transplanting success into different containers and perhaps applied to other species, such selection criteria could help with the integration of SE laboratory protocols with greenhouse protocols [For Scots pine, survival results varied , suggestrotocols .p < 0.0001) on all growth characteristics measured   had a significant effect (iameter) . Althougiameter) . It is iThe root development of seedlings is also a critical factor for their successful establishment when out-planted . TypicalPicea glauca somatic plantlets resulted in numerous root deformations compared to zygotic seedlings where no transplantation was required [When embryos were germinated and acclimatized under Treatment 2 (cold storage/short germination/nursery), 72% of emblings fell into the “good” root architecture category, compared to only 15% of emblings from Treatment 1 (short germination/micro-plug) and 10% of emblings from the control treatment . This imrequired . Growingrequired . It is pReducing the requirements for strict environmental conditions for acclimatization would greatly decrease the overall cost of the SE technology, as this is one of the challenges with integrating tissue-cultured plantlets into normal seedling production . To be aP. radiata, this protocol significantly improved the chances of survival as well as improving the overall quality of resulting emblings. An even more simplified protocol involving a cold storage step, shortened germination period and direct transfer to nursery conditions also showed some promise, especially with respect to root architecture in radiata pine.In conclusion, a germination and acclimatization protocol designed for Norway spruce which includes a shortened germination period and a micro-plug system for in vitro to ex vitro transfer of somatic emblings, has successfully been applied to two pine species. In P. sylvestris, the current results show that shortened in vitro germination can give as good survival of the emblings as the traditionally used longer, two-step in vitro germination method. This needs to be confirmed, however, using broader material. Further, for this species, the earlier phases, especially initiation, are challenging and require improvements for SE to be practically applicable in this species.For The measurement regime of embryos and emblings applied to Scots pine could be refined and applied to other species. Root length or overall length after in vitro germination may work as an indicator for post-transplant survival, or it could be applied as a threshold to determine if embryos are qualified for transplanting and/or to determine the optimal length of the germination period. However, a larger dataset is required for this, and the measurements would need to be optimized for each growing system (container plug size) and species.P. radiata, we believe that the improvements to the protocol presented here could be optimized further. Both the time on germination and the time spent in Vivi™ trays have the potential to be reduced. Both adjustments are likely to improve the quality of the root system. It could also be advantageous to investigate replacing the loose fill in the Vivi™ trays used in this study with small miniplugs, therefore avoiding or limiting root disturbance during transplant from these trays to larger containers. Further conditions in the germination stage could also be investigated for radiata pine in our protocols, such as nitrogen content and light quality. Long-term field trials of somatic seedlings resulting from these protocols, as well as comparisons with currently acceptable planting stock, will be required to ensure the issues with quality historically seen by industry using direct SE are not still an issue. Cold storage (both conditions and timing) should be investigated further. Although some changes seen while in cold storage may be beneficial, it is important to reduce the likelihood of negative changes .For P. radiata embryogenic cell lines were used for germination experiments. These cell lines were initiated at Scion, Rotorua, NZ in 2010  and 2021  using protocols described by Hargreaves [Mature somatic embryos from 18 rgreaves . Brieflyrgreaves , as modirgreaves .P. radiata [All cell lines, whether recovered from cryopreservation using protocols developed for  radiata  (A–D) or radiata  supplemeFollowing a proliferation phase, embryogenic cell lines were then transferred as lumps (approximately 5 × 100 mg per Petri plate) to a pre-maturation medium (proliferation medium minus plant growth regulators) for two weeks. Tissue was then suspended in a proliferation liquid medium containing no growth regulators at a rate of 1 g of embryogenic tissue to 10 mL of liquid medium. Then, 1 mL of this suspension (100 mg of embryogenic tissue) was dispensed to filter paper discs  on a maturation medium  for the −2 s−2 for a further 7 days and then full light intensity of approximately 90 µmol m−2 s−2 for the remainder of the germination period (3 weeks).Mature cotyledonary somatic embryos were harvested from a maturation medium and placed on a modified Boulay  germinatFor the somatic embryos which were not cold stored (18 cell lines), two germination treatments were compared; in the control treatment, somatic embryos were cultured on KNV87 for a total of 5 weeks. This is our standard germination treatment for radiata pine somatic embryos. Germinated embryos were then carefully removed from germination media. Roots were trimmed where necessary and transferred to the glasshouse facility , where they were planted into seed propagation trays containing a screened bark fiber medium: perlite: vermiculite: 3 mm pumice: peat mix (40:10:10:20%). Propagation trays were covered with plastic vented lids (vent closed) and covered with 50% shade cloth for one week, and misted daily. The shade cloth was then removed, and emblings were slowly hardened using the vent and the eventual removal of the lid over 4–6 weeks.In Treatment 1 (short germination/micro-plug protocol), somatic embryos were cultured on KNV87 for 2 weeks, as described above, before being planted into Vivi™48  trays containing a sterile loose mix of peat: perlite (30:70%). Trays were placed inside Vivi™ outer boxes and covered with perforated foil (15 rows). Trays were then returned to the light rig under full light conditions for a further 3 weeks. Boxes containing trays were then transferred to a propagation room in a glasshouse facility . Three × 15–20 cm slits were made in the foil at this stage, and emblings were hand