Saudi Arabia and Yemen are the only two countries in the Arabian Peninsula that are yet to achieve malaria elimination. Over the past two decades, the malaria control programme in Saudi Arabia has successfully reduced the annual number of malaria cases, with the lowest incidence rate across the country reported in 2014. This study aims to investigate the distribution of residual malaria in Jazan region and to identify potential climatic drivers of autochthonous malaria cases in the region.A cross-sectional study was carried out from 1 April 2018 to 31 January 2019 in Jazan region, southwestern Saudi Arabia, which targeted febrile individuals attending hospitals and primary healthcare centres. Participants’ demographic data were collected, including age, gender, nationality, and residence. Moreover, association of climatic variables with the monthly autochthonous malaria cases reported during the period of 2010–2017 was retrospectively analysed.Plasmodium falciparum and Plasmodium vivax, respectively. In general, subjects aged 18–30 years and those aged over 50 years had the highest (42.7%) and lowest (5.9%) percentages of malaria cases. Similarly, the percentage of malaria-positive cases was higher among males than females (86.2 vs 13.8%), among non-Saudi compared to Saudi subjects (70.6 vs 29.4%), and among patients residing in rural rather than in urban areas (89.8 vs 10.2%). A total of 407 autochthonous malaria cases were reported in Jazan region between 2010 and 2017. Results of zero-inflated negative binomial regression analysis showed that monthly average temperature and relative humidity were the significant climatic determinants of autochthonous malaria in the region.A total of 1124 febrile subjects were found to be positive for malaria during the study period. Among them, 94.3 and 5.7% were infected with Malaria remains a public health problem in most governorates of Jazan region. The identification and monitoring of malaria transmission hotspots and predictors would enable control efforts to be intensified and focused on specific areas and therefore expedite the elimination of residual malaria from the whole region.The online version contains supplementary material available at 10.1186/s12936-021-03846-4. Plasmodium falciparum is considered the most virulent and prevalent Plasmodium species, accounting for 99.7, 69 and 62.8% of the reported malaria cases in the AFR, EMR and SEAR regions, respectively [Plasmodium vivax and that 3.3 billion people are at risk of vivax malaria infection worldwide [Malaria, one of the most common life-threatening infectious diseases, is still a major public health problem worldwide, particularly in tropical and sub-tropical regions. According to the World Health Organization (WHO), about 229 million new cases of malaria were reported worldwide in 2019 and over 3.4 billion people are at risk of infection [In Saudi Arabia, the national malaria control programme, which was established in 1948, has achieved a tremendous reduction in the annual number of malaria cases, and malaria is now restricted to the southwestern parts of the country, which includes the Aseer and Jazan regions. The number of autochthonous/indigenous  malaria cases in Saudi Arabia decreased dramatically between 2000 and 2014, from 511 in 2000 to just 30 in 2014, and the country has been included in the E-2020 WHO initiative, which is focused on achieving a target of zero autochthonous cases by 2020 , 7. In tProgress toward malaria elimination in Saudi Arabia until 2014 has been investigated by several researchers , 9. HoweIn the first part of this study, a health facility-based survey was carried out between April 2018 and January 2019. Malaria cases were detected through passive case detection among symptomatic febrile patients who visited government hospitals in different governorates of Jazan region. In the second part, association between monthly autochthonous malaria cases and climatic variables was assessed retrospectively for the period 2010 to 2017.2 and a total population of about 34 million [The Kingdom of Saudi Arabia lies in the furthermost part of southwestern Asia, bordered by the Red Sea to the west, the Arabian Gulf, United Arab Emirates, and Qatar to the east, Yemen and Oman to the south, and Kuwait, Iraq and Jordan to the north. It is the largest country in the Arab Peninsula, occupying about four-fifths of the Peninsula with a total surface area of approximately 2,000,000 km million . Geograp million . Althoug million .2 and having a total population of about 1.8 million. The region consists of three distinct zones: a highland zone  at an elevation of 2500–3000 m above sea level and with rainfall of more than 200 mm/year; a hill zone at an elevation of 400–600 m above sea level and with rainfall of less than 200 mm/year; and, a coastal plain  at an altitude of less than 400 m above sea level and with little of sporadic rain [Jazan region is located in the southwest corner of the Kingdom of Saudi Arabia between longitude 42.7076° E and latitude 17.4751° N. Jazan region is considered the smallest region in the Kingdom, but it has the highest population density in the country, occupying a total land area of approximately 11,671 kmdic rain , 14. Thedic rain . Jazan rAnopheles superpictus and Anopheles stephensi are still present as possible vectors); (3) western region ; and, (4) southern and southwestern regions  [Anopheles arabiensis is the main vector for malaria in the region; however, other species, which are known to be competent malaria vectors, have also been recorded, including Anopheles sergentii, Anopheles dthali, Anopheles fluviatilis, An. stephensi, An. superpictus, and Anopheles culicifacies [Anopheles usually peaks following the rainy season that falls during October to December [With regards to the malaria profile of the country, Saudi Arabia can be classified generally into four topographical categories as follows: (1) centre of the country ; (2) northern and eastern regions  , 8. The e rates) . Jazan re rates) , 17. Anocifacies , 19. AltDecember , 21.Saudi Arabia adopted artemisinin-based combination therapy (ACT) as its anti-malarial drug policy in 2007 . AccordiPlasmodium species and parasite density for malaria-positive patients, as well as the date of blood examination were also extracted. Moreover, whole blood samples and Giemsa-stained blood films of all malaria-positive subjects examined during the study period and who gave their consent to participate were collected from the respective healthcare facilities for further examination. In addition, blood samples of randomly selected malaria-negative subjects were also collected and re-examined.From 1 April 2018 to 31 January 2019, blood samples were collected from febrile patients presenting for healthcare at participating healthcare facilities in Jazan region. All patients who were found positive for malaria were invited to take part in the study regardless of their gender, age and nationality. Demographic data about the participants including age, gender, nationality, and residential address (village or town and governorate) were collected by using a standard semi-structured questionnaire or from patients’ medical records. The data were extracted from the patients’ medical records by trained research assistants on a weekly basis and entered into standard Excel spreadsheets prepared for the study. Laboratory diagnosis data including malaria status, and About 2–3 ml of venous blood was collected from each participant into an EDTA tube clearly labelled with the patient’s unique reference number, name, age and gender. Next, a drop of blood was subjected to a rapid diagnostic test (RDT) using the AMP Rapid Test-Malaria p.f./pan kit . Then, thin and thick blood films were prepared and stained with 3% of buffer-diluted Giemsa stain for 45 min, and then examined by light microscopy for the presence of malaria parasites. Infection status and parasite density were assessed using the thick films while parasite species and stages were assessed on thin films. A thick blood film was considered negative if no malaria parasites were detected after examining 200 high power fields under a 100 × objective .Parasite density (parasitaemia), expressed as the number of parasites per µl of blood, was determined by counting the asexual stages against 200 white blood cells (WBCs) on the thick blood film, and then multiplying by 40, based on the WHO-recommended average of 8,000 WBCs per μl of blood in every human being . The levhttps://mewa.gov.sa/ar/Pages/default.aspx) [https://www.stats.gov.sa/en/258) [Data were double entered by two different research assistants into Microsoft Office Excel 2010 spreadsheets. Then, a third researcher crosschecked the two datasets for accuracy and created a single dataset for analysis. Climatic data for Jazan region, including mean minimum, maximum and average temperature, mean relative humidity, mean wind speed, mean atmospheric pressure, and monthly aggregate rainfall, sandstorm and dust haze events were provided by Sabya governorate’s weather station located in central Jazan, and from the annual statistical documents published by the Ministry of Environment, Water, and Agriculture – Saudi Arabia (lt.aspx) , 25, andData analysis was performed using IBM SPSS Statistics version 27.0—Essentials for R . The demographic characteristics of the participants were treated as categorical variables and presented as frequencies and percentages, while the quantitative variables were presented either as mean  or median . Pearson’s Chi-square test was used for 2 × 1 contingency table analysis to test the associations between malaria infection and demographic characteristics including age, gender, nationality, and residence .For each climatic variable, a monthly average or aggregate was calculated. Malaria cases were grouped by month of onset. In Jazan region, malaria is mostly imported, however, only locally transmitted cases reported between 2010 and 2017 were considered in this study in order to justify potential relationship with local climatic variables. Pearson’s correlation coefficients were calculated to determine the correlation between malaria cases and the climatic variables. For biological reasons such as the development and survival of mosquitoes and the development of the parasite within the mosquito, different lags  were considered for climatic variables to determine the maximum significant correlations. Since malaria cases was a countable variable with excessive zero counts, and the cases were over-dispersed with a variance greater than the mean, zero-inflated negative binomial regression (ZINB) was applied to identify the relationship between malaria cases and climatic factors at different monthly lags .P value of < 0.10 in the best ZINB models favored by AIC were included in the final reduced model (Model C). To quantify the effects of the included climatic variables, influences or percentage change (eβ − 1) were computed [P value of < 0.05 was considered statistically significant for all statistical tests.The covariates were selected based on multicollinearity (r > 0.80) and the strength of their statistical correlation