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Resilience of cassava (Manihot esculenta Crantz) to salinity: implications for food security in low-lying regions

Highlights Cassava tolerates low but not high levels of salinity via ion exclusion and higher foliar cyanide. Mechanisms for resistance are related to the decreasing value of leaves as plants mature.

Introduction
Rising sea levels and salinization of groundwater is becoming an increasing problem in many parts of the world, reducing the area of land available for agricultural crops (Church et al., 2013;Nunn, 2013). Globally, one-fifth of irrigated land is salt affected (450 000 km 2 ), resulting in huge losses in productivity (Munns and Gillman, 2015). The ablity to tolerate salt varies within and between species. Barley (Hordeum vulgare), for example, is one of the more salt-tolerant crops, while lupins (Lupinus albus) is one of the most sensitive (Munns, 2002). Little is known about the impact of salinity on some of the world's major staples.
Cassava (Manihot esculenta Crantz) is the most widely grown root crop in the world and the tuberous roots are the principle source of calories for many of the world's poorest people (Nweke et al., 2002;FAO, 2014). Cassava has the ability to cope with a wide range of environmental stresses and continues to produce tubers under poor growing conditions, such as low nutrients and drought (El-Sharkawy, 1993;Jørgensen et al., 2005;Burns et al., 2010), yet little is known about how it responds to salt stress. Its classification as 'moderately sensitive' by the FAO is based on three early studies (Anon, 1976;Hawker and Smith, 1982;Indira, 1978). More recently, Carretero et al. (2008), in a study of pre-tuberous plants, found large effects on growth at 68 mM sodium chloride (NaCl) and only 30% survival at the highest concentration (136.8 mM NaCl).
Even less is known about the impact of salinity on the nutritional value of cassava. Unlike all other staple foods, cassava can be lethal if not correctly processed due to the high levels of the cyanogenic glucosides, linamarin and lotaustralin, that break down to release cyanide when mixed with specific β-glucosidases (McMahon et al., 1995;McKey et al., 2010). Consumption of cassava that is high in cyanide may cause diseases in humans, such as Konzo (an irreversible paralysis of the lower limbs), tropical ataxia, and even death (Cliff et al., 1997;Burns et al., 2010;Bradbury et al., 2011). There is a strong link between Konzo epidemics and the increase in cyanide concentrations during droughts (Ernesto et al., 2002), but there are no published studies on whether salinity-induced physiological droughts also result in higher levels of cyanide, and possible threats to health and food security. Ballhorn and Elias (2014), in the only published study on the effect of salinity on the production of cyanogenic glucosides, found for Trifolium repens that the cyanogenic glucoside concentration did increase in proportion to increasing salinity. Salinity is also known to affect the micronutrient concentration in plants. Given that children consuming cassava as a staple food are at risk for micronutrient deficiencies, particularly zinc, iron, and vitamin A (Gegios et al., 2010), it is important to assess the possible impact of salinity on their availability in cassava food products.
Cyanogenic glucosides are an effective defence again herbivores , but they represent a significant drain on resources that might otherwise be used for growth (e.g. Simon et al., 2010). This is balanced by the alternative roles they may play in primary metabolism, such as the transport and storage of nitrogen and mitigation of oxidative stress Kongsawadworakul et al., 2009;Selmar and Kleinwächeter, 2013;Gleadow and Møller, 2014). Thus the partitioning of resources is a balance between defence, particuarly when growth is limited, and, for example, stress tolerance and nitrogen turnover.
The aim of this study was, therefore, to determine the effect that salinity had on biomass and nutritional composition at two different life stages of cassava. We tested the tolerance of established plants, with well-developed tubers, to a wide range of NaCl solutions. The second experiment involved a detailed study on young clonally propagated plantlets through to tuber initiation. Photosynthetic parameters, growth indices, mineral nutrient composition, and cyanogenic glucoside concentration were determined and used to estimate the impact of salinity on plant production and nutritional value.

Materials and methods
Plant material and growing conditions Cassava (Manihot esculenta Cranz) cv. MAus7 was grown in a greenhouse at the School of Biological Sciences complex Monash University, Clayton. Average temperature was 25 °C/23.5 °C day/ night with a relative humidity of 75%. Glasshouses received natural light. The photoperiod was extended to 14 h from May to September 2014 using sodium lamps (MK-1 Just-a-shade, Ablite Australia, Sunnfield Enterprises, Allambie Heights, NSW, Australia). Plants were rotated weekly to reduce microenvironment effects.

Experiment 1: effect of a range of salt concentrations on established cassava plants
The effect of salt on cassava plants with established tuberous roots ('tubers', hereafter) was tested. Cassava (one plant per pot) was grown in 8 litre pots containing commercial potting mix with slowrelease fertilizer (Osmocote ® ) for 8 months (June 2013-January 2014) and then treated with four different concentrations of NaCl for 4 weeks. Eight-month-old cassava plants were matched according to height and leaf number to form five sets with seven plants in individual pots in each group. One group was harvested for initial biomass determination (n=7 plants). The other groups were assigned to one of four different salt treatments (0, 50, 100, or 150 mM NaCl) (Hawker and Smith, 1982;Carretero et al., 2008) in a randomized matched-pair design. Plants were watered with 1.4 litres of solution two times per week, such that the solution ran through the pots. Pots were flushed weekly with 2 litres of water to prevent the build-up of salt in the soil. All plants were destructively harvested after 28 d for biomass determination and chemical analysis.