watered through these slits for a further 3 weeks. The foil was then removed completely, and emblings were then misted with a hose. A total of between 10 and 72 individual somatic embryos for each cell line were used for each of the treatments depending on the embryo availability of each cell line.After approximately 6–7 weeks under the above conditions, all surviving emblings from both treatments were repotted into Transplant systems TS48 trays containing 30% No. 8 composted pine bark, 35% C.A.N Fines (fine-screened composted pine bark) and 35% Coco Fiber with 6 kg per cube of osmocote exact control release fertilizer, 0.75 kg per cube micronutrients. At this time, an assessment of survival was carried out. Two weeks later, the trays were moved to another room in the glasshouse with natural light conditions. A shade cloth was used for approximately 2 weeks to allow for adaptation to these new conditions. A final assessment was done on emblings approximately 13–15 weeks after repotting. The following characteristics were monitored at the final assessment; survival (%), shoot height (mm), root length (mm), root collar diameter (mm) and root quadrant score (0–4). The definitions for root quadrant scoring were 0 = primary root only  occupied with secondary/tertiary roots.For 10 of the 18 cell lines tested in the control treatment and Treatment 1, a third treatment was also tested, Treatment 2 (cold storage/short germination/nursery). In this treatment, embryos were harvested from a maturation medium following a period in cold storage (5–6 months) and placed on KNV87 as above for 2 weeks. Embryos were then transferred to the nursery, removed from the culture, and planted into Ellepot 4 cm plugs (125 ccs) containing no.8 bark media .The root quadrant score (RQS) is a measurement of how balanced the root architecture is and, therefore, how likely the plant will be able to support itself. An RQS of 0 means that only the primary root is present. Scores from 1 to 4 represent further growth of the root system, with the number representing the area of further root growth if the center of the primary root was split into 4 quadrants. A rating of 2 is unbalanced, with all further root development occurring in only two of these quadrants, and a rating of 4 is very well-balanced, with all four quadrants containing further root growth. For statistical evaluation of the root quadrant score, scores (0–4) were grouped into two categories, poor root architecture  and good root architecture (a score of 3 or 4). For statistical evaluation of the root quadrant score, scores (0–4) were grouped into two categories, poor root architecture  and good root architecture (a score of 3 or 4). Differences in the frequency of categories among treatments and among cell lines were tested using a log-likelihood χ2 test .P. sylvestris) embryogenic cell lines were thawed in August 2020 following established procedures. Briefly, samples were rapidly thawed in a water bath, and the cryoprotectant was rinsed off with liquid proliferation media. Samples were then plated on proliferation media with filter paper and transferred three times, gradually decreasing the sucrose content in the proliferation media [−1 Phytagel [Cryopreserved samples of Scots pine  which was then dispensed onto filter paper  using a Büchner funnel as described by Klimaszewska and Smith [ied from  for a peied from  for a peIn December 2020 , 216 cotyledonary embryos from four of the cell lines (54 embryos per cell line) were germinated in vitro directly following maturation carried out in October.−2/s−1 in time proportions of 43.0, 28.5, and 28.5%, respectively.In vitro, germination for both trials was carried out in a +20 °C growth room ,16. LEDsPicea abies L. Karst) in increasing light intensity following the procedure successfully used for Norway spruce [Three different germination treatments were studied in Scots pine. Control treatment consisted of germination on MB5 Petri plates for two weeks with increasing light intensity as described above, and two weeks in MB6 jars in “full light” following the established Scots pine procedure . In MB 2y spruce ,16.3, cell surface area: 18.3 cm2, growing density: 546 cells m−2; BCC, Iso-Vimma, Finland) filled with semi coarse, pre-fertilized, light peat substrate (Kekkilä FPM 420 W F6) and placed into a commercial greenhouse unit where the sowing of a seed lot had just been finished [Following germination treatments, emblings were transplanted to a peat-based growing substrate. In the Dec20 trial, the transplanting was carried out to miniplugs  and the emblings were grown for ten weeks in the growth room, according to the protocol by Tikkinen et al. . The finfinished . The finIn both trials, all emblings, independent of their quality or germination vigor, were transplanted to containers to enable tracking of the development of an individual embryo within a treatment, cell line and container location. Partially due to this, the proportion of viable plants after the growing season appears low and does not fully represent the conversion rate of germinated embryos.Cotyledonary embryos were photographed (Canon Powershot G5 PC1049) in Petri plates containing a scale at the beginning of and after two weeks of in vitro germination. Additionally, the emblings were measured after one week of germination in the Dec20 trial to determine the growth rate of the emblings during the first and second weeks of germination. At the beginning of in vitro germination, the overall length of the cotyledonary embryo was measured. After one and two weeks, the length of the shoot, root and overall length were measured. All the measurements were carried out in pixels , which were then converted to millimeters by applying the scale in individual photos.To compare survival of emblings in different size classes the embling were grouped into following categories: 1 mm classes for shoot length , 2 mm classes for root length  and 3 mm classes for total length .All measurements and inventory results were analyzed using SPSS Statistics version 27. Normality could not be assumed among embryo and embling measurements. Thus, the Mann–Whitney U nonparametric test was applied to compare the size of embryos and emblings between trials, cell lines and treatments. The Kruskall–Wallis nonparametric test was used to compare survival differences among different embling size classes. When the December 20 and June 21 trials were compared, the analysis was made for only the cell lines included in both trials.