with malaria cases  had P. vivax  parasitaemia of vivax malaria was 2084  parasites/μl of blood.A total of 1124 febrile subjects were found to be positive for malaria: 1060 (94.3%) were infected with Figure Figure P < 0.001). Also, subjects aged 18–30 years had the highest percentage (42.7%) of confirmed malaria cases followed by those aged 31–40 years (23.8%) while those aged over 50 years had the lowest (5.9%) when compared with other age groups. With regards to nationality, 70.6% of confirmed malaria cases were non-Saudis and 29.4% were Saudi subjects and the difference was statistically significant (P < 0.001). Similarly, a significantly higher percentage of malaria cases was found among subjects from rural areas (89.8%) compared to those from urban areas (10.2%). With regards to species, vivax malaria cases were not found among young children (aged below 10 years) and older persons (aged ≥ 65 years). Interestingly, four cases (6.3%) only of vivax malaria were among female patients, while the rest of the cases  were found among their male counterparts. As regards nationality, three cases (4.7%) only of vivax malaria were among Saudis, while 61 cases (95.3%) were found among non-Saudis. More than three-quarters  of the vivax malaria cases were among rural subjects compared to 21.9% among their urban counterparts.Table P < 0.001). On the other hand, the correlation was significantly positive with mean relative humidity  and atmospheric pressure . Similarly, Table P < 0.01). On the other hand, the correlation of malaria cases with wind speed (r = − 0.171), aggregate rainfall (r = − 0.157) and number of dust haze events (r = -0.070) was not significant (P > 0.05).Overall, a total of 8,718 malaria cases were reported in Jazan region between 2010 and 2017. Of these, 95.3% were imported cases and 4.7% were autochthonous  cases. Table P > 0.05).Correlation of malaria cases with lagged climatic variables was assessed Table . The sigThe results of ZINB regression showed both the count model coefficients and the inflation model that contained logit coefficients for predicting excess zeroes Table . In ModeBased on AIC, Model C (AIC = 327.260) was best suited to predict the monthly autochthonous malaria cases in Jazan region compared to Model A (AIC = 330.893) and B (AIC = 337.659) and other models. Thus, ZINB logit coefficients of Model C were the final results of this regression model Table . The resThe current study revealed that malaria remains a public health problem in Jazan region, with a total of 1124 confirmed cases were reported among the febrile patients presented at healthcare centres during the study period. Since the introduction of a malaria elimination strategy in 2004, the burden of malaria in Saudi Arabia has been markedly reduced, and the country has successfully decreased the burden and geographic extent of malaria nationwide . HoweverIn the current study, the percentage of febrile subjects found positive for malaria parasites was higher than that reported by previous studies conducted in Jazan and some other regions of the country, including the Makkah region and in the Al-Ahsa governorate in Eastern province –33. El HMoreover, the current study revealed that malaria transmission is still active in Jazan region with malaria cases identified in 14 governorates (out of 17) during the study period. The findings also showed that malaria in Jazan region occurs throughout the year without any obvious seasonal patterns, a finding that coincides with that reported by a previous study conducted in Aseer region . LikewisP. falciparum was the predominant cause of malaria in Jazan which is consistent with previous reports [P. falciparum is the predominant species in the neighbouring endemic country, Yemen [The current findings also showed that  reports , 41. They, Yemen , 43, theAs regards the distribution of cases according to demographic factors, the current findings showed that malaria was present among all age groups including children below 5 years. However, the percentage of positive for malaria was the highest among patients aged 18–30 years, which corresponds with previous reports . This fiIn addition, the findings showed significantly higher percentage of malaria among non-Saudi patients as compared with Saudi patients, with more than half (54.2%) of the cases found among Yemeni patients. These findings are consistent with those previously reported for Jazan , 35. Mor/settlements and rural areas [The porous international borders between countries represent a major challenge to the elimination of malaria, with the Saudi Arabia border with Yemen being a typical example , 48. Theal areas .The association between climatic factors and malaria transmission has been extensively studied and the results are quite varied, thereby indicating that this association is complex. Nonetheless, such information from Saudi Arabia and Jazan region is lacking. Although rainfall is considered a critical factor that provides suitable breeding sites for mosquitoes, temperature is a key driver of many vital traits of the development and the life cycle of both the parasite and the mosquito –54. The The current study has some limitations that should be considered when interpreting the above findings. First, data on the distribution of malaria cases between April 2018 and January 2019 came from passive case detection among febrile patients presenting for healthcare at participating health facilities and this limited the ability to detect malaria infection among parasitaemic but asymptomatic individuals. It should be mentioned that according to the Saudi Ministry of Health’s statistics, 184,734 individuals were examined for malaria in Jazan region during 2018, and 1516 of them were found positive with a positivity rate of 0.82% . As a reThis study revealed that Saudi Arabia still has a long way to go to eliminate malaria from the entire country. Despite the continued intensive efforts being made by the Saudi health authorities to combat malaria, numbers of malaria cases are still being reported in most governorates of Jazan region. However, the identification and monitoring of malaria transmission foci would enable control efforts to be intensified and focused on specific areas, and therefore might also ultimately lead to the elimination of residual malaria in the whole region.Promotion of operational research is crucial to identify and determine the feasibility of innovative and integrated measures that would maximize the effectiveness of control strategies against cross-border malaria as well as to generate and make best use of data to guide malaria elimination efforts. Moreover, further studies on modelling the relationship of malaria and its vectors with metrological and climate change-related factors in Jazan region are essential to predict malaria outbreaks and to understand the transmission dynamics of the disease in southwestern Saudi Arabia.Additional file 1: Table S1. Pearson’s correlation coefficient matrix of unlagged monthly climatic variables and autochthonous malaria cases reported in Jazan region between 2010 and 2017


In this paper, we analysed the complete for sequence in eight clones of the peach potato aphid Myzus persicae, reporting the presence of nonsense and frameshift mutations in three studied clones characterized by a reduced number of winged females and by the absence of exploratory behaviours. Quantitative PCR experiments evidenced similar results in clones possessing for genes with a conserved coding sequence, but low expression levels. The comparison of the for transcriptional level in Myzus persicae persicae and Myzus persicae nicotianae showed very different expression in the two studied M. p. nicotianae clones so that our data did not support a previous hypothesis suggesting that a differential for expression was related to ecological specialization of M. p. nicotianae. In view of its role in both the dispersal of winged females and exploratory behaviours, the screening of the for sequences could be useful for predicting invasions of cultivated areas by peach potato aphids.The  In the last decades, several well-studied examples that shed light on genes and pathways underlying insect behavioural plasticity have been described ,2,3,4,5.foraging gene (for), which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), suggesting that it may regulate different behaviours in several insects, including Diptera, Hymenoptera, Coleoptera and Hemiptera [Several recent achievements in this research field have been related to the emiptera ,7,8,9,10Drosophila melanogaster [for gene has been successively deeply studied in social insects, where it has been observed that its differential expression plays a key role in the division of labour in diverse castes [for regulates worker transitions between behavioural tasks and specific behavioural traits associated with morphological castes [Originally identified as a behaviourally polymorphic gene that drives alternative foraging strategies in the fruit fly nogaster , the fore castes ,6,12. Inl castes .for in the insect brain is currently unknown, studies in some insect species indicated that PKG signalling plays a conserved role in the neuronal plasticity of sensory, cognitive and motor functions, which underlie behaviours related to appetitive learning, aggression, stress response, phototaxis and response to pheromones [Although the specific neurological role of eromones .for gene orthologue has also been identified in the pea aphid Acyrthosiphon pisum (Apfor) [Apfor may trigger the shift from sedentary to exploratory behaviour [A  (Apfor) ,14, wherehaviour .for orthologue has also been studied in the peach potato aphid Myzus persicae (Mpfor) comparing generalist biotypes with tobacco-specialized races , suggesting that Mpfor influences the process of host searching and the evolution of ecological specialization [for expression are related to a reduced PKG expression, resulting in reduced mobility, foraging activity and dispersal [A lization . At the ispersal .for expression observed in M. p. nicotianae could reinforce differences in diet breadth and ecological specialization by, for instance, reducing resource-searching behaviours and mobility. This hypothesis has been supported by the presence of a higher for transcription in alates that are responsible for host searching and dispersal in generalist clones in respect to specialized ones [Interestingly, Tapia and colleagues  suggestezed ones .for sequence in eight M. persicae clones in order to evaluate if the screening of the for sequence could be useful to predict invasions of cultivated areas by peach potato aphids. Lastly, we compared the for expression in M. p. persicae and M. p. nicotianae to confirm the role of Mpfor in promoting ecological specialization.Considering that the dispersal of winged females is a key element to determine invasiveness of aphids, here, we compared the M. persicae were obtained from eight aphid lineages maintained as a colony of parthenogenetic females on pea Pisum sativum  plants at 19 °C with a light–dark regime of 16 h light and 8 h darkness that ensures parthenogenic reproduction . Aphids were