Experiment 2: long-term effects of salinity on growth and tuber initiation
In the second experiment, longer term effects of salt on plants prior to tuber initiation were tested. In this experiment, young plants were established from cuttings (January 2014) and transplanted after 2 months (~15 cm tall, three leaves) into 2 litre pots containing potting mix (as above) and mixed 5:1 (v/v) with soil containing mycorrhizal colonies, from the Jock Marshall Reserve, Monash University following Vandegeer et al. (2013). During this time, they were watered twice a week, once with a commercial full nutrient solution (Aquasol ® , 10 ml l −1 H 2 O), and once with plain water. Before treatments were imposed, a set of 10 plants were harvested for dry mass determination. Plants were then watered with three different concentrations of salt [0, 40, and 80 mM NaCl (n=10) based on the results of Experiment 1]. In order to avoid a shock response, the salt concentration was increased gradually, starting at 20 mM and increasing by 20 mM every 3 d until the levels of 40 mM and 80 mM were reached. These levels were then maintained for the remainder of the experiment, a total of 70 d. The plants were watered with 300 ml of their appropriate solution twice a week. This volume was enough to saturate the soil. Pots were flushed weekly with 500 ml of water to prevent accumulation of salt in the soil. To ensure plants did not become nutrient limited, they were watered with Aquasol ® on days 49, 52, and 56 of the study at a rate of 10 ml l −1 .
The stem height and leaf number of each plant were recorded each week. Stem height was measured from the soil surface to the point where the newest leaf was expanding. The number of lobes of the third fully expanded leaf and the length of the middle lobe of that leaf was also recorded weekly. The lobe length was the distance from the point where the leaf blade was attached to the petiole to the tip of the lobe. All plants (n=30) were destructively harvested after 70 d (see below).

Harvesting protocol
The biomass of plants was separated into above-ground (stem, petioles, leaves) and below-ground (cutting, roots, tubers). The above-ground biomass of plants was removed by cutting the stem at the soil surface. Leaves were removed from the plant and separated into three classes: fully expanded, unexpanded, or senescent. Expanded leaves were defined as fully developed leaves, unexpanded leaves were young, soft leaves, and senescent leaves were defined as leaves that were yellow or brown in colour on >50% of the leaf surface. Leaf fresh weight was recorded for all classes and leaf area was taken for expanded and unexpanded leaves using a leaf area meter (LI-3000 Portable Area Meter, Li-Cor, Lincoln, NE, USA). Three leaf discs (diameter=5 mm) were taken from the third fully expanded leaf and one disc from tubers and frozen at -20 °C for later analysis for cyanogenic glucosides. Height, and stem and petiole weight were recorded. Roots and tubers were washed and all soil removed where possible. For Experiment 1, tubers and roots were weighed separately, oven-dried at 60 °C for 14 d, and dry weights determined. For Experiment 2, roots were separated into fine roots and thick roots (tuberous roots). Thick roots were defined as roots that were white in appearance and >2 mm in diameter. All leaf and root tissue was frozen in liquid nitrogen and freeze-dried for 5 d, and weighed. The remaining biomass (stems and petioles) was oven-dried at 60 °C for 14 d and dry weights determined.

Growth indices
The root:shoot ratio was calculated by dividing the total belowground biomass (DW) by the total above-ground biomass (DW). The relative growth rate (RGR) was calculated as follows: RGR=log(W 2 )log(W 1 )/(t 2 -t 1 ), where W 1 and W 2 are the initial and final dry mass at time 1 (t 1 ) and time 2 (t 2 ). The specific leaf area (SLA) of fully expanded leaves was calculated as leaf area per g dry leaf mass.

Photosynthetic parameters: assimilation rates, F v '/F m ', greenness, and chlorophyll
Light-saturated measurements of the photosynthetic rate and lightadapted chlorophyll fluorescence, F v '/F m ', were made using a Li-Cor 6400 gas exchange system on all living plants in Experiment 2 in the week before harvesting (20 August 2014). Three concordant measurements were made on the third fully expanded leaf from each living plant at 700 µmol m −2 s −2 PAR; 400 ppm CO 2 , 25 °C (growth temperature), and a relative humidity of 50%, following Gleadow et al. (2009).
Total chlorophyll concentration was measured following Gleadow et al. (1983) as modified by Burns et al. (2002). Freezedried leaf tissue (100 mg) was extracted twice in 2 ml of 80% acetone and the absorbance measured at 647, 665, and 750 nm using a FLUOstar Galaxy plate reader (BMG, Australia). Greenness measurement readings were made of the third fully expanded leaf using the GreenIndex+ Ap (Spectrum Technologies Inc. ® ) by taking a photograph of the middle lobe against a background of known colour and using in-built algorithms. To our knowledge, this is the first time GreenIndex+ measurements have been made on cassava.