As the population ages, total hip arthroplasty has become more common in elderly patients including patients over the age of 90 years. Efficacy in this age group has been established, though literature regarding safety of total hip arthroplasty in nonagenarians is mixed. The anterior-based muscle-sparing (ABMS) approach, which exploits the intermuscular plane between the tensor fasciae latae and the gluteus medius, has proposed benefits of fast recovery, excellent stability, and reduced bleeding and may be adventitious among elderly, more fragile patients.A total of 38 consecutive nonagenarians undergoing elective, primary total hip arthroplasty via the ABMS approach for any indication from 2013 to 2020 were identified, and information regarding operative outcomes and patient-reported outcomes was gathered from review of medical records and our institutional joint replacement outcomes database.Included patients ranged from 90 to 97 years of age with the majority classified as American Society of Anesthesiologists score 2 (50%) or American Society of Anesthesiologists 3 (47.4%). The mean operative time was 74.6 minutes ± 13.6 minutes. Of all patients, 5 required a transfusion, 2 patients were readmitted within 90 days, and there were no major complications. The mean hospital length of stay was 2.8 days ± 0.8 days with 22 patients (57.9%) discharged to a skilled nursing facility. Limited patient-reported outcomes data showed statistically significant improvements in most outcomes scores at 6 months to 1 year postoperatively compared to preoperative scores.The ABMS approach is safe and effective in nonagenarians who may benefit from decreased amounts of bleeding and recovery times associated with the ABMS approach, which is evident from the low complication rates, relatively short hospital lengths of stay, and acceptable transfusion rates compared to previous studies. Total hip arthroplasty (THA) is a common and successful procedure, which offers the possibility to greatly improve mobility and quality of life. As the population ages, THA has been increasingly performed in older patients. Nonagenarians, people aged 90 years and older, are of particular interest as they comprise some of the oldest patients, and their numbers are expected to double to nearly 10% of the population by 2050 , literatHistorically, the posterior (Southern) approach has been a popular choice for THA due to its expansile nature, although the modified Hardinge approach has been frequently utilized due to a low incidence of dislocation. The direct anterior (DA) approach is gaining popularity with patients and surgeons with proposed benefits faster recovery and lower early dislocation risk. Another notable approach is the anterior-based muscle-sparing (ABMS) approach, ABLE, or Rottinger approach. Repopularized by Bertin and Rottinger in 2004, the ABMS approach to the hip is an intermuscular approach using the interval between the tensor fascia latae and gluteus medius ,16. WhilThe study protocol was deemed exempt by our institutional review board who determined the project was exempt from institutional review board review. The electronic medical record was queried for patients aged 90 years and older at time of surgery undergoing elective THA using the ABMS approach for any indication from 2013 to 2020. Surgeries were performed by 1 of 3 arthroplasty surgeons experienced in the ABMS approach with use of cemented or press-fit implants performed at the surgeon’s discretion. The electronic medical record medical and functional outcomes measures, including Hip Dysfunction and Osteoarthritis Outcome Score for Joint Replacement (HOOS JR) scores, University of California, Los Angeles (UCLA) Activity Scores, Single Assessment Numerical Evaluation (SANE) scores, and Patient-Reported Outcomes Measurement Information System (PROMIS) physical and mental health scores, were extracted for the included patients from the center’s joint replacement outcomes database.A total of 38 patients were included in the study, all undergoing elective unilateral THA . The patOperative times were typically between 60 and 90 minutes with a mean operative time of 74.6 minutes ± 13.6 minutes (range: 45-106 minutes) . There wThe mean length of stay (LOS) was 2.8 days ± 0.8 days (range 1.2-6.6 days). Patient disposition is outlined in Patient-reported outcomes including SANE scores, HOOS JR scores, UCLA Activity scores, and PROMIS mental and physical health scores were gathered as secondary outcomes and are reported in Nonagenarians are a growing portion of the population and the demand for THA in this age group is growing for both elective and femoral neck fracture surgery. Our study population was similar to that of other studies looking at nonagenarians in terms of gender, medical complexity, and BMI  3,4,6,,20. Nona. Nona20.Our mean operative time was 74.6 minutes ( ± 13.6 minutes). This was 15 minutes less than the mean operative time published by Shah et al  in theirWe observed a transfusion rate of 13.2%. This was lower than a majority of studies , with soLiterature regarding the safety of THA in nonagenarians is mixed with reported complication rates varying widely from near 0 to over 50% with medical complications alone reaching 56% in some studies . This vaHospital LOS is a complicated outcome and may be affected by regional differences in placement times for patients requiring a skilled nursing facility or acute rehabilitation facility, patient preferences regarding discharge disposition, and the availability of therapy services in addition to speed of patient recovery. In our study, we found an average LOS of 2.8 days ± 0.8 days which is, to our knowledge, the shortest LOS reported following THA among nonagenarians , with 57Our secondary outcomes showed improvement in postoperative HOOS JR scores, UCLA Activity Scores, SANE scores, and PROMIS physical and mental health scores, supporting the efficacy of the ABMS approach to THA and efficacy of THA in nonagenarians. Multiple studies have shown significant improvements in patient-reported outcomes following THA in nonagenarians and the amount of improvement appears comparable to younger patients ,5,6. WitOur study had several limitations. Lack of a control group to allow for assessment of outcomes using alternative approaches limits the ability to draw approach comparison conclusions from our study; and there is no way to control for institutional differences when comparing to previously published outcomes. Small sample size also limits our study, though our sample size is in-line with other single-center studies. Our patient-reported outcomes were particularly limited by small sample size with low response rates and variable follow-up times. The retrospective nature of our study provides a further limitation and makes our study vulnerable to selection and information bias. Modern electronic medical records allow for extensive information extraction and inclusion of patients of age 90 years and older undergoing THA for any indication and this may help mitigate these biases.Our study shows that the ABMS approach to elective THA is safe and efficacious in nonagenarians. In comparison to other studies, our group showed decreased LOS and transfusion rates as well as lower complication rates and operative times. Given its potential benefits with no clear drawbacks compared to other approaches, the ABMS approach should be considered when performing THA in elderly patients who may be at greater risk of complications and longer lengths of stay. Further research comparing the ABMS approach to other approaches, especially the DA approach which may have similar results, is warranted.George Babikian, Adam Rana, and Brian McGrory are authors who are paid consultants for Smith & Nephew, Inc., and the data set for this paper came from a Smith Nephew–funded research project, data sharing agreement. The other authors declare no potential conflicts of interest.https://doi.org/10.1016/j.artd.2023.101125.For full disclosure statements refer to