transferred to fresh pea plants once a week.Specimens of Aphids were reared in parallel at low and high population densities. In particular, five wingless parthenogenic females were transferred in an insect box and their progenies collected after a week. This low population density condition provided good food quality and a large space for female reproduction. High population density was obtained by transferring thirty females in a box containing a single pea plant and left there, giving rise to several generations until, after a week, crowded conditions were achieved.M. persicae clone 1GK was collected from Greece and kindly supplied by John Margaritopoulos (Greece); clones K1, K10, D type and 229 were collected from Scotland and were kindly supplied by Brian Fenton (Scotland); clones 1ITA, 33H and 7 were collected from Italy and were kindly supplied by Emanuele Mazzoni . Clones 1GK and K1 were previously identified as M. p. nicotianae using microsatellite-based and chromosomal analyses [Aphid clones were collected from three countries: analyses ,16. A deIsofemale clonal lineages were obtained from lines of aphids issuing from a single female and maintained in culture in the laboratory on pea plants in order to obtain homogeneous aphid samples. Clonal lines are periodically checked with different biochemical, molecular and biological assays in order to ensure the stability of the biological traits  observed at their first identification.Aphids reared in high population densities have also been used to evaluate the presence of winged adults and wingless foragers. In particular, the number of both winged adults and unwinged walking aphids exploring the insect box was evaluated after a week of breeding in crowded conditions. This approach evidenced an abundant presence of both alate females and walking foragers in response to crowding clones 1ITA, 1 GK, K10, D-type and 229, whereas clones 33H, K1 and 7 consisted of unwinged females only, even if maintained on crowded pea plants.SV Total RNA Isolation System (Promega), according to the supplier’s suggestions. RNA samples were quantified by spectrophotometric absorbance measurements using a NanoDrop™ ND1000 Spectrophotometer .RNA extraction was performed with the for gene was performed by RT-PCR with the Access RT-PCR System (Promega) and the primers F-for (5′-GAGACGTTCTACAATGCTGGA) and R-for (5′-AGCAAAACCAAAGTCGACCA) at an annealing temperature of 57 °C for 1 min using 0.5 µg of polyA+ mRNA. Primers were designed using the webtool Primer3  using the template sequence JF776573.1, annotated as for orthologue in the pea aphid A. pisum. RACE amplification was carried out to complete the for sequence using the 5′/3′ RACE Kit (Roche), according to the supplier’s instructions.Amplification of an internal 807 bp-long portion of the ® kitTA Cloning (Invitrogen) and transformed into electrocompetent ® TOP10One shotEscherichia coli cells (Invitrogen). Recombinant plasmids from ten positive colonies for each aphid clone were extracted, purified and sequenced at BMR Genomics .Amplified fragments were cloned with the ® CLC sequence viewerBiogen  and with the nBLAST and pBLAST tools available at NCBI . The analysis of the conserved domains in the coded foraging proteins was carried out using the Conserved Domain Search tool available at NCBI .Sequence alignments were performed using the software for genes in the eight M. persicae clones was accomplished via quantitative real-time PCR experiments (qPCR). In particular, RNA samples for qPCR were extracted from a pool of ten M. persicae wingless parthenogenic adults collected after a week of breeding in crowded conditions using the Quick-RNA™ Miniprep Kit  following the manufacturer’s protocol. All RNA samples were double-checked for purity and quantified using both Qubit® RNA HS Assays  and NanoDrop™ ND1000 Spectrophotometer .The comparison of the expression level of the ® cDNA Synthesis KitiScript  according to the manufacturer’s instructions. The qPCR reaction was performed using a ® Green SupermixSsoAdvanced™ Universal SYBR  with the specifically designed primers F-for_qPCR (5′-TGGAGTCTTGTCTTGGTGGT) and R-for_qPCR (5′-ATGCTTCAAGACACATCCG), following instructions provided by the manufacturer and using 1 µg of polyA+ mRNAs.The mRNA was reverse-transcribed to cDNA using the CFX96 Touch Real-time PCR detection system . Once the amplification reaction was completed, the melting curves were inspected for all the amplicons. Relative quantification of qPCR data was obtained through the 2−(ΔΔCt) method, according to Livak and Schmittgen [p < 0.05).The applied thermal profile was as follows: 95 °C for 10 min (1 cycle), 95 °C for 2 min, and 58 °C for 30 s (30 cycles). All the reactions were performed in triplicate on a hmittgen . Statistfor gene in the eight studied M. persicae clones, showing the presence of a 2310 bp long coding sequence for all M. persicae clones. The length of the Mpfor coding sequence is in agreement with data reported in the pea aphid A. pisum, where two orthologues have been identified with respective sizes of 2331 bp for Apfor1 and 2112 bp for Apfor2 [The combined use of RT-PCR and RACE allowed the amplification of the complete r Apfor2 .Apfor orthologues differ in the first two exons, whereas they perfectly overlap in their following sequence. In particular, exons 1 and 2 of Apfor2 are located within the intron 2 of the Apfor1 gene suggesting that these exons are spliced in the Apfor1 variant. The presence of a single for gene in M. persicae supports the occurrence of a specific duplication in the A. pisum lineage.As reported by Tares et al. , the ApfM. persicae coded foraging protein showed a 99% and 96% identity with the A. pisum Apfor2 and the D. melanogaster foraging respectively, whereas a lower identity (86.7%) was obtained for the Apfor1 protein, suggesting that this probably represents a paralogous copy of the for gene.The M. persicae clones 1ITA, 1GK, 229, K10 and D-type, with sequence identity ranging from 99.69 to 100%  all showed low for expression levels.The analysis of the qPCR experiments  evidencefor expression was observed in clones 1GK, 229 and K10, whose wingless adults and nymphs moved greater distances on crowded plants (exploratory behaviour) in respect to the other clones  so that our data did not support this proposal. Even if we agree that the adaptation to new host plants and/or the specialization for some of them may result from the simultaneous contribution of several genes, our data suggest that other mechanisms, such as the upregulation of detoxification-related genes [M. p. nicotianae.Lastly, we compared the lization . Our fined genes  and geneed genes ,29, coulfor gene has been suggested as a possible target for RNA interference-based field control strategies for the hemipteran Bemisia tabaci [Interestingly, the a tabaci , furtherM. persicae populations. As a consequence, the evaluation of the propensity of aphids to disperse in the fields, is essential to plan proper strategies for their control. In view of its involvement in the presence of both alate aphids and explorative behaviours, the screening of the for sequence could be useful as a tool for predicting invasions of cultivated areas by peach potato aphids.The dispersal of winged females and the presence of exploratory behaviours represent key elements at the basis of the invasiveness of the


Catastrophically, the older age groups lost ∼80% of their maximum force after one eccentric contraction (EC) of 20% strain with the greatest loss of ∼92% recorded in senescent 22-month-old mdx mice. In old age groups, there was minimal force recovery ∼24% after 120 min, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-phase model where a “tipping point” is reached when branched fibers rupture irrevocably on EC. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient  The initial wave of necrosis is followed by regeneration of the skeletal muscle tissue and subsequent cycles of necrosis and regeneration . The samrcolemma . In the controls . It is is of age . The EC-s of age .mdx mouse as a preclinical model to examine if the pharmaceutical or genetic intervention strategies prevent or reduce this EC force loss in dystrophin deficient fast-twitch skeletal muscle . These experiments were conducted in compliance with the animal ethics checklist and ethical principles under which the journal operates.The majority of previous dystrophic muscle function studies used separate colonies of wild-type control and dystrophic mice which have been inbred for over 25 years, introducing the possibility of new mutations to the groups.In this study, littermates are bred to act as control animals for dystrophic mice. These are genetically more appropriate controls for dystrophin studies as both dystrophin negative and positive animals are from identical genetic backgrounds .mdx and male C57BL/10ScSn mice. Littermate controls were distinguished from dystrophic mice by genotyping.Male dystrophic mice with littermate controls were obtained from the Western Sydney University animal facility. The colony of dystrophic mice and littermate controls used in this study were second generation offspring crossed between female C57BL/10ScSn-Dmdad libitum. Basic enrichments such as nesting crinkle material and polyvinyl chloride pipe tube were provided.Mice from five age groups were used in this study: 4, 9, 15, 18, and 22 months old where dystrophic muscles have undergone at least one round of necrosis/regeneration . These amdx mice and 32 littermate control mice). One hundred thirty-five EDL and 117 TA muscles collected for this study, not all of which were used in every procedure.A total of 71 male mice were used in this study  to maintain pH at 7.4. The muscle was stimulated by delivering a current between two parallel platinum electrodes using an electrical stimulator . All contractile procedures were designed, measured, and analyzed using the 615A Dynamic Muscle Control and Analysis software (Aurora Scientific Instruments). At the start of each experiment, the muscle was set to optimal length (Lo) which produces maximal twitch force. Muscle dissection and experiments were conducted at room temperature (∼20–22°C).Mice were placed in an induction chamber and overdosed with isoflurane delivered at 4% in oxygen from a precision vaporizer. Animals were removed when they were not breathing, and a cervical dislocation was immediately carried out. Both the fast-twitch EDL and TA muscles were dissected from the hind limb which was submerged in oxygenated Krebs solution at all times during the dissection. TA muscles were dissected tendon to tendon, trimmed (excess tendons), and weighed. The dissected EDL muscle was tied by its tendons from one end to a dual force transducer/linear tissue puller  and secured to a base at the other end using 6–0 silk sutures . Each muscle was then placed in a bath containing Krebs solution  with a composition of (in mM): 4.75 KCl, 118 NaCl, 1.18 KHPo and the maximum force output of the muscle as Lo. Pairs of EDL muscles from each animal were divided into two test groups that undergo either force frequency or isometric contraction protocols. Immediately