Plant composition: cyanogenic glucosides, total nitrogen, and micro-and macronutrients
The cyanogenic glucoside concentration was measured by the evolved cyanide method. Freeze-dried tissue (three leaf discs diam-eter=1 cm 2; or one tuber disc, 2 mm thick) was placed into sealed vials with 270 µl of 0.1 M pH 6.4 phosphate buffer, and a separate 0.2 ml inner tube containing 200 µl of 1 M NaOH. Vials were then frozen and thawed at room temperature twice to disrupt the cells, and incubated for ~19 h at 37 °C. The cyanide evolved from the cyanogenic glucosides trapped in the NaOH was assayed colorimetrically following Blomstedt et al. (2012). To ensure complete conversion of the cyanogenic glucosides to cyanide, latex [30 µl, in 0.1 M phosphate buffer pH 6.4, 1:100 (v/v)] was added to the reaction vial, as latex contains the degradative β-glucosidases and α-hydoxynitrile lyases necessary to degrade both linamarin and lotaustralin (Jørgensen et al., 2005). Tissue in vials was rinsed, dried, and weighed for biomass determination to allow the calculation of the hydrogen cyanide potential (HCNp) of the tissue analysed.
Total elemental nitrogen and carbon were measured on finely ground, freeze-dried leaf and tuber samples using a LECO CNS2000 analyser. The remaining nutrient analyses were performed using inductively coupled plasma (ICP)-MS on microwave-digested samples (Environmental Analysis Laboratory, Southern Cross University, NSW, Australia).

Statistical analysis
Data were analysed with Prism Graphpad 6 ® using ANOVA and t-tests. Matched-pair ANOVAs were used for analysis of Experiment 1 as plants were grouped according to size. Means were compared using post-hoc Tukey's tests (P<0.05) where a statistical significance was detected between groups. Data were log transformed if required in order to satisfy the assumptions of normality.

Experiment 1: effect of a range of salt concentrations on established cassava plants
Plants were grown at four concentrations of salt for 28 d. Above-ground biomass decreased with increasing salinity and was significantly lower in plants grown at 100 mM and 150 mM NaCl (Fig. 1A) compared with controls. Mean above-ground dry weight in control plants was 169 ± 14.5 g, but was only 67.9 ± 8.9 g in plants grown at 150 mM NaCl (F 3,24 =20.35, P<0.0001; Fig. 1A). The root:shoot ratio was higher as a consequence of increased salinity, driven by the difference in above-ground biomass, as there was no significant difference in tuber biomass between treatments (F 6,18 =3.7, P=0.85; Fig. 1B, C). Leaf area of plants decreased as the salinity level increased, primarily through plants shedding leaves, with significant differences detected between control and 100 mM, control and 150 mM, and 50 mM and 150 mM NaCl treatment groups (F 3,24 =16.64, P=0.021, 0.003, and 0.005, respectively; Fig. 1D).
The HCNp in the leaves decreased with increasing salt concentration (Fig. 1E). In the tubers, HCNp initially increased and was significantly higher at 50 mM and 100 mM NaCl (F 3,22 =6.46, P=0.006 and 0.015, respectively). At the highest salt treatment, the HCNp decreased in the tubers, but there was no significant difference compared with any other treatment.

Experiment 2: long-term effects of salinity on young cassava plants and tuber initiation Plant growth, biomass, and phenology
The number of leaves present on each plant was recorded once a week for the duration of the study. After 5 weeks, plants in the 80 mM NaCl group began to lose leaves at a steady rate, whereas plants in the control and 40 mM NaCl groups had steady increases in their number of leaves over the course of the experiment ( Fig. 2A). Six of the 10 plants grown at 80 mM NaCl died (i.e. all leaves abscised and there was no subsequent sign of recovery), four at day 56 and the other two at day 60 of the study. Surviving plants were shorter ( Fig. 2B), had fewer fully expanded leaves, and had more senescent leaves than plants grown at 40 mM NaCl ( Fig. 2; Supplementary Table S1 at JXB online). There was a significant decrease in leaf area of plants grown at both 40 mM and 80 mM NaCl (Fig. 2C). Total biomass was highest in plants grown without salt, with an average mass of 7.68 ± 0.42 g compared with the plants grown at 40 mM (5.73 ± 0.32 g) and 80 mM NaCl 2.09 ± 0.43 g, respectively (F 2.27 =52.24, P<0.0001; Fig. 2D). Control plants had greater above-ground biomass than plants from either of the salt treatments (F 2.27 =72.40, P<0.0001, Fig. 2D), but the below-ground biomass was not significantly different between the control and 40 mM NaCl groups. Thus, the higher root:shoot ratio in the 80 mM plants of 2.98 ± 0.64 compared with 1.33 ± 0.08 in the 40 mM-grown plants (Supplementary Table S1; F 2,27 =7.58, P=0.0038) was driven primarily by the lower above-ground biomass, as the fine root mass was small and only one plant produced a tuber ( Fig. 2D; Supplementary Table S1). There was no significant difference in the RGR between plants grown at 0 mM and 40 mM NaCl, but there was a significant decrease in plants at 80 mM salt (Fig. 2E).
Total chlorophyll concentration also decreased with increasing salt concentration. Plants grown under 40 mM NaCl had a lower chlorophyll concentration than control plants but the difference was not significant (F 2,23 =411.10, P=0.0036; Fig. 3C) and the concentration of chlorophyll in leaves from 80 mM-grown plants was significantly lower than  Table S1).