Algal materials, sediment samples, zebra mussel viscera, periphyton from shells, as well as fluids and tissues from the digestive tract of C1 were investigated for the following cyanotoxins: anatoxin-a, homoanatoxin-a, dihydroanatoxin-a (dhATX), cylindrospermopsin, saxitoxin, and microcystins. Necropsy results of C1 indicated neurotoxicosis with significant levels of dhATX in the duodenum tissues (10.51 ng/g dry weight (DW)), jejunum tissue (6.076 ng/g DW), and stomach contents (974.88 ng/g DW). Algae collected near the site of C1’s death contained levels of dhATX, ranging from 13 to 33 µg/g. By comparison, dhATX was detected at much lower concentrations in sediment samples (310.23 ng/g DW) and the periphyton on zebra mussel shells (38.45 ng/g DW). While dhATX was suspected in the deaths of canines from an event in Texas in 2019, this is the first report linking dhATX neurotoxicosis through pathological findings in Texas and potentially in the United States.The frequency of dogs becoming ill or dying from accidental exposure to cyanotoxins, produced by cyanobacteria, is increasing throughout the United States. In January and February of 2021, two dogs died and five dogs became ill after swimming in Lake Travis, central Texas, USA; one deceased dog (C Cyanobacteria  are common in aquatic systems worldwide where they exist in many different forms, and some species produce harmful cyanotoxins. Cyanobacteria can be found floating in the water column (planktonic), growing on the bottom of lakes or rivers (benthic), attached to other algae (epiphytic), attached to substrates (periphyton), or in visible composites that aggregate on the water surface to form floating mats (metaphyton). These varied forms exist year-round in low numbers but can reproduce quickly in favorable conditions to form planktonic or benthic blooms .During bloom events, the rapid proliferation of cyanobacteria can result in elevated levels of cyanotoxins, which can lead to animal intoxication incidents ,2,3,4,5.Abiotic factors such as climate change, invasive species colonization, and changing nutrient availability can shift energy production in a system to favor benthic growth, in a process called “benthification” . Benthic1) swam in Lake Travis, located near Austin, Texas, and died from suspected neurotoxicosis. Symptoms were consistent with ingestion of cyanobacteria, i.e., trembling, inability to stand, panting, vomiting, tremor, and stumbling. While investigating the death of C1, there were five more reports of illness related to cyanotoxin exposure with one additional canine death reported. All dogs had been swimming in the same area where C1 died, and the first death (C2) occurred 20 January 2021.On 20 February 2021, a dog  does not currently have any reference values for dhATX, but a recent study showed that dhATX is roughly 3- to 4-times more toxic when ingested orally than anatoxin-a (ATX) [Manning et al.  reported-a (ATX) . The WHO-a (ATX) .1, environmental cyanotoxin data found throughout the site of C1’s death, including the presence of cyanotoxins at the site over time, as well as additional cyanotoxin and cyanobacteria data from nearby lakes. Between the death of C1 and C2, this part of central Texas experienced an unprecedented winter storm. Beginning on 11 February 2021, temperatures ranged from 3 to −17 °C for eight days prior to the death of C1.The current study presents data from the necropsy of C1) approximately 3 years old and weighing 51 pounds died after swimming on a freshwater reservoir (Lake Travis) located near Austin, Texas. The location, known as Hudson Bend   , has wat1’s death became public, several other residents came forward to report that their dogs also experienced similar symptoms following exposure to the water and shoreline of Hudson Bend. A survey was sent to the owners who reported these symptoms, which included a report of an unconfirmed canine death that occurred on 30 January 2021. Results from this survey can be found in the corresponding data set [1 showed filamentous green algae and an unidentified cyanobacteria present in the gut at the time of death , homoanatoxin-a (HTX) and cylindrospermopsin were all non-detect in the digestive tissues of C1. High levels of dhATX were found in multiple digestive tissues, including duodenum tissues , jejunum tissue , and from mixed stomach content . Toxins in samples were verified by high-performance liquid-chromatography–high-resolution mass spectrometry (HPLC-HRMS) using methods described in Manning et al. 2020 [+, which agrees with previous results [During the necropsy, additional samples were collected from the digestive tract and tested for cyanotoxins; dhATX concentration is reported on a dry weight (DW) basis . A trace1 was neurotoxicosis caused by dhATX.Overall, water samples had significantly lower quantities of dhATX when compared to complex extracts from metaphyton materials. Further, the retention time (RT) of dhATX in water samples was consistently observed slightly earlier in HPLC-HRMS runs when compared to extractions from more complex materials, e.g., biomass. As such, slight lags were observed for the RT of dhATX detected in extracts of metaphyton and necropsy materials. Raw and processed data from all analyses are reported in replicate and can be found in the corresponding data set . The pren = 10) found dhATX present in all composite samples collected, ranging from 7.80 to 8270.00 ng/mL , Composite samples containing both metaphyton and periphyton material collected from Hudson Bend on 22 February 2021, contained relatively high levels of dhATX (630 ng/mL), with low levels of microcystins/nodularins (1.13 ng/mL), saxitoxin (0.08 ng/mL), ATX (1.0 ng/mL), and non-detects for cylindrospermopsin and HTX. Results from an expanded survey around Lake Travis  conducted on 3 March 2012 (00 ng/mL . HTX was00 ng/mL  with low1’s death) showed that toxins were still present in metaphyton materials from 21 