after, all muscles were left to rest for 5 min, during which muscle length was reset to Lo, followed by the EC and recovery procedures.An initial supramaximal stimulus was given at 125 Hz (1 ms pulses) for 1 s and force produced was recorded as P) to the stimulation frequency (f) was fitted to these data.Force frequency curves were generated for one set of muscles to measure contractile function. Trains of 1 ms pulse stimuli performed at different frequencies  were given for 1 s. The forces produced were measured and a 30 s rest was allowed between each frequency. A sigmoid curve relating the muscle force (The curve had the equation:Pmin) and maximal (Pmax) stimulation at the conclusion of the force frequency curve. Half-frequency (Kf) is the frequency at which the force developed is halfway between (Pmin) and (Pmax) and Hill coefficient (h), which quantifies the slope of the muscle force frequency sigmoidal curve. These were used for population statistics.From the fitted parameters of the curve, the following contractile properties were obtained: force developed at minimal  supramaximal tetanic contractions, each lasting 1 s, separated by a 60 s rest, and then given a recovery contraction 5 min after the protocol . Stimulus pulses within the 1 s tetanus were 1 ms in duration (width) and were delivered at a frequency of 125 Hz. This protocol is a modified version of the sequence used by Lo. At t = 0 s, the muscle was stimulated via supramaximal pulses of 1 ms duration and 125 Hz frequency. At t = 0.9 s, after maximal isometric force was attained, each muscle was stretched 20% longer than their optimal length and held at this length for 2 s before returning to Lo. Electrical stimulus was stopped at t = 5 s. The EC procedure was repeated six times with 3 min rest intervals. This is followed immediately by a recovery protocol. The force measured at each EC was expressed as a percentage of the force produced during the first  contraction. From the first EC data trace, baseline force was taken before and after the first EC to quantify baseline changes as a result of the first EC.A series of eccentric (lengthening) contractions were then performed on each EDL where the contracted muscle was stretched 20% from Po measured before the six ECs. The recovery protocol is a modified version of the sequence used by Upon completion of the six EC contraction sequence, recovery force was measured immediately (Post 0’) through isometric contractions given at 125 Hz (1 ms pulses) for 1 s and at the 20, 40, 60, 80, 100, and 120 min time points . These force values were then expressed as a percentage of the Twitch kinetics were measured at three time points throughout the contractile protocol to compare differences between (1) pre-EC, (2) post EC, and (3) post-recovery kinetics. The twitches were performed at 2 Hz, for 1 ms (width), and 1 s duration. The first twitch taken from this response was used to measure kinetics immediately after initial maximum force (Pre) after the EC protocol (Post) and recovery protocol (Recov). The following parameters were collected: twitch force, half relaxation time (HRT), and time to peak (TTP).Lo) during the first EC.Stiffness is the resistance of an elastic body to deflection or deformation by an applied force. As an indicator of active muscle stiffness (during contraction), the change in force as the muscle is lengthened was measured during the first EC. Force was expressed as a percentage of the isometric force before stretching, and length was expressed as a percentage of optimum length. To estimate muscle stiffness, the change in muscle force was divided by the change in muscle length  and weighed using an analytical balance .Lf). We chose not to in the present study because:Physiological cross-sectional area (PCSA) was calculated by dividing the muscle mass by the product of its length and mammalian muscle density. Specific muscle force was obtained through dividing raw force values by cross-sectional area (CSA). When normalizing force using a calculation of PCSA in mouse EDL, many studies use a correction factor to allow for fiber length  There is considerable variation in the literature as to the value of ing used . Dependif/LoL across the whole muscle might not be valid due to the different geometry of branched fibers and unbranched fibers. It is not clear how the length of the fiber would be defined. It is possible that some other method of estimating total fiber CSA is necessary in a muscle containing branched fibers.(ii) As we show here, branched fibers are found in the dystrophic muscles, and the measurement of fiber length becomes especially problematic in branched fibers. The application of a uniform Lo × D), where MM is the muscle mass, Lo is the optimal length, and D is the density of skeletal muscle (1.06 g/cm3) to enable us to compare muscle of differing sizes and weights , and maintained at 37°C. After 25 min, the muscle was removed from solution, rinsed in Krebs solution containing 0.1% fetal calf serum, and placed in a relaxing solution with the following composition (mM): 117 K+, 36 Na+, 1 Mg2+, 60 Hepes, 8 ATP, and 50 EGTA . Each muscle was then gently agitated using pipette suction, releasing individual fibers from the muscle mass. Using a pipette, 0.5 ml of solution was drawn, placed on a glass slide for examination, and photographs of dissociated fibers taken. During counting, each fiber and its associated branches are counted as one fiber. In our earlier paper  containing 3 mg/ml collagenase type IV A , gently bubbled with carbogen .Data was presented as means ± SD. Differences occurring between genotypes and age groups were assessed by two-way ANOVA with genotype being one fixed effect and age groups the other. mdx EDL muscles is shown in mdx animals age, the number and complexity of branched fibers increases dramatically. Single enzymatically isolated EDL fibers from littermate control animals showed between <1% and ∼3% branching in all age groups , P < 0.0001] and 22 months  age groups. These findings and a lack of age effects on muscle mass are consistent with dystrophic TA studies published previously , P = 0.0045] and 22 month  age groups. Muscle hypertrophy in mdx muscle is a recognized feature  for all age groups is presented in mdx mice produced ∼25 and ∼57% less force, respectively, than controls  . Interestingly, the 22-month-old dystrophic cohort produced significantly less force compared to all other age groups . When each EDL muscle’s force was corrected for PCSA  = 7.85, P < 0.0001]. In all the age groups looked at, mdx EDL muscles produced significantly less specific force compared to littermate control animals. At 4 months of age, the mdx muscles produce ∼20% less force compared to controls . This deficit increases at 15 months with mdx muscles producing ∼54% less force compared to controls  and peaks at 22 months of age where dystrophic EDL muscles generated ∼73% less force than controls . This decline in specific force correlates with the increase in number and complexity of branched EDL fibers in the dystrophic mice , P < 0.0001], 18 months ∼45%  and 22 months ∼51% . The twitch force in 22-month-old dystrophic mice was significantly less when compared to other age groups . F  = 20.8, P < 0.0001]. This decrease in specific twitch force correlates with the increase in number and complexity of branched fibers , P < 0.0001]. It is important to note that dystrophin is absent in both 4 and 22-month-old mdx EDL muscles, and the age-matched littermate controls discount an aging effect, leading us to propose that once there are a significant number of complexed branched fibers present in the mdx EDL, there is a causative connection between the isometric force loss and the degree of fiber branching Extensor digitorum longus muscles from each age group and genotype were subjected to a series of 10 maximal isometric contractions at 1-min intervals, and the final “recovery” contraction (no 0.11) was given after a 5-min rest interval. To enable change in force comparisons to be made between EDL muscles from different age groups and genotype, the isometric force was normalized to the first isometric contraction. All the age group and genotype data are shown in mdx, however, in mdx mice from 9 to 22 months, the majority of the EC force deficit in EDL occurred on the first contraction. The isometric force drops after each EC is normalized to starting force for all age groups , P < 0.0001]. In contrast to the 4-month mdx EDL, dystrophic EDL muscles from the older 9 to 22-month age groups had ≥80% force deficit after the first EC in the series, with the greatest lost at ∼92% for 22-month-old mdx mice compared with ∼16% in age matched littermate controls  , P = 0.0016], 9-month mdx animals lost ∼93% of their starting twitch force compared to ∼55% in controls after the EC protocol . In the aged mdx cohorts, EDL muscle twitches barely produced any force reflecting the consequence to force loss due to EC-induced damage in mdx mice losing ∼97% of the pre-EC twitch force compared with ∼56% in littermate controls .In line with the tetanic force data, EDL twitch force from the older 9 to 22-month protocol . EDL musP < 0.0001], whereas the 22-month-old mdx EDL muscles recovered the least with ∼25% of their starting compared to ∼65% in age matched littermate controls . The recovery in mdx EDL decreases as the dystrophic animal ages, resulting in a decline in end recovered force as shown in P < 0.0001], 15 months recovered ∼29%  and 18 months recovered ∼33%  of starting force. Given that both 4 and 22-month-old mdx mice contain 100% regenerated dystrophin-negative muscle fibers , P < 0.0001], 15 months ∼20% for mdx compared to ∼45% for controls , 18 months ∼22% for mdx compared to ∼44% for controls , and 22 months ∼17% for mdx compared to ∼51% for controls . Once again, it is striking that although 4 to 22-month-old dystrophic EDL muscles contain regenerated dystrophin negative fibers, the force loss and EC deficit is strongly correlated with the degree and complexity of branched fibers , P < 0.0001], 15 months , 18 months , and 22 months .Stiffness is an inverse indication of muscle compliance. A stiffer muscle would exhibit a greater change in force for a given change in length. Our earlier skinned fiber studies of single non-branched muscle fibers from proteins . Here, tranching  and is lmdx and controls for each age group are as follows: 4 months , 9 months , 15 months , 18 months , and 22 months . It is interesting to note that less work is done on the mdx EDL compared with the work done on age matched littermate control EDL.Several studies have reported that the work done to stretch the muscle during the lengthening phase of an EC was the best predictor of the magnitude of the force deficit produced . We calcmdx mice produced a significantly higher resting force after an EC across all age groups, which increased in magnitude and spread with age. Differences in baseline change between mdx and controls for each age group are as follows: 4 months , 9 months , 15 months , 18 months , and 22 months .The change in resting baseline absolute force after the first EC is shown in in vitro, in vivo muscles