Cyanogenic glucoside and nutrient analysis
Cyanide assays were performed on freeze-dried leaf and tuber tissue. HCNp was highest in the leaves of plants grown at 40 mM, with lower levels in control plants and those grown at 80 mM NaCl (F 2,26 =9.32, P=0.0149 and 0.0008, respectively, Fig. 4A). There was no difference between control and 40 mM groups in the amount of HCNp found in tubers (Fig. 4A). Statistical tests on HCNp were unable to be performed for tubers grown at 80 mM NaCl as only one plant grown at this salt level contained tuberous roots. Overall, there was no significant difference in the HCNp:total biomass ratio between treatments (F 2, 27 =0.43, P=0.6536), indicating that observed changes in HCNp were not driven by changes in biomass.
Total nitrogen concentration was higher in the leaves of control plants compared with 40 mM plants (F 2,22 =8.14, P=0.0019; Table 1; Fig. 4B), although no difference between control and 80 mM plants or 40 mM and 80 mM plants was found. Carbon was lower in leaves of 80 mM-grown plants so there was no overall difference in the C:N ratio between control and 80 mM-grown plants. However, the leaves of plants grown at 40 mM NaCl had a higher C:N ratio compared with control and 80 mM plants (F 2,22 =14.86, P=0.0014 and 0.0002, respectively; Table 1).
Analysis of leaves (all treatments) and tubers (0 and 40 mM NaCl treatment only due to lack of tuber initiation at 80 mM NaCl) showed that there were significant differences in concentrations of key nutrients and trace elements between treatments (Table 1; Fig. 4C, D). The most striking difference is seen in the concentration of sodium. Plants at 80 mM NaCl had higher amounts of sodium in their leaves (1.80%) compared with control (0.01%) and 40 mM (0.02%) plants (F 2,22 =50.58, P<0.0001 and 0.0073, respectively; Table 1). The sodium concentration in the tubers in plants grown at 40 mM NaCl was nearly 20 times higher than in control plants (P=0.0022). The amount of potassium was also Downloaded from https://academic.oup.com/jxb/article-abstract/67/18/5403/2485531 by Monash University user on 24 July 2020 significantly higher in the tubers of plants grown at 40 mM salt, but the magnitude of the increase was much less, with a 1.4-fold increase (Table 1). Iron and zinc concentrations were significantly higher in leaves of plants grown at 80 mM NaCl (Fig. 4C, D). Concentrations of these two nutrients were somewhat lower in the tubers of salt-stressed plants, but the differences were not significant. Of the remaining macronutrients, the concentration of manganese in leaves also increased with rises in salinity levels and ranged from 43 mg kg −1 in control plants to 107 mg kg −1 in 80 mM plants (F 2,22 =47.13, P<0.0001, <0.0001, and 0.0146, respectively; Table 1). The amount of magnesium increased with increasing NaCl concentration in leaf tissue, and was significantly different between all treatments. Leaf phosphorus was marginally higher in the leaf tissue of plants grown at 80 mM NaCl and also 10-fold higher in the tubers of 40 mM plants (Table 1). Leaf sulphur levels in 40 mM plants were lower than in control and 80 mM-grown plants. The levels of trace elements in addition to iron and zinc, such as copper and cobalt, increased in leaf tissue with increasing salt (Table 1). In general, salt stress results in an increase in the nutrient levels in leaves and a decrease in the tubers (Table 1).

Discussion
The impact of salinity on growth and nutritional value (i.e. the cyanogenic glucoside and micronutrient concentrations) depended on the age of the plant. Older plants that had already developed tubers were more salt tolerant than younger, pre-tuberous plants in terms of survival and growth.
The key effect of salinity on cassava was a reduction in biomass, leaf area, and photosynthetic rate. There was an increase in HCNp in the leaves of young cassava plants under moderate stress (Fig. 4), but in the leaves of mature cassava plants, HCNp decreased step-wise with increases in salinity (Fig. 1). The age-affected differences may be related to: (i) the propensity for cassava to shed leaves in response to

Growth and photosynthesis
We found that tuberous plants were able to tolerate fairly high concentrations of salt, up to 150 mM NaCl (Fig. 1).
In contrast, growth and survival of pre-tuberous plants was severely retarded at 80 mM NaCl, with only one plant developing a tuber (Fig. 2). In both experiments, there was a decrease in mass of ~1% for every 1 mM NaCl increase, relative to the initial biomass (y=-1.0197x+456.6; R 2 =0.20; Experiment 1; Supplementary Fig. S1) and (y=-0.0699x+7.966; R 2 =0.77; Experiment 2; Supplementary Fig. S2). These results are broadly consistent with earlier studies that report severe  stunting and death of cassava plants between 50 mM and 135 mM, depending on variety, length of treatment, and soil environment, with older plants and those with mycorrhizae generally being more tolerant (Indira, 1978;Hawker and Smith, 1982;Carretero et al., 2008). The large reduction in biomass in both pre-and post-tuberous plants can be attributed to the loss of photosynthetic area and reduced rates of carbon assimilation. Weekly phenological measurements allowed us to track individual plants over the entire treatment period. In young plants, there was no significant reduction in leaf number until nearly 8 weeks in plants grown at 40 mM NaCl and even in the 80 mM treatment leaves were retained until 4 weeks after treatments commenced. This is consistent with the characteristic shedding of older leaves when plants are under stress, for example when droughted (Setter and Fregene, 2007;Vandegeer et al., 2013). Final harvest data showed that leaf area was even more sensitive to salt than leaf number, with a significant reduction in pre-and post-tuberous plants at 40 mM and 100 mM NaCl, respectively, (Figs 2 and 1, respectively).
The concentration of chlorophyll, greenness, and F v '/F m ' (measured on the third fully expanded leaf) were not significantly lower in plants grown at 40 mM, indicating that these leaves were not under severe stress. Plants from the 80 mM treatment, on the other hand, did show signs of stress with a lower F v '/F m ', and decreased chlorophyll concentration. Vandegeer et al. (2013) showed that the health of these highly productive, expanded leaves is maintained well after drought has affected other parts of the plant metabolism, and leaves are only discarded after prolonged drought. The significant leaf loss after 4 weeks growth and the reduction in photosynthetic capacity suggest that at 80 mM salt cassava is severely stressed.