February 2021 through 23 June 2021, when monitoring was concluded after two measurements were below the limits of detection, i.e., non-detects  . Secchi Turbidity measurements remained consistent with the observed historical average with only a slight deviation in October 2021. Total suspended solids (TSSs) followed historical trends, Secchi disc depth was slightly higher than average, while turbidity was slightly lower than average leading up to and immediately following the incident . Tempera1 as well as the high levels found in both metaphyton/periphyton material and water samples at the site of exposure provide strong evidence that C1 died from dhATX neurotoxicosis. While ATX was also found at the site, recent studies have demonstrated that dhATX is more toxic than ATX when ingested orally with a lethal dose of 8 mg/kg for dhATX and 25 mg/kg for ATX [Incidents of dogs becoming ill or dying after exposure to cyanobacteria have increased in recent years; however, very few of them are directly linked to cyanotoxins due to lack of supporting necropsy evidence, or incidents are reported too late to collect relevant samples ,3. The p for ATX .1 and before the onset of the winter storm that impacted Texas . This iing data . While oing data ,6,9,14.The survey of Lake Travis showed that high levels of dhATX can be present in mat material, while whole water samples collected directly next to mats can be non-detects for dhATX. This observation supports previous work suggesting that consumption of lake water alone is often not enough to cause intoxication events, as cyanotoxins can have low stability in the water matrix, and cyanotoxin-related deaths are most likely caused by consumption of mat material ,14. Addi1’s death, historical routine water quality data in Lake Travis showed that water temperatures in the months before, during, and after the death of C1 were consistent with observed trends from the previous five years. Reinl et al. 2023 defined a cold water cyanobacterial bloom as a cyanobacterial bloom that occurs in freshwater lakes when water temperatures are below 15 °C [1, ambient air and water temperatures in Texas were historically low (<15 °C), with an extreme temperature difference potentially leading to turnover in Lake Travis. Physical disturbances or extreme storms can disturb the benthos and cause benthic proliferations to float to the surface [Water quality parameters collected from Lake Travis before, during, and after the dhATX exposure event can shed light on how these events may be impacted by water quality. While water and ambient air temperatures were unusually cold in the week leading up to C surface ,15, whicDreissena polymorpha. Zebra mussels were first detected in Lake Travis in 2017 [The decrease in turbidity and increase in secchi depth values in Lake Travis over the past five years may be a result of the continuing invasion and colonization by zebra mussels,  in 2017  and have in 2017 . The est1 ultimately died from dhATX neurotoxicosis. In addition to confirming the first canine death related to dhATX in Texas, this study supports the growing body of literature concerning not only the rise in benthic cyanobacterial blooms but provides evidence that benthic blooms can occur in winter following extreme temperature shifts. Since the event of C1’s death in Lake Travis, the managing authority over the waterbody has implemented a year-round monitoring program for common benthic and planktonic cyanotoxins. However, there is little to no guidance for monitoring programs regarding benthic proliferations, with WHO threshold values only applying to whole water samples and no such guidelines for dhATX in water or metaphyton materials. This lack of recreational threshold guidelines for benthic proliferations can make lake and river closures difficult to justify. Therefore, development of actionable guidelines for dhATX is a crucial next step to maintain public health and help inform waterbody management strategies.Necropsy evidence coupled with environmental samples containing dhATX provide strong evidence that CLake Travis  is an impoundment of the Texas Colorado River created after the construction of Mansfield Dam was completed in 1942. It is a water supply reservoir and is the fifth reservoir in the Highland Lakes chain. Hudson Bend is the most downstream major bend in Lake Travis. Residence time and water levels in Lake Travis fluctuate based on inflows and management of the reservoir according to a stakeholder-driven and state-approved water management plan. The Lower Colorado River Authority collects routine water quality data from Lake Travis and the Texas Colorado River basin as a partner in the Texas Clean Rivers Program (CRP) administered by the Texas Commission on Environmental Quality (TCEQ) according to Texas Water Code, Section 26.0135. Historical data used to identify five-year trends in Lake Travis were collected from a CRP site located in front of Mansfield Dam .Whole water samples were collected at the surface (approximately 0.3 m depth) and contained no proliferations or algal material. Composite samples consisted of any floating mats, or epiphytic and/or periphytic algal material found at the site. Both whole water and composite samples were collected in amber bottles and frozen until processed. Historic water quality data from Lake Travis were collected following protocols outlined by the TCEQ Surface Water Quality Monitoring Procedures, Volume 1 . Potenti1 was performed by Texas A&M Veterinary Medical Diagnostic Laboratory, which included examining the musculoskeletal, respiratory, circulatory, digestive, hepatobiliary, hemolymphatic, urinary, reproductive, endocrine, and nervous systems. Necropsy included a toxicology screen for a broad range of organophosphates, some carbamates and organochlorine insecticides, as well as permethrin, petroleum products, and some drugs. Necropsy also included a screen for botulism A, B, and C. Twenty-three tissue samples were collected for histopathology. The full necropsy report can be accessed in the corresponding data set [A full necropsy of Cdata set . Additiodata set ). Microcdata set .g to sediment debris. Water and solid/biomass extracts were processed by C18 solid-phase chromatography to enrich cyanotoxins and analyzed by HPLC-HRMS, according to procedures described in Manning et al., 2020 [2O) with sonication. Zebra mussels (six bodies) were shucked and extracted in ddH2O with 1% HCl  assisted by heating (60 °C) and sonication for 6 h; and 12 zebra mussel half shells were extracted in ddH2O for 2 h with sonication. Necropsy liquids and tissues were extracted in methanol and 1% HCl with sonication for 6 h. Extracts were dried and resuspended in 5% MeOH in preparation for analysis by HPLC-HRMS. All samples were performed in duplicate.Water samples and composite metaphyton/periphyton material samples were analyzed by GreenWater Laboratories, sample analysis methods are described in the corresponding data set, and the full