likely contract at sub-maximal levels. To address this, we looked at twitch kinetics as an outcome parameter. Twitch kinetics were measured at (1) pre-eccentric, (2) post-eccentric, and (3) after recovery from the EC protocol with statistical analysis presented in mdx EDL, which took significantly longer to relax. Following ECs, (2) post-eccentric twitch relaxation time for mdx EDL muscles increased in all age groups and were significantly higher than control values. When measured (3) after recovery from the EC protocol, dystrophic EDL muscles had reduced half relaxation time closer to starting values but still took significantly longer to relax relative to age matched littermate controls  After recovery from the EC protocol, dystrophic EDL muscles showed a significantly slower TTP than control animals at 4-, 9-, and 22-month age groups and remained similar for remaining cohorts  pre-eccentric twitch time to peak measures showed that dystrophic EDL muscles had a significantly faster twitch time to peak compared to age matched controls in all age groups. In the 4-month-old group, both  cohorts .mdx EDL muscles across all groups and remained elevated when measured (3) after recovery from the EC protocol.mdx and age-matched littermate controls suggest the fiber type profiles of the EDL remains unchanged in the mdx, while the higher HRT/TTP ratio reported for dystrophic muscle fibers post EC shows dystrophic muscles contracting at a similar rate to relaxing. This might be due to the presence of stressed branched fibers with reduced excitability and slower rates of contraction and relaxation throughout the branched syncytium.The similar HRT/TTP ratio, (1) pre-eccentric in mdx EDL  on a light microscope, demonstrate various degrees of complex fiber branching in the senescent (22 month) mdx EDL . Figure mdx EDL muscle fibers before EC. The branch pattern in these mdx fibers range from offshoots from the main trunk of the fiber shown in mdx fiber branching in relation to mechanical findings post EC. These photomicrographs illustrate, in a qualitative manner, breaks in branched fibers, which we propose occurred because of the EC. In mdx mouse is the muscle most used to study the pathophysiology caused by an absence of dystrophin from the inner surface of the sarcolemma. The mouse EDL is a mix of fast fiber types; ∼79% type 2B (fast glycolytic), ∼16% type 2X and ∼4% type 2A . This confirms studies that have reported no significant changes in fiber types with age in mdx muscle , is controlled by a single neuromuscular junction  there is a graded force loss over the six EC which recovers by ∼47% after 120 min compared to only a ∼25% recovery in the old dystrophic EDLs with extensively complexed branching. It should be noted that EC stretches are specified relative to Lo while uncertain of fiber length, the fiber strain during these stretches may be greater than the 20% indicated where our EC protocol is severe enough to produce a non-recoverable force deficit of 20–35% in age matched littermate controls. 2+]in which produces an increase in resting tension. Our current findings, along with the review by In ranching , 9 have controls . We attrols (mdx  to the sols (mdx , 9 preseols (mdx . In Figufirst EC , 12. Olt recover . Here, wmdx EDL muscles from adult to senescent age groups have the largest component of eccentric damage which occurs as an abrupt loss of force on the first EC (mdx muscle have a graded drop in force during each phase of the EC protocol (mdx mice which are prone to rupture following EC induced injury becoming functionally obsolete. Dependent on the EC strain, degree of branching and complexity of branching, the capacity for recovery varies in mdx muscle and can explain the variability reported in literature (mdx dystrophin-deficient mouse at all age points throughout the dystrophic animals’ life span. Many studies report skeletal muscle pathologies in mdx mice between 6 and 12 weeks of age yet fail to address why these dystrophin-deficient animals can live largely asymptomatically past 108 weeks of age. It is only in the old mdx mouse that we start to see them die earlier than age matched controls (In the present study, we have demonstrated that first EC . As prevfirst EC . In contprotocol , resemblprotocol . When dyprotocol . Given tprotocol . In agedprotocol , indicatterature . Our curcontrols  and we cThe raw data supporting the conclusions of this article will be made available by the authors, upon reasonable request.The animal study was reviewed and approved by Western Sydney University Animal Care and Ethics Committee.LK, SH, PH, JM, and KN conceived and designed the experiment. LK and SK collected the data. LK and SH performed the analysis and wrote the manuscript. LK, SH, SK, PH, JM, and KN revised and approved the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Leucocoprinus gongylophorus, mediates nutrition of leaf‐cutting ants with carbons from vegetal matter. In addition, diazotrophic Enterobacteriales in the fungus garden and intestinal Rhizobiales supposedly mediate assimilation of atmospheric nitrogen, and Entomoplasmatales in the genus Mesoplasma, as well as other yet unidentified strains, supposedly mediate ant assimilation of other compounds from vegetal matter, such as citrate, fructose, and amino acids. Together, these nutritional partners would support the production of high yields of leafcutter biomass. In the present investigation, we propose that three phylogenetically distinct and culturable diazotrophs in the genera Ralstonia, Methylobacterium, and Pseudomonas integrate this symbiotic nutrition network, facilitating ant nutrition on nitrogen. Strains in these genera were often isolated and directly sequenced in 16S rRNA libraries from the ant abdomen, together with the nondiazotrophs Acinetobacter and Brachybacterium. These five isolates were underrepresented in libraries, suggesting that none of them is dominant in vivo. Libraries have been dominated by four uncultured Rhizobiales strains in the genera Liberibacter, Terasakiella, and Bartonella and, only in Acromyrmex ants, by the Entomoplasmatales in the genus Mesoplasma. Acromyrmex also presented small amounts of two other uncultured Entomoplasmatales strains, Entomoplasma and Spiroplasma. The absence of Entomoplasmatales in Atta workers implicates that the association with these bacteria is not mandatory for ant biomass production. Most of the strains that we detected in South American ants were genetically similar with strains previously described in association with leafcutters from Central and North America, indicating wide geographic dispersion, and suggesting fixed ecological services.The obligate mutualistic basidiomycete fungus,  In leaf‐cutting ant pests, an obligate mutualistic basidiomycete mediates the ants’ nutrition with carbons from vegetal matter using a fermenter‐like system inside the nest, called a “fungus garden.” Diazotrophs such as Enterobacteriales in the fungus garden and intestinal Rhizobiales were proposed to mediate the incorporation of atmospheric nitrogen into ant proteins; Entomoplasmatales were proposed to power the fungus metabolism. Leafcutters likely rely on a microbial community to access the carbon and nitrogen necessary for the production of the millions of individuals found in a single giant colony. In the present investigation, we found that mutualism between the leafcutter and Rhizobiales occurs in a wide geographic range and mainly between three closely related species within the Rhizobiales genera Liberibacter, Bartonella, and Terasakiella. However, the association between the ant and the Entomoplasmatales genus Mesoplasma is likely parasitic or commensal, since giant leafcutter colonies may be either massively invaded by or completely devoid of these bacteria, suggesting that these endosymbionts are not essential for high yields of ant biomass. We also found three new candidate mutualists, frequently isolated from the ant abdomen, including nearly identical diazotrophs of the genera Methylobacterium  and Ralstonia  and nondiazotrophs of the genus Acinetobacter. These results point to multiple noncompetitive and complementary strategies for atmospheric nitrogen nutrition within the leafcutter system. Itirapina colony was used for microbial isolation procedures for checking the occurrence of isolates in a different geographic site as compared to Rio Claro colonies. Rio Claro colonies were used for assessing microbial diversity in a single geographic region but in four different leafcutter species, as well as molecular studies. Samples were generally collected at the entrance of the nest; Acromyrmex coronatus samples were collected on the trail due to the difficulty of accessing the arboreal nest. The species were identified using barcode sequencing of mitochondrial genes COI and COII . The generated sequences were edited with the BIOEDIT 7.0.5 program  to search for adapters and primers and verify sequence quality per base. We found no adapter or primer sequences and trimmed out low‐quality raw reads (Phred Q score <20) using SeqyClean  per number of reads (X‐axis). Principal coordinates analysis (PCoA) was implemented using OmicsBox with the Bray–Curtis distances to compare bacterial community composition in different libraries. Bacterial alpha‐diversity indices were estimated on the same platform.Eight libraries were prepared, two for each of the Rio Claro colonies, either before or after sanitizing the ant samples. The ant abdomen from 2 to 4 ant soldiers was removed and macerated into a fine powder in liquid nitrogen using a mortar and pestle; then, approximately 20 mg (wet weight) was subjected to DNA extraction with the Cells and Tissue DNA isolation kit (Product # 53100) from Norgen Biotek Corp according to the manufacturer's instructions. The extracted DNA was sent to Macrogen Inc., where amplicon libraries of the V3–V4 region of the 16S rRNA gene were prepared, multiplexed, submitted to next‐generation sequencing (NGS) on the MiSeq Illumina platform (paired‐end 2× 300 bp), and demultiplexed. We then performed quality control in the FastQC program version 0.11.5 , to identify bacterial genera that were shared between ant colony samples.Venn diagrams were generated with Bioinformatics & Evolutionary Genomics tools  compared with Acromyrmex (15) from each sampling. Each soldier was washed, the abdomen was separated and macerated with a pestle in a 1.5‐ml Eppendorf tube, and portions of the macerate were streaked on solid culture media surfaces. Washing removed many of the fast‐growing yeasts, making bacterial isolation more efficient. For the isolation of Rhizobiales, the culture medium used was YMA  from Sigma‐Aldrich (cod. 101417911) and 0.25% Congo red . The media were incubated for approximately 3–4 days at 30°C and colonies were shown stained in red  and 1492R , 407R (AAICCRCCRCAIACIACRTC) conditions described by Ueda et al. , with so2.8Klebsiella pneumoniae as the positive control. The test was performed in tubes containing 5 ml of semi‐solid, nitrogen‐free NFb culture medium for approximately 72 h at 28°C. This assay was performed in duplicate. The formation of a growth film in the