Nutrient analysis and evidence for ionic exclusion at low salinity
Evidence that cassava is able to tolerate low to moderate concentrations of salt comes from the ionic composition of the tissues. Foliar sodium concentration was the same at 40 mM NaCl as in plants grown under control conditions, indicating that survival is from ionic exclusion, rather than tissue tolerance. This ability to exclude sodium breaks down at the higher concentrations, with a 100-fold increase in foliar sodium in plants grown at 80 mM. This type of response is typical of plants that are sensitive to salt. Salt-tolerant species, in contrast, are able to tolerate quite high concentrations of tissue salt (Munns and Gillman, 2015). Some plants cope with excess available Na by accumulating K as a balancing cation and this may influence K + uptake (Mattius, 2014;Munns and Gillman, 2015). We found no evidence for a change in ionic balance in salt-stressed cassava in the leaves. However, in the tubers, there was a significant increase in both Na and K with salt stress, though the relative increase was much greater for Na. This agrees with the results of Carretero et al. (2008) for roots/tubers but not leaves where it was unchanged (Carretero et al., 2008), and there are also reports of potassium being lower (Hawker and Smith, 1982) than controls due to salt stress. It is not clear why there is such a difference in the effect of salinity on potassium concentration reported in these experiments, but it may be the result of plant age or that particular leaves were tested, rather than pooling material, as we did here.
While tubers are an excellent source of carbohydrates, they are very poor in nutrients (Burns et al., 2010). On the other hand, leaves are an important source of protein and micronutrients for many subsistence farmers in Africa (Latif and Müller, 2015). The concentration of foliar and tuber micro-and macronutrients measured here were in the same range of concentrations as plants grown in the field in Africa (Burns et al., 2012). Any change in foliar nutrients, therefore, could have a major impact on human health. In this study, there was a significant reduction in above-ground biomass in both young and old plants, and nutrient analysis of the young plants shows a significant change in important nutrients, such as iron and zinc. The concentration of these increase in leaf tissue but in terms of total nutrients available for food there is a major reduction due to salt stress. In tubers grown under moderate salt stress, there is a reduction in the concentration of macro-and micronutrients, indicating that cassava under even moderate salt stress is not a nutritious food source. There was also a small but significant decrease in foliar nitrogen concentration.

Impact of salinity on cyanogenic glucosides and nutritional status
Changes in the concentration of cyanogenic glucosides in our study were very different in both magnitude and direction in old and young plants. In established plants, foliar cyanogenic glucoside concentration decreased with each increment of salt from 0 to 150 mM. In contrast, in pre-tuberous plants, the cyanogenic glucoside concentration was somewhat higher in the leaves of plants grown at 40 mM than in those of the control plants, but much lower in plants grown at 80 mM NaCl (Fig. 4A). One explanation for this difference is to consider the relative cost of the investment in defence and leaf infrastructure in the two age groups. The primary mechanism for coping with salinity stress in older plants is to discard leaves and rely on the tubers for storage, thus any investment in foliar defence is unnecessary. The significant increase in cyanogenic glucosides in the tubers of these plants is consistent with this hypothesis and also the fact that in cassava cyanogenic glucosides are synthesized in the leaves and transported to the tubers (Jorgensen et al., 2005). On the other hand, in younger plants, the leaves represent a relatively large proportion of total plant biomass. Moreover, as there are no tubers, there is no sink for the storage of cyanogenic glucosides and little prospect of recovery should the shoots die. In younger plants, therefore, it is to the plant's advantage to protect and maintain the leaves that it has already produced. Further detailed analysis of plants over time based on the hypotheses illustrated in the schematic in Fig. 5 are required.
The higher concentration of cyanogenic glucosides in the young plants from the 40 mM treatment and the significant decrease in HCNp in severely salt stressed (80 mM NaCl) plants may also be directly linked to the plant's ability to tolerate stress. Salt stress promotes the production of reactive oxygen species (ROS), such as H 2 O 2 and superoxide anions, which are toxic (Mattius, 2014;Roy et al., 2014). One way to deal with stress is to have a ROS-scavenging system, for example via the up-regulation of antioxidant enzymes such as superoxide dismutase. Such enzymes are induced within minutes of salt being applied-mainly in the form of H 2 O 2 (Cabello et al., 2012;Mattius, 2014). It has been proposed that the synthesis of cyanogenic glucosides may also act to mitigate oxidative stress: NADPH + is essential for the conversion of precursor amino acids to the intermediate oxime, plus the putative in vivo cyanogenic glucoside turnover pathway consumes H 2 O 2 (Møller, 2010;Gleadow and Møller, 2014;Pičmanová et al., 2015). The nitrogen levels in the leaves of plants at both moderate and severe salt stress do not differ significantly but the levels of HCNp do. We hypothesize that at moderate stress cyanogenic glucosides are acting in defence whilst at the higher salinity levels the cyanogenic glucosides are turned over and protect the plant from oxidative stress and ROS scavenging (Fig. 5B). However, details of the putative pathway need to be elucidated before this hypothesis can be confirmed.