reports can be viewed . Environ2O. Then, the water or algal sample supernatant was slowly pushed through the column to adsorb metabolites. The eluate was retained as the sample load, and 5 mL of 100% MeOH was pushed through the cartridge to collect metabolites adsorbed on the column. Methanolic fractions were dried under nitrogen at 40 °C in pre-weighed glass vials. Once dried, extract residues were resuspended in 1 mL of 5% MeOH (aqueous), representing a 25- to 50-times enrichment from the original samples. All samples were prepared for analysis containing 0.1 ng/µL of caffeine internal standard  to monitor injection consistency and detection sensitivity; 100 µL of each sample with standard was transferred to a glass insert in a 2 mL amber glass vial in preparation for HPLC-HRMS.Clarified lake water (50 mL) and extracts (25 mL) were passed over Waters Sep Pak C18 solid-phase extraction (SPE) cartridges  to enrich targeted compounds. SPE cartridges were activated with 5 mL of 100% MeOH  and conditioned using 5 mL of ddHSamples were analyzed on an Agilent 6530 QTOF instrument . Samples (10 µL) were injected and separated on an Agilent Zorbax Eclipse Plus C18 column  using a water:methanol gradient with 0.1% formic acid addition . The gradient was ramped from 1.0 to 95.0% MeOH over 12 min and held at 100% MeOH for 6 min. Mass spectrometry data were collected following electrospray ionization (ESI) operated in positive mode to produce the total ion chromatogram (TIC) and related extraction ion chromatograms (EIC) for target toxins.n = 2).Due to an absence of commercial reference materials, quantitative analysis of dhATX in samples was estimated using the caffeine internal standard as a proxy analyte of similar mass and polarity. The area underneath each peak was combined to represent the quantity of dhATX present in each injection and extrapolated to estimate the amount of dhATX per L or water or per g or biomass sampled  was used to assess composite sample dhATX concentrations alongside multiple water quality parameters collected in open water locations within Lake Travis using the prcomp function in the R Statistical Software  . Toxins 2 values of the imputation equations ranged from 0.1537 to 1.0; however, 54% of the equations had an R2 value above 0.9 and an additional 27% of the equations had an R2 over 0.7. The majority of the imputation equations fit the data well with only a few instances where no equation fit the data well.Composite samples were not collected on the same day as the water quality samples, so water quality parameters were imputed and estimated based on mathematical formulas, using equations for Julian day. The formulas were derived from water quality samples collected every other month at each CRP site from 25 February 2021 to 14 December 2021, for a total of six samples at each site. The frequency of observed values would not allow for the evaluation of acute changes to water quality, but it is appropriate to allow the authors to evaluate general trends of water quality within the site location during the event. Quadratic to quintic equations were chosen as imputation equations for each parameter and site based on the fit of the equation. The Rn = 46. There were quality control issues with one TKN sample and the sample size for TKN was n = 45. The average for each parameter by day was calculated by transforming the date to Julian day and applying locally estimated scatterplot smoothing , also k


Partial mitochondrial DNA sequences from 25 dogs revealed eight haplotypes within canine haplogroup A , C , D  and B . Based on shotgun sequencing, four Roman mitogenomes were assembled, representing sub-haplogroups A1b3, A1b2 and C2. No canine pathogens were identified, weakening the assumption of infectious disease as a cause for dog disposal. The genetic and morphological diversity observed in dogs of Augusta Raurica and Vindonissa is similar to modern dog diversity.Several dog skeletons were excavated at the Roman town of Augusta Raurica and at the military camp of Vindonissa, located in the northern Alpine region of Switzerland (Germania Superior). The relationships between them and the people, the nature of their lives, and the circumstances of their deaths are unclear. In order to gain insight into this dog population, we collected 31 dogs deposited almost simultaneously in two wells , three dogs from burial contexts (70–200 CE and third to fifth century CE) at Augusta Raurica, and two dogs from burial contexts at Vindonissa (ca. first century CE). We detected a mixed population of young and adult dogs including small, medium and large sized individuals. Three small dogs had conspicuous phenotypes: abnormally short legs, and one with a brachycephalic skull. Stable isotope analysis of a subset of the dogs showed that their diets were omnivorous with a substantial input of animal proteins and little variation, except one with a particularly low δ While accompanying people across the world a close relationship was established and as consequence of their various exploitations by humans, dogs gained the ability to digest starchy food and underwent bodily changes, e.g. coat colour and floppy ears9.Since their domestication before the emergence of Neolithic farming, dogs diversified in appearance, behaviour and function, a process still ongoing10, in Europe dogs increasingly diversified during the Roman period, a process which likely began around the 3rd century BCE in Italy. This is well documented in texts, depicted in mosaics, and supported by objects  and/or with abnormally short limbs , have been described in the archaeozoological record from a variety of Roman sites in Britannia22, Hispania23, Italia24 and Pannonia25. Together, the data suggest dog breeding for the purpose of selection for a specific phenotype, such as size, shortened limbs or slenderness.Although various body sizes and coat colours are already attested for in the Magdalenian time period (18′000–12′000 BCE) and throughout prehistory26. In some cases, their dead bodies were even carefully buried, for example, at the Roman pet cemetery in Berenice, Egypt27. However, more often their bodies were deposited in rubbish pits or in wells out of use29. However, dog health, feeding and welfare issues and their complex relationship to people have rarely been addressed or brought into focus31.Normally dogs were not part of the human diet in the Roman period and were not slaughtered32 and have been widely used to study faunal dietary diversity33. Therefore, stable isotope analysis of dog bones may shed light on their diet and living conditions. Roman authors actually recommended specific diets depending on dogs’ tasks and life stage and presumably the composition of dog food in Roman times may have varied due to the individual feeding preferences of dog owners31.In addition to archaeozoology, stable isotopes and ancient DNA offer further perspectives for addressing social relationships between humans and dogs. Stable