culture medium indicated the capacity to fix nitrogen  against Sanger sequences obtained from every isolate. When no hit was found, the isolate was considered absent from the 16S rRNA library. If hits were found, identities were calculated using Blastn for the first 500 best hits. We also mapped reads from each of the 16S rRNA libraries to the Sanger 16S rRNA sequences, using the Bowtie2 , and Entomoplasma  obtained from Acromyrmex  isolates from two Atta laevigata and one Atta sexdens colonies; six (85.8%–99.7%) Ralstonia isolates  from Atta laevigata, Atta sexdens, and Acromyrmex coronatus, and two nearly identical Pseudomonas  isolates (99.3%) from Acromyrmex rugosus. The 85.8% identity was a relatively low value, so genus classification within Ralstonia may look dubious in this case. However, genus classification was confirmed by RDP with 95% confidence interval and, in addition, Ralstonia sequences retrieved from NCBI  showed 82 to 99.6% identity to each other, so strains currently classified within Ralstonia show a more diverse 16S rDNA sequence.Twenty‐one representative isolates were sequenced and classified in five different bacterial genera. Of these 21 isolates, 12 were found to fix nitrogen as they had the Acinetobacter  isolates (97%–99% identity) were found in every analyzed ant colony/species and, in addition, one Brachybacterium  was found in Atta laevigata. Methylobacterium, Ralstonia and Acinetobacter strains were prevalent, since they were found, respectively, in three, four, or five out of the five nests sampled  in sequences of the 16S rRNA libraries with maximal identity variation from 93% to 99.1% Table .3.5Based on the 16S rRNA sequence, strains in the major genera that we found in Brazil have similar or nearly identical relatives in the United States and Republic of Panama Table , being tLiberibacter were similar (96% identity), and representative sequences from dominant Bartonella and Terasakiella were nearly identical (~99% identity) to that of the RhiAcro1 symbiont (GenBank accession # KR336619), which was found in the guts of Acromyrmex ants from Gamboa, Panama  to EntAcro1 (GenBank accession # KR336618) from Central America or Mesoplasma lactucae OTU‐1544 (GenBank accession # KT248001) from North America  from Central America  and Simpson (9%) indices, as well as the average read number (15% decrease) in libraries , Ralstonia , and Pseudomonas  were detected in libraries of all ant species , which possibly domesticated these microbes as endosymbionts that recycle nitrogen , obtained from Atta sexdens, Atta laevigata, and Acromyrmex coronatus, with 16S rRNA sequence showing 91.5%–93.9% identity to other Ralstonia sequences detected in Atta sexdens libraries .Diazotrophic isolates within Pseudomonas isolates in Acromyrmex rugosus, and bacteria in this genus have been previously isolated from the cuticle of Acromyrmex coronatus . They are genetically similar with RhiAcro1, a dominant extracellular Rhizobiales strain forming an intestinal biofilm confined to the lumen of three Acromyrmex species, with potential to fix  is the fourth most frequent dominant in the core bacteriome, but we found no other Rhodovulum sequences associated with leafcutters in public databases. Acinetobacter isolates were found in the four analyzed ant species and detected in the 16S rRNA library of Atta laevigata and Acromyrmex coronatus. Isolates of this genus have already been reported in association with the cuticle of Acromyrmex coronatus  mediation of leafcutter nutrition on atmospheric nitrogen provided by Enterobacteriales in the genera Ralstonia , Methylobacterium , and Pseudomonas , so leafcutters seem to rely on multiple complementary and phylogenetically distinct nutritional partners to produce biomass on a large scale.Our current results add other partners, which likely contribute to leafcutter nutrition on nitrogen: culturable abdominal diazotrophs in the genera The authors declare that they have no competing interests.Renata de Oliveira Aquino Zani: Investigation ; methodology . Milene Ferro: Investigation ; methodology . Maurício Bacci Jr: Resources (supporting); supervision (supporting); writing‐review and editing (supporting).Table S1‐S4Click here for additional data file.


ESCO2 gene; however, prenatal diagnosis of RBS has never been reported in Chinese families. Additionally, fetal-specific phenotypic characteristics associated with ESCO2 variants have not been reported.Roberts syndrome (RBS) is a rare autosomal recessive disorder caused by variations in the ESCO2 gene . Both of the couples were heterozygous carriers for this variant.A fetus in a healthy, nonconsanguineous Chinese family with multiple serious congenital malformations was diagnosed prenatally. Two consecutive fetuses in this family presented with tetraphocomelia, growth restriction, cleft lip and palate bilaterally, and other abnormalities. The main phenotypic characteristics of this case were strongly suspected to be associated with RBS. Finally, whole exome sequence analysis revealed the insertion of a homozygous base pair in exon 6 of the We are the first to report a prenatal case of RBS diagnosed in a Chinese family. Here, we have confirmed that the rare variant is a definite pathogenic variant, and we provide detailed phenotypic characteristics for the prenatal diagnosis of RBS due to this causative variant. ESCO2) gene [Roberts syndrome  is a rare autosomal recessive disorder caused by variants in the establishment of the cohesion 1 homolog 2  and her husband were referred to our fetal medicine department for genetic counseling. On the 22nd week of her first pregnancy, the female fetus had multiple serious congenital anomalies, including limb malformation, bilateral cleft lip and palate, and severe growth restriction. They opted for termination of pregnancy, and no other survey was conducted for the aborted fetus. The healthy, nonconsanguineous couple of Chinese origin denied any other family history of congenital diseases and had no history of exposure to medication during pregnancy.During her current pregnancy, the woman did not have regular physical examinations. At 25 weeks of gestation, ultrasound examinations performed at the local hospital again showed multiple similar congenital malformations of the fetus. At the same time, a detailed ultrasonic examination conducted in our hospital showed a singleton male fetus with severe growth restriction, with a biparietal diameter (BPD) of 55.02 mm , occipito-frontal diameter (OFD) of 67.6 mm , head circumference (HC) of 195.4 mm , abdominal circumference (AC) of 155.23 mm  and estimated fetal weight (EFW) of 378 g   and 31.3 mm (− 4.5 SD), while the length of the right and left femurs was 33.3 mm (− 4. 4SD) and 33.9 mm (− 4.1 SD), respectively. The bilateral radius, ulna and fibula were apparently not presented , and both couples were heterozygous carriers for this variant. Sanger sequencing was performed and confirmed this variant  and trio-whole exome sequencing (Mingma Technologies Co. Ltd) to detect copy number variations and gene variants, respectively. No pathogenic or likely pathogenic copy number variants were identified in the fetus. A trio-whole exome sequencing analysis revealed a homozygous frameshift insertion of a homozygous base pair in exon 6 of the ESCO2 gene [ESCO2 gene located at 8p21.1, which encodes a protein with the function of acetyl-transferase in establishing sister chromatid cohesion during S phase and mitosis [ESCO2 gene defects lead to the loss of acetyltransferase activity, sister chromatid polymerization failure and centromeric separation, and they result in very slow or even interrupted cell division and abnormal gene expression patterns [RBS, also known as Roberts-SC phocomelia syndrome or ESCO2 spectrum disorder, is a rare autosomal recessive disorder caused by variants of the CO2 gene . PatientCO2 gene , 6. The  mitosis . In RBS patterns .For prenatal diagnosis of RBS, there must be careful differentiation between cases where the conditions are clinically similar. For example, limb anomalies and growth retardation can be observed in RBS, Cornelia de Lange syndrome, CHARGE syndrome, Tetra-melia syndrome 1 and thrombocytopenia-absent radius syndrome. Therefore, identification of the causal genetic factor plays a critical role in the clinical diagnosis or future preimplantation genetic testing of these diseases. In this case, a fetus in a healthy, nonconsanguineous Chinese family with multiple serious congenital malformations was diagnosed prenatally. Two consecutive fetuses in this family had tetraphocomelia, growth restriction, bilateral cleft lip and palate and other anomalies. Most of the clinical parameters observed are below the 3rd percentile. The key phenotypic features of our case are strongly suspected to be associated with RBS.ESCO2 variants listed in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) or in the literature [ESCO2 gene is not present in population databases (such as the ExAC and gnomAD databases). Although this variant has been reported three times in individuals with RBS, for which it has been classified as a pathogenic or likely pathogenic variant (https://www.ncbi.nlm.nih.gov/clinvar/variation/21232/), the fetal-specific phenotypic characteristics correlated with this variant have not been reported.Although approximately 150 cases of RBS have been reported, the exact prevalence of RBS is unknown. To date, there are approximately 40 pathogenic or likely pathogenic terature , 9, and We reviewed the relevant literature and databases, and unfortunately, the prevalence of the disease and this variant in the Chinese population is not known. Although the couple denied consanguinity, we cannot rule out the possibility of a common ancestry between them, given that both members of the couple are carriers of the same very rare variant.According to the gene variant interpretation guidelines  and the ESCO2 gene could cause a severe and classical phenotype of RBS.Although the c.1111dupA variant has already been described once previously , the cliESCO2 gene, the parents were informed that they have a 25% risk of having another affected child and were discussing options regarding a future pregnancy. In addition, fetal cells can be obtained by amniocentesis extraction of amniotic fluid, and cytogenetic analysis of fetal DNA, which is the gold standard for the diagnosis of RBS during pregnancy, can confirm the diagnosis [Genetic testing of carrier status for the parents was indicated to calculate the risk of having another child with RBS in a future pregnancy. As the genetic analysis revealed that both parents are carriers of the c.1111dupA variant of the In conclusion, we report, for the first time, a case of prenatally diagnosed RBS in a Chinese family. We have confirmed that the rare variant is a definite pathogenic variant and provided detailed phenotypic characteristics for the prenatal diagnosis of RBS due to this causative variant. Studies on the pathogenesis of such recurrent congenital malformations in whole exome sequencing provide the basis for genetic counseling to avoid the risk of recurrence and complications in future pregnancies.