Conclusions and implications for food security
The study of climatic variables and the consequences for cassava growth is key to evaluating the effect of global change on food security. Cassava is able to tolerate a wide range of conditions and so it is often assumed that the impact of climate change on cassava will be minimal (e.g. Lobell et al., 2008;Burns et al., 2010). Rising sea levels and reliance on saline irrigation water in increasingly hot, dry climates poses risks that have not been previously explored for many crops.
Changes in cassava tissue chemistry in response to salinity reflect a combination of factors, including differences in the balance in trade-offs in resource allocation between growth and defence in plants at different stages of development and the possible reduction in oxygen radicals. This is summarized in the model presented in Fig. 5. This model presents a number of testable hypotheses. It is noteworthy that all the measurements of cyanogenic glucosides are above the FAO/WHO Codex (1989) standard for edible cassava flour of 10 ppm cyanide, underscoring again the importance of processing before consumption We conclude cassava to be sensitive to low to moderate concentrations of salt, particularly at early stages of development and, therefore, that cassava is not suitable for planting in regions contaminated with even relatively low levels of salt. In coastal areas, impacts may be minimized by irrigating with less saline water, or during periods of high rainfall to allow time for plants to become established before they are exposed to higher concentrations of salt. Given that alternative tuberous crops such as sweet potatoes are even more salt sensitive Fig. 5. Proposed schema detailing the responses of cassava to salinity stress. During growth and development, cyanogenic glucosides (CNglcs) are synthesized in the leaves and transported to the roots and appear to play crucial roles in defence, nitrogen storage, and turnover, particularly during early plant establishment, and in mitigating oxidative stress (Jørgensen et al., 2005;Selmar and Kleinwächter, 2013;Gleadow and Møller, 2014;Sendker et al., 2016). (A) Under optimal growing conditions, light energy is converted to ATP and NAPDH+ and used to drive carbon fixation and other reducing reactions, such as nitrate reductase. (B) Under stress, plants shift the balance; the Calvin cycle is reduced, the xanthophyll cycle and photorespiration increase, and there is an increase in the production of secondary metabolites, such as CNglcs (Selmar and Kleinwächter, 2013. Stress enhances the synthesis of secondary plant products: the impact of stress-related over-reduction on the accumulation of natural products. Plant and Cell Physiology 54, 817-826, by permission of the Japanese Society of Plant Physiologist). The CNglcs can be turned over to release the N as ammonia or amino acids and also has been shown to be involved in the detoxification of reactive oxygen species (ROS), which also increase during stress.
Downloaded from https://academic.oup.com/jxb/article-abstract/67/18/5403/2485531 by Monash University user on 24 July 2020 than cassava (Shannon and Grieve, 1999), breeding for more salt-tolerant varieties is necessary if cassava is to continue to expand its role as a staple in a future, more saline world.

Supplementary data
Supplementary data are available at JXB online. Table S1. Height, leaf area, biomass, growth indices, and chlorophyll measurements of cassava plants grown at three levels of salinity (0, 40, and 80 mM NaCl) for 70 d. Figure S1. Total biomass of 6-month-old, tuberous cassava plants after 4 weeks of treatment with different concentrations of salt (Experiment 1). Figure S2. Total biomass of young, pre-tuberous cassava plants plotted versus salinity (Experiment 2). Figure S3. Correlation between total chlorophyll and greenness measured using the GreenIndex Ap.


extfieldsofstudy: Biology Medicine

text:
Visualization of the Epiblast and Visceral Endodermal Cells Using Fgf5-P2A-Venus BAC Transgenic Mice and Epiblast Stem Cells

Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.