isotope ratios in skeletal remains reflect dietary compositions36 with a dominance of A lineages in modern times, in contrast to a dominance of haplogroup C lineages during the Neolithic, suggesting a prehistoric turnover (introductions/replacements) of mtDNA lineages in Europe37. However, haplogroup A lineages have continuously dominated in Iberia since the Mesolithic and other lineages in central Europe have also likely survived since the Neolithic38. For the Roman era genetic information about dogs is scarce. Only two genetic studies addressing Roman dogs mtDNA diversity in Iberia/Morocco20 and Bulgaria5 are currently available. Their results show high mtDNA diversity with uneven geographic distribution of haplotypes belonging to the major dog haplogroups A and D (Iberia) and A, B and D (Bulgaria), denoting different histories of maternal dog lineages between the different regions of the western and eastern part of the Roman Empire, and possibly suggesting diverse centers of dog “breeding” activities.Genetically dogs are highly diverse. Based on mitochondrial DNA (mtDNA), European dogs belong to one of the four major haplogroups A-D40 . Large socio-economical changes occurred such as the introduction of horticulture, new crops and spices, new domestic animals and new technologies to Switzerland. The region was prospering and trading intensified41. People were very mobile in the Roman Empire43 and they transported not only objects related to daily life but they were also accompanied by their animals such as dogs. Therefore, the region considered here probably became a melting pot not only for people but also for animals. In the 2nd half of the third century CE, while the once prosperous city Augusta Raurica gradually deteriorated, several dogs were deposited in different wells, a common praxis of animal disposal during the Roman period in Europe44. In the case of the wells of Augusta Raurica, however, the archaeological evidence points to quick depositions within a few years or even within a few months suggesting that the animals were living at the same time and deposited almost simultaneously. Potential circumstances accompanying this event are still under debate48. The dogs from Vindonissa were from burial contexts and date to the 1st century CE.With the northward expansion of the Roman Empire beyond the Alps and the establishment of the imperial province Germania Superior, economic, cultural and military centers such as the colonial town of Augusta Raurica and the military camp of Vindonissa in the north of Switzerland were foundedThis research combined osteometry, mitochondrial DNA D-loop typing, mitogenome analysis, shotgun sequencing to screen for pathogens and stable isotope analyses of dogs from Augusta Raurica and Vindonissa. The aim was to gain insight into the size, age and skeletal particularities, into the genetic variation in terms of mtDNA haplogroups and haplotypes and potential diseases as well as the diet of dogs from a local and near contemporaneous context of two wells at Augusta Raurica. The study was extended to include dogs from different time periods of Augusta Raurica and Vindonissa Fig. .47. Numerous artefacts were also found in the wells, such as short term dated coin casting molds and pottery. Matching ceramic fragments were found scattered throughout the wells, proving that the filling is to be considered as a unit and that it took place in a short time or even in one event44 and for both wells at the same time span. This is also supported by the nearly completeness of the animal skeletons  and from the legionary camp of Vindonissa (n = 2) and were made available for examination by the archaeological services of Augusta Raurica and the canton of Aargau. Both sites are located in the northern Alpine margins of the Roman Imperium (today Switzerland) Fig. . Most ofons Fig. .Table 1I51  dog within an officer’s house close to two neonate burials40 .Osteological analyses were carried out at IPAS, Basel, Switzerland. Withers height calculations (cm) were attempted for all individuals according to Clark55 with modifications as described in56 .Stable isotope analyses were carried out at IPAS, Basel, Switzerland. Stable carbon and nitrogen isotope analysis was performed on 18 dog individuals from Augusta Raurica, of which 9 were also analysed for aDNA. The same bone specimens were used for both analyses Table . AnalytiAll aDNA data of the bones were produced independently in Basel, Switzerland and Kiel, Germany.59. For details see Supplementary Information.To prevent contaminations, handling and processing of ancient samples followed strict standards in aDNA research established at Integrative Prehistory and Archaeological Science (IPAS)1 was PCR-amplified and sequenced to determine mtDNA haplotypes in 28 dog individuals  was abnormally short-legged . Further two dogs HAU12 and HAU14 from well house in Insula 8 (“Brunnenhaus”) were brachymelic too, but additionally the skull of subject HAU14 showed the characteristic traits of a brachycephalic (“short-headed”) dog Table . To our 65 and van Klinken66. They yielded between 2.9 and 17.0% of collagen, 28.6 to 44.2% of C, 10.5 to 16.0% of N, and had atomic C/N ratios of between 3.1 and 3.4. The analytical data are listed in Table 13C and δ15N values of the dog samples from Augusta Raurica varied between − 20.7 and − 18.2‰ and 7.4 and 9.8‰ respectively, and are in the range of published stable isotope data for dogs : δ13C: Z = 2.708, p = 0.038; δ15N: Z = -2.260, but p = 0.265). This large adult individual displayed a combination of low δ15N and high δ13C  = 2.698; p = 0.017) suggesting potential differences between these time periods.Collagen of all 18 samples fulfilled the quality criteria for ancient collagen as suggested by Ambrose36. Haplotype RS-a5 (HAU7) is a private variant matching no identical sequence in public databases. Further, haplotype RS-a3 found in three dogs from Augusta Raurica  is not shared with any other Roman dog  was successful in 24 dogs (out of 26) from Augusta Raurica and in one dog (out of two) from Vindonissa. Attempts to obtain a longer D-loop fragment  failed in all samples positive for the shorter fragment. In total, 8 haplotypes could be assigned to one of the four major haplogroups A-D Table , 4. Fivedog Fig. . Three h20 and Bulgaria5 are available. Consequently, dog mtDNA sequences from Augusta Raurica and Vindonissa were compared to mtDNA sequences from these two studies , all their haplotypes are shared with dogs from Augusta Raurica and Vindonissa   Fig. . Four doBy comparing Roman with pre-Roman sequences, shifts in haplotype frequencies were observed. For example, while most haplogroup A sequences affiliate with cluster “RS-a4” in pre-Roman Iberia/Morocco, a second cluster “RS-a2” appears with increased numbers of sequences and haplogroup D appears for the first time in Roman Iberia/Morocco Table , Fig. S438; a decrease in haplogroup A and an increase in haplogroup D frequencies in Roman