The performance of spectral reflectance indices (SRIs) with different algorithm forms, partial least square regression (PLSR), and stepwise multiple linear regression (SMLR) in estimating the three units of Chl content was compared. Results show that most indices within each SRI form performed better with Chl area and Chl plant and performed poorly with Chl SPAD. The PLSR models, based on the four forms of SRIs individually or combined, still performed poorly in estimating Chl SPAD, while they exhibited a strong relationship with Chl plant followed by Chl area in both the calibration  and validation  datasets. The SMLR models extracted three to four indices from each SRI form as the most effective indices and explained 73–79%, 80–84%, and 39–43% of the total variability in Chl area, Chl plant, and Chl SPAD, respectively. The performance of the various predictive models of SMLR for predicting Chl content depended on salinity level, genotype, season, and the units of Chl content. In summary, this study indicates that the Chl content measured in the lab and expressed on content (μg cm−2) or concentration (mg plant−1) can be accurately estimated at canopy level using spectral reflectance data.Although plant chlorophyll (Chl) is one of the important elements in monitoring plant stress and reflects the photosynthetic capacity of plants, their measurement in the lab is generally time- and cost-inefficient and based on a small part of the leaf. This study examines the ability of canopy spectral reflectance data for the accurate estimation of the Chl content of two wheat genotypes grown under three salinity levels. The Chl content was quantified as content per area (Chl  The greatest challenge for the agriculture sector in arid and semiarid regions is the scarcity of freshwater, which is increasing year after year due to population rise and the negative impacts of global warming. The use of non-conventional water resources such as drainage water, recycled water, and saline groundwater has been suggested as an alternative source of extraordinary interest to ensure the sustainable production of food crops in these regions ,2,3. HowThe sub-components of salinity stress interact together and lead to an increase in the level of reactive oxygen species (ROS) in plant cells, which in turn leads to accelerating chlorophyll (Chl) degradation or reduces Chl synthesis ,14. PrevAlthough the assessment of leaf Chl content through the traditional laboratory methods is very accurate, on the other side it is destructive, expensive, time-consuming, laborious, and often inappropriate for tracking the dynamic changes in Chl content when the measurements are made for a large number of genotypes at a large scale. The Chl meter devices such as SPAD-502 can be used to estimate the leaf Chl content for a large number of genotypes in an expeditious and non-destructive manner. Several previous studies have reported that the SPAD numerical values showed strong relationships with the leaf Chl content under both normal and stress conditions ,16,17. HThe ground-based hyperspectral reflectance tool has enabled us to capture the canopy spectral reflectance in the full range of the spectrum 400–2500 nm), and therefore it can provide important information on several canopy biophysical and biochemical characteristics. For example, the spectral reflectance from the canopy in the range of the visible spectrum , mainly in the blue 420–470 nm) and red (640–680 nm) regions, depends on the concentrations of leaf pigments, especially Chl content. The spectral reflectance in both regions is low with a peak at the green region (520–580 nm) when the leaf Chl concentration is high and vice versa ,19,20,21–470 nm a00 nm, an2) and high leaf area index (LAI > 3) [721 − R744)/(R721 − R714) is in MND form, it was more effective than SD and SR in estimating Chl content over several plant species and at various growth stages. This is because this index incorporates three wavelengths from the RER, which are effective in eliminating the effects of leaf surface properties on the spectral reflectance [750/R700) or ND form (R750 − R705)/(R750 + R705) incorporated the most sensitive wavelength (700 nm) for Chl content, both indices showed a weak correlation with the Chl content when applied across a wide range of species. However, when this wavelength was combined with a third wavelength and incorporated in modified form (RNIR − R705)/(RNIR + R705 − 2R445), this new algorithm showed good performance in Chl estimation across a wide range of species [To employ the spectral reflectance data collected from the canopy for estimating the leaf Chl content, different spectral reflectance-based indices (SRIs) have been developed by incorporating two or more wavelengths in a simple mathematical formula. Several SRIs have recently received considerable attention and have been found to be effective in assessing the status of Chl content in plants. The wavelengths in the formula of SRIs are usually built in different forms such as normalized difference (ND), modified normalized difference (MND), simple difference (SD), simple ratio (SR), modified simple ratio (MSR), or integration of more forms ,34,35,36LAI > 3) ,23,26. WLAI > 3)  found thlectance ,37. Alth species ,36. Ther−1) or a unit of area (μg cm−2). As both units are often used interchangeably in the relevant literature, the choice of the best algorithms of SRI for remotely estimating Chl content based on both units of Chl appears to be important. Grossman et al. [Commonly, Chl content is expressed either as a unit of mass  using different published algorithm forms of SRIs separately or coupled with PLSR and SMLR models. The SMLR method was used to extract the most important indices within each form of SRIs that explain the most variability in each unit of measurement of Chl content. The capability of different SMLR models based on these important indices in predicting the different units of measurement of Chl content was examined under various conditions .The primary goal of this study was to assess the relationship between canopy spectral reflectance measurements and the different units of measurements of Chl content  during 2017/2018 and 2018/2019 growing seasons . Two spr−3, organic matter of 0.46%, electrical conductivity of 1.12 dS m−1, available N of 45.2 mg kg−1, available P2O5 of 2.44 mg kg−1, and available K2O of 186.9 mg kg−1 [At the research station and during the growing season of spring wheat (from December to April), the rainfall ranged from 8.0 to 25.0 mm, the mean temperature ranged from 12.9 °C to 32.2 °C, and the humidity ranged from 17.7% to 47.5%. Soil texture is sandy loam with a pH of 7.85, bulk density of 1.48 g cm mg kg−1 .−1), moderate salinity level (6.0 dS m−1), and high salinity level (12.0 dS m−1), and the two genotypes were assigned to the main plots, whereas the two genotypes were distributed randomly in subplots.The experiments were conducted in a split-plot design with three replications. The three salinity treatments, namely control (0.35 dS m−1. The two genotypes were fertilized with a nitrogen–potassium–phosphorus (NPK) fertilizer at a rate of 180 kg ha−1 of N, 60 kg ha−1 of P2O5, and 60 kg ha−1 of K2O. The entire amount of potassium  and phosphorus  and 50 kg ha−1 of nitrogen (as ammonium nitrate) were applied before sowing. The plants were fertilized again at tillering and booting growth stages with 50 kg ha−1 of nitrogen.The seeds of each genotype were planted on 5 December 2017, and 2018, in sex 6 m-long rows spaced 0.2 m apart at a seeding rate of 150 kg ha−1 solution, respectively, until the final irrigation. The irrigation water was applied using a low-pressure surface irrigation system. This system consisted of a main line (76 mm in diameter) that delivers water from plastic water tanks (5.0 m3) to each subplot. To control the amount of water delivered to each subplot, this main line was branched off to the sub-main hoses at each subplot and equipped with a manual control valve. The genotypes were irrigated with fresh water 10 times in the control treatment, while in the two salinity treatments, they were irrigated with fresh water 2 times and with saline water 8 times. The irrigation frequency and rate was adjusted according to plant growth stage and environmental conditions.The two genotypes were irrigated with non-saline water for the first two weeks after sowing in the three salinity treatments. Thereafter, the control treatment continued to be irrigated with non-saline water, while the treatments treated with moderate and high salinity levels were irrigated with artificial saline water containing 60 mM and 120 mM NaCl L2 of plant canopy. Before capture the spectral reflectance for the plants in each subplot, a white barium sulphate panel  was used to calibrate the device in order to avoid any changes in atmospheric conditions and sun irradiance on the spectral measurements [Canopy spectral reflectance was captured at the middle anthesis growth stage  , from twurements . The finData of spectral reflectance were used to derive 83 published two- and three-band SRIs, using the wavelengths  in the 400–850 nm regions . These SSPAD) were obtained from 10 fully expanded leaves, randomly selected from the spectral collection area using a chlorophyll meter . The SPAD reading for each leaf was an average of three SPAD-502 readings, taken from three places from leaf base to apex. Additionally, to ensure consistency between the SPAD readings and leaf Chl content, quantitatively determined by a spectrophotometer in the laboratory, leaf discs were collected immediately after the SPAD-502 readings from an approximate position on the leaf sample at which the SPAD measurements were obtained. Ten leaf discs (~0.28 cm2 each) were taken by hole puncher and collected in a micro-centrifuge Eppendorf tube, immediately wrapped in aluminium foil and stored in ice. The samples were transported to the laboratory within 45 min and ground in the dark with 95% (v/v) ethanol using a clear mortar. Subsequently, the leaf pigment mixture was transferred to a 50 mL volumetric flask with 80% ethanol and one part of the homogenous solution was placed in a plastic tube and centrifuged for 