Introduction
Mouse embryonic stem cells (mESCs) are the first pluripotent stem cell type that was derived from the inner cell mass of the developing blastocyst [1,2]. Self-renewal and pluripotency are the defining features of mESCs, meaning that these cells can be maintained indefinitely in culture while retaining their ability to differentiate into all cell lineages of an adult organism. It is well-known that the core pluripotency transcription factor network formed by Oct3/4, Sox2, and Nanog is connected with extracellular signaling pathways, such as leukemia inhibitory factor (LIF), bone morphogenetic protein, and Wnt, which shields mESCs from differentiating stimuli [3][4][5]. mESCs can be grown either in conventional medium supplemented with LIF and serum or in serum-free medium containing dual inhibitors (known as 2i) for mitogen activated protein kinase (Mapk) and glycogen synthase kinase-3 (Gsk3) [6].
Subsequent studies led to the establishment of another pluripotent stem cell type, termed epiblast stem cells (mEpiSCs), which are isolated from the postimplantation mouse epiblast [7,8]. Unlike mESCs whose pluripotency relies on LIF/Janus-associated kinase-signal transducer and activator of transcription 3 (Jak-Stat3) signaling, mEpiSC self-renewal is dependent on basic fibroblast growth factor (bFGF) and Activin/transforming growth factor beta (TGFβ) signaling. In addition, when injected back into the host blastocyst, mESCs highly contribute to chimera formation while only a very small fraction of mEpiSCs analogous to the early postimplantation epiblast can do so [9]. However, a recent study reported that mEpiSCs could readily form chimeras including germ cell lineage provided they were grafted to gastrulating embryos that retained pluripotency of the postimplantation epiblast [10]. Thus, the inherent discrepancies in colony morphology, molecular and epigenetic status and chimera formation support the notion that mESCs and mEpiSCs are representatives of distinct pluripotent states termed naïve and primed pluripotency, respectively [11]. Interestingly, these naïve and primed pluripotent states can be interconverted in defined culture conditions. Naïve mESCs can achieve a primedlike state by stimulating bFGF and Activin/TGFβ signaling while mEpiSCs can be reprogrammed back into a naïve-like state by a combination of 2i/LIF and forced expression of pluripotency-related factors, such as Nanog, Esrrb, Klf2, Klf4 or Klf5 [12][13][14][15][16].
Heterogeneity is an inherent feature of mESCs when grown in the conventional culture condition containing LIF and serum [17][18][19][20]. mEpiSCs also exhibit heterogeneous expression of Oct3/4 and T (also known as Brachyury), resulting in differences in differentiation potential of subpopulations [9,21]. Oct3/4-negative mEpiSCs could not incorporate into the host blastocyst for chimera contribution, whereas a very small fraction of Oct3/4-positive mEpiSCs harboring distal enhancer activity of Oct3/4 could efficiently form chimeras [9]. Furthermore, while Tpositive mEpiSCs were prone to differentiate towards mesoderm and endoderm fates, a feature similar to that of in vivo epiblast cells that ingress through the primitive streak during gastrulation process, T-negative mEpiSCs had a propensity to give rise to the neuroectoderm cell lineage [21].
Fibroblast growth factors (FGFs) are structurally related proteins comprising 22 members in mammals [22]. The interactions between FGFs and FGF receptors (FGFRs) play important roles in regulating a wide variety of biological processes, ranging from modulation of tissue repair, inflammation, cell proliferation, survival and differentiation [23] to pluripotency and lineage specification [24] and regulation of energy expenditure [25]. Among the FGF family members, Fgf5 is transiently expressed at different stages of the developing embryo [26]. Subsequent studies proposed a potential role of Fgf5 in the process of gastrulation through stably maintaining the mobility of cells subjected to become the prospective embryonic germ layers [27][28][29][30]. Fgf5 has since been used as a marker for epiblasts in pre-streak and streak stages of mouse embryos [31][32][33]. Fgf5 is also strongly expressed in mEpiSCs [7,15,34,35], whereas it is hardly detectable in mESCs [36]. These findings implicated Fgf5 as a valuable marker for differentiation study of various tissues and epiblast cells in vivo and in vitro. Therefore, generation of an animal model mimicking Fgf5 expression in vivo and in vitro would be useful for a better understanding of epiblast cells and other biological events occurring during development as well as cell fate decision made by mEpiSCs.
Here we report for the first time the generation of Fgf5-P2A-Venus BAC (bacterial artificial chromosome) transgenic (Tg) mice to trace Fgf5 expression during early embryonic development. Our results show the recapitulation of endogenous Fgf5 expression governed by Fgf5-P2A-Venus BAC Tg in the postimplatation epiblast and visceral endodermal layer of E6.5 and E7.5 embryos as well as in mEpiSCs. Furthermore, surprisingly, while most Tg mEpiSCs expressed Venus abundantly, Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells. This observation indicates that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. These will serve as valuable tools for marking Fgf5-expressing cells during development and studying lineage commitment initiated by mEpiSCs.

Material and Methods
Construction of the Fgf5-P2A-Venus BAC Tg The BAC clone (RP23-153I24) harboring the Fgf5 gene was purchased from Invitrogen (Carlsbad, CA, USA). For generation of the reporter cassette, the PGK-gb2-neo sequence flanked by FRT sites was ligated to a DNA fragment encoding P2A-Venus-ipacpA (Gene Bridges, Heidelberg, Germany). The cassette was then inserted into the first or third exon of Fgf5 by PCR amplification. The resulting BAC targeting vector and RED/ET expression plasmid (Gene Bridges) were co-transformed into Escherichia coli. After screening with Kanamycin, recombinants were identified by PCR analysis.