Bulgaria5. Notably, Bulgaria preserves the highest ratio of haplogroup B (ca. 30%) over time 81, and Hispania Tarraconensis24. The detection of three small-sized adult and abnormally short-limbed dogs  one of which with a brachycephalic skull (HAU14), and a slender dog (HAU2), denotes distinctive morphotypes of small dogs co-occurred at Augusta Raurica. A similar short-limbed small brachycephalic dog was reported from the Roman cemetery of Llanos del Pretorio 23, implying that such combined traits were not unique and restricted to one particular region. This raises the question of where this dog type was first bred and whether they were indeed lapdogs, as previously suggested for these kinds of small individuals82. Two dogs, one juvenile (HAV1) and one adult (HAV2) from the military camp of Vindonissa were small and shared no particular phenotype but were found in burial contexts, potentially being esteemed household dogs of legionary people.The present study used an interdisciplinary research approach to analyze a large number of Roman dog individuals including dogs deposited in two wells that likely lived contemporaneously around the mid-third century CE in the town of Augusta Raurica, Germania Superior, a unique assemblage of bone deposits in Northern Switzerland. Osteometric analysis of all individuals (n = 33), revealed a broad range of sizes providing the first morphometric information from a large population of dogs living on the edge of the Roman Empire. The dogs were classified into three size categories, e.g. small (n = 10), medium (n = 17) and large (n = 6) and included young and adult individuals. Overall, the dogs show a similar size variation to dogs across other Roman regions and sites, e.g. Pompeii 47, raising questions around the way that these deposits should be interpreted. Various theories are being discussed for this phenomenon such as “only” waste disposal, victims of war or epidemic, political crisis, the contamination of water or ritual deposits48. The short period of time during which the animals were deposited and the relatively large number of dogs and other animals disposed in the wells suggest that an epizootic disease was a potential cause. Shotgun DNA sequence analysis of dog viral and DNA based pathogens was, however, negative. The general low quality and quantity of endogenous nuclear DNA of the samples might explain the negative screening results for viral and bacterial pathogens. Or, pathogens attacking primarily soft tissues remained unperceived. Therefore, we cannot completely rule out infectious disease or other pathologies as cause of dog death and disposal.In contrast, it is rather surprising to find a brachycephalic dog with such a particular trait disposed in a well. Was this dog not considered as something special (e.g. prestige pet?) and worth a proper burial? Was it subject to a ritual act, or was it a precipitous disposal with another background? The same questions may also apply to the slender dog. The remains of several other wild and domestic animals  were also disposed of in the wells along with the dogs83 suggesting that dogs followed similar diets as humans with substantial proportions of vegetal food components besides animal proteins. It is known that the Romans were feeding their dogs differently according to a number of factors, including function and age, but most likely the majority of Roman dogs were relying primarily on table scraps31. Stable carbon and nitrogen isotope ratios obtained from dogs of Augusta Raurica of mixed size, age and haplotype composition , a site located close by Augusta Raurica , and a minor component of meat, bones, milk and whey (Harvey31). The substantial difference in δ13C and δ15N values between herbivores from Augusta Raurica, i.e. horses73 suggests that the proportion of animal proteins in the dogs’ diet should not be underestimated. The low isotopic variability may support the hypothesis that size, age or function, may have only moderately impacted stable isotope compositions at Augusta Raurica. However, stable isotope compositions as found in the dogs from Augusta Raurica do not allow for reliable assignments of functions to individual dogs. The isotopic signals show no significant differences between the small dogs with particular morphologies  and the others  components low in δ13C that eventually became scarce by the 2nd half of the third century CE when the town of Augusta Raurica supposedly was in decline, another explanation could be diachronically varying climatic, e.g. more humid, conditions. The only distinct outlier, HAU5, fed on an exceptionally low-protein and more plant based diet  as reflected in dog lineage diversity in Augusta Raurica and Vindonissa.Mitochondrial DNA variation in a 97 bp fragment of the control region in 25 individuals points to a genetically diverse dog population at Augusta Raurica and Vindonissa. Eight haplotypes representing all four widely recognized major haplogroups A-D86. Pre-Roman and Roman Bulgaria is a known centre of haplogroup B5 which is also reflected in the haplogroup frequency distribution in Europe , but not with Roman Bulgaria suggesting a connection between dog lineages from both regions. From the Median-Joining Network analysis of pre-Roman dog lineages Fig.  other liope Fig. . Compariope Fig. . The sit36). The four Roman mitogenomes differed from dog mitogenomes from Palaeolithic Switzerland , underpinning the region as a trade and transport hub north of the Alps under the rule of Rome.This study has provided the first archaeozoological and biomolecular insights into a hitherto unstudied dog population from Augusta Raurica and Vindonissa, Germania Superior, emphasizing the utility of wells and burials as sources for the reconstruction of dog diversity in Roman times in general. Based on our data no function was assigned to the dogs. However, it is a reasonable assumption that dogs from wells were a mixture of free-roaming individuals and animals cared for by people. The dogs were highly variable in size and include dogs with short-limbs, slenderness and one extreme case of brachycephaly.30.Overall, the dogs of Augusta Raurica were similarly omnivorous with a tendency towards animal proteins. Whatever the living conditions were, the dogs showed no sign of undernourishment  in line with general observations from other Roman regionsMitochondrial D-loop and mitogenome diversity suggests that diverse maternal lines were kept in Augusta Raurica and Vindonissa and probably had different geographical origins and haplogroup frequencies in the Roman period, showing a specific pattern compared to other European regions. Some of the dogs shared the same maternal lineage and, since they were living almost contemporaneously and all very likely in Augusta Raurica, they might have been closely related, raising questions of kinship and potential local breeding without excluding the possibility of mixing by chance.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.