20 min at 5000× g. The supernatant was transferred from a plastic tube to a cuvette to immediately measure its absorbance using a UV-VIS spectrophotometer  at 645, 663, and 470 nm wavelengths. The total leaf Chl content was calculated using the formula provided by Lichtenthaler and Wellburn [area μg cm−2) using the following equations:After spectral measurements, the leaf SPAD readings  was calculated from the total leaf area per plant (LA plant) and the Chl content per leaf area (Chl LA) using the following equation [The total Chl content per plant  during two growing seasons, the analysis of variance (ANOVA), appropriate for a randomized complete block split-plot design, was used. To compare the differences between the mean values of the three units of Chl content between salinity levels and genotypes, Duncan’s test at a p ≤ 0.01 and 0.05 significance level was applied. Pearson’s correlation coefficient (r) was calculated under each salinity level (n = 24), for each genotype (n = 36), and across all salinity levels and genotypes (n = 72) in order to evaluate the relationship between the three units of Chl contents. The relationship of 83 SRIs with each unit of Chl content across all conditions  was evaluated by simple linear regression.To test the impacts of salinity levels, cultivars, and their interaction on different units of Chl content (Chl 2). Random 10-fold cross-validation was employed to the datasets from the two seasons to increase the robustness of the results. In this study, PLSR modelling was performed using Unscrambler 10.2 software .Because the PLSR algorithm has an inferential capability, it was used in this study to model a possible relationship between different forms of SRIs (predictor variables) and each unit of Chl content (response variable). This algorithm is an effective method that specifies a relationship between a set of predictor variables and response variables. In this algorithm, the optimum number of latent variables (ONLVs) entering a final model needs to be carefully identified to represent the calibration data without excessive overfitting or under-fitting. The ONLVs were determined by employing a leave-one-out cross-validation (LOOCV) scheme. The best ONLV is that yielding the smallest root mean square error (RMSE) and the largest coefficient of determination (Rn = 72) were applied to SMLR as independent and dependent variables, respectively. To predict three units of Chl content under specific conditions , the different models of the best indices of each form of SRIs were used. The model with the lowest values of RMSE and the highest values of R2 of the linear relationship between the observed and predicted values of each unit of Chl content was designated the model with the higher prediction accuracy. The SMLR was performed using Sigma Plot for Windows .To determine the most influential indices within each form of SRIs accounting for the highest variability in each unit of Chl content, the indices of each form of SRIs and three units of Chl content of pooled data (−1) did not differ in Chl SPAD, while the other two units of Chl content (Chl area and Chl plant) significantly decreased with increasing salinity levels. Averaged over the two seasons, decreases in both Chl units for 6 dS m−1 and 12 dS m−1 treatments relative to control treatment were 16.4% and 39.5% for Chl area, and 28.9% and 57.5% for Chl plant, while it was only 2.4% and 18.5% for Chl SPAD, respectively , each genotype (n = 36), and when data for the salinity levels, cultivars, and seasons were pooled together (n = 72) , while the Chl SPAD had a moderate correlation with Chl area (r = 0.52) and Chl plant (r = 0.62)  . All uni = 0.62) .plant and Chl area better than Chl SPAD. Nearly all indices of each form of SRIs exhibited a moderate to strong relationship with Chl plant and Chl area and a weak to moderate relationship with Chl SPAD  and 10-fold cross-validation  datasets of the PLSR models for estimating the different units of Chl content using the pooled data (n = 72). The PLSR models were determined based on the indices of each form of SRIs, as well as all indices of the four SRIs forms together. In general, the different PLSR models also provided a more accurate estimation for Chl plant and Chl area than for Chl SPAD in both the Cal. and Val. datasets. In addition, all PLSR models generated a strong estimation performance in both Cal. and Val. datasets for Chl plant (R2 ranged from 0.77 to 0.86), while they exhibited a strong estimation performance in Cal. datasets (R2 ranged from 0.65 to 0.82) and a moderate to strong estimation performance in Val. datasets (R2 ranged from 0.63 to 0.77) for Chl area. The different PLSR models showed a weak to moderate estimation performance in both Cal. and Val. datasets for Chl SPAD (R2 ranged from 0.29 to 0.58)  . The PLSChl SPAD .n = 72) in order to extract the most influential indices within each form of SRIs that contributed the major variation for each unit of Chl contents. In general, the different SMLR models also provided a more accurate estimation for Chl area (R2 ranged from 0.73 to 0.79) and Chl plant (R2 ranged from 0.80 to 0.84) than for Chl SPAD (R2 ranged from 0.39 to 0.43)  . The RGIectively . Among tChl SPAD .SPAD under moderate and high salinity levels, except the indices of ND and MND forms, which exhibited a weak relationship with Chl SPAD under high salinity levels (plant (R2 ranged from 0.56 to 0.82) and the Chl area (R2 ranged from 0.54 to 0.74) under moderate and high salinity levels  and Chl plant (R2 ranged from 0.80 to 0.84) than they did for Chl SPAD (R2 ranged from 0.33 to 0.55)  .area, Chl plant, and Chl SPAD) to salt stress depended on salinity levels and the degree of salt tolerance of the two genotypes , Chl content per leaf dry mass (g g−1), or Chl content per whole plant (g plant−1) are the most common units to quantify the amount of Chl in the leaf and whole plant [plant (g plant−1) was more correlated to Chl area (μg cm−2) than to Chl SPAD under each salinity level, for each genotype, or when the data of salinity levels and genotypes were pooled together (−1), Chl SPAD had non-significant correlation with Chl area but it was significantly correlated with Chl plant (plant (g plant−1) was a more suitable unit to express plant Chl content than the other two units is probably because Chl plant was obtained by using total leaf area per plant multiplied by Chl content per leaf area. Therefore, the Chl plant takes into account the vertical heterogeneity of Chl content within wheat canopies.However, to understand the effect of salinity stress on total Chl content in plants, it is important to take into account the units that are used to express the content. Chl content per leaf area , which is in MSR form, performed better than the MND form (R800 − R750)/(R750 − R670) in estimating the carotenoids content expressed in g m−2 (r = 0.71), while the latter form showed the best correlation with carotenoids content expressed in μg cm−2 (r = 0.71) and mg g−1 (r = 0.67). Yi et al. [−2 and g m−2 as compared with mg g−1. Kattenborn et al. [−2) rather than as leaf mass-based concentration (g g−1). The results of this study found that nearly all indices of the four forms of SRIs estimated Chl content expressed as mg plant−1 (Chl plant) and μg cm−2 (Chl area) better than expressed as SPAD value (Chl SPAD). These indices exhibited a moderate to strong relationship with Chl plant and Chl area and a weak relationship with Chl SPAD . As previously stated, because there are several physical and chemical canopy characteristics that affect the efficiency of canopy spectral reflectance for accurately estimating the plant traits of interest, the most important factors for determining the optimal SRIs are the formula and the sensitive wavelengths incorporated in this formula. The formula and wavelengths should be applied to a suitable algorithm to remove the effects of physical and chemical characteristics on canopy reflectance, as well as to increase the sensitivity of the SRIs to the plant traits of interest ,28,37,67i et al.  also repn et al.  also conChl SPAD . These fi et al.  reportedplant and Chl area than of Chl SPAD. Additionally, the PLSR models coupled with ND and MND forms or four forms together had the best performance in the estimation of the three units of measurements of Chl content in both the calibration and validation datasets , whereas they exhibited moderate to strong relationships with Chl plant (R2 = 0.56–0.82) and Chl area (R2 = 0.54–0.71) under moderate and high salinity levels  and Chl area (R2 = 0.67–0.84) for both genotypes, while they provided a more accurate estimation of Chl plant than they did for Chl area for Sakha 61 and vice versa for Sakha 93 (SPAD for Sakha 61 (R2 = 0.36–0.48) than for Sakha 93 (R2 = 0.29–0.41)  . This rearea (μg cm−2) and Chl SPAD ) and whole plant level (Chl plant (mg plant−1)). The results allowed the following conclusions: 1-plant and Chl area were highly greater than those for Chl SPAD.The different algorithm forms of SRIs showed the same pattern for their relationship with the three units of Chl contents, but the coefficients of determinations for Chl 2-plant and Chlt area.Nearly all MND forms were found slightly more efficient than other SRIs forms for estimating Chl 3-The PLSR models coupled with ND and MND forms, or four forms together, had the best performance in the estimation of the three units of measurements of Chl content, both in the calibration and validation datasets.4-area and Chl plant, and only 39–43% in Chl SPAD.The indices that were extracted from each form of SRIs by SMLR explained 73–84% of the variability in Chl 5-The ability of different models of SMLR for predicting the three Chl measurements depended on salinity levels, genotypes, and seasons.6-area), can be accurately estimated in a rapid and non-destructive manner using canopy spectral reflectance data, when the Chl content is also expressed in the whole plant (Chl plant).Finally, our results indicate that the Chl content, measured on a laboratory basis at leaf level  separately or combined with PLSR and SMLR models for estimating Chl contents that were expressed in different units at leaf level (Chl