Generation of Tg mice
Recombinant BAC DNAs were purified with a NucleobondXtra BAC Kits (Macherey-Nagel, Düren, Germany) and then linearized by Pl-SceI digestion. Pronuclear injection was performed in fertilized eggs isolated from C57B6/J females, followed by transplantation into pseudo-pregnant ICR females (SLC Inc., Shizuoka, Japan). Tg mice were confirmed by PCR genotyping with the following primer sequences: 5 0 -TTCAAGGACGACGGCAACTACAAGAC-3 0 and 5 0 -GCTTCTCGTTGGGGTCTTTCTCAG-3 0 . The Tg mice were maintained on an ICR or B6 genetic background. This study was approved and conducted in accordance with the Regulations for Animal Experimentation of Shiga University of Medical Sciences.
Immunohistochemical analysis for mESCs and mEpiSCs was performed as previously described [16].
For reprogramming experiments, 2-4 × 10 4 cells were seeded onto fibronectin-coated 6-well plates in EpiSC culture conditions. After 24 h, the medium was switched to 2i/LIF conditions; the 2i inhibitors included 1 μM Mek inhibitor PD0325901 (Wako Pure Chemical Industries) and 3 μM Gsk3 inhibitor CHIR99021 (Wako Pure Chemical Industries). After 7 days, immunofluorescence analysis was performed. To check the characteristics of the resulting miPSCs, several miPSC colonies were picked up, expanded, and then cultured in 2i/LIF conditions for further experiments.

Reverse transcription (RT)-quantitative (q) PCR analysis
Total RNAs were extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany), followed by cDNA synthesis using the ReverTra Ace (TOYOBO CO., LTD, Osaka, Japan) according to the manufacturer's instructions. Real-time PCR was performed with the Thermal Cycler Dice Real Time System (Takara Bio Inc., Otsu, Shiga, Japan) and SYBR Premix EX Taq II (Takara Bio Inc.). Data were normalized against the expression of β-actin gene. Primer sequences are listed in S1 Table. Cell sorting Cells were dissociated by 0.25% trypsin-EDTA and resuspended in DMEM supplemented with 10% FBS. To exclude dead cells, the single cell suspension was incubated with propidium iodide (Cat #P3566, Molecular Probes Inc.) for 10 min on ice. Flow cytometry analysis was performed with FACSCalibur (Becton Dickinson Biosciences, San Jose, CA, USA). Cell sorting was performed with a FACSAria Fusion (Becton Dickinson Biosciences).

Statistical analysis
Student's t-test was applied for statistical analysis. Data are presented as means and standard errors. Statistical significance was determined at P < 0.05.

Generation of Fgf5-P2A-Venus BAC Tg mice
To generate transgenic (Tg) mice recapitulating endogenous Fgf5 expression, we took advantage of the enhanced yellow fluorescence protein Venus, which possesses valuable features for visualization, such as quick maturation and resistance to acidosis [40]. As the first trial, a BAC clone was used to cover the entire genomic region of Fgf5 with a modification at the first exon in which an in-frame fusion Venus was implemented right after the start codon (S1 Fig). Although we obtained six Tg lines, Venus expression was not found in the epiblast of E6.5 and 7.5 embryos (data not shown). Because we anticipated that the reporter cassette perturbed potential regulatory regions located around the first exon and intron, we inserted the P2A (porcine teschovirus-1 self-cleaving peptide)-Venus reporter cassette into the third exon of Fgf5 in the BAC clone (Fig 1A), and generated six lines of Fgf5-P2A-Venus BAC Tg mice. To check Venus expression, we dissected embryos at E6.5 and 7.5 from 6 Tg lines, and found strong Venus expression in the epiblast of all Tg lines (Fig 1B and 1C, data not shown), indicating that Fgf5-P2A-Venus BAC Tg construct can efficiently direct expression in the epiblast.

Venus expression in Fgf5-P2A-Venus BAC Tg embryos
The observation that Venus expression was detectable in the postimplantation epiblast of Tg embryos prompted us to examine detailed expression of Venus directed by the Tg. Embryos collected at E6.5 and E7.5 were stained for the epiblast marker Oct3/4 and endodermal marker Gata4 or mesodermal marker T (Figs 2 and 3). We found uniform expression of Venus in the epiblast of the Tg embryo at E6.5 (Fig 2A), consistent with previous reports [27][28][29][30]. Venus was also weakly seen in the visceral endodermal layer, in accordance with previous observations [27,28,30]. Higher magnification confirmed our observation that Venus and Oct3/4 expression were expressed uniformly throughout the epiblast, whereas Gata4 protein was only discernible in the visceral endoderm (Fig 2A). Importantly, fluorescent mRNA labeling by cytoplasmic FISH revealed the presence of endogenous Fgf5 mRNA signals in the epiblast and visceral endodermal layer of Tg embryos at E6.5 (Fig 2B). These results indicated that the Fgf5-P2A-Venus BAC Tg construct recapitulated endogenous Fgf5 expression in Tg embryos at E6.5. In Tg embryo at E7.5, Venus and Oct3/4 expression was found to be overlapping in the epiblast regions while Venus was also detected abundantly in the anterior visceral endoderm layer (Fig 3A), consistent with previous reports [28,41]. We also confirmed endogenous Fgf5 mRNA expression in the epiblast and visceral endodermal layer of Tg embryos at E7.5 (Fig 3B). Collectively, these results demonstrated that Fgf5-P2A-Venus BAC Tg is capable of recapitulating endogenous Fgf5 expression in the postimplantation epiblast and visceral endodermal layer.
While epiblast cells that ingress through the primitive streak can form the mesoderm and endoderm, epiblast cells that do not traverse the primitive streak can give rise to the ectoderm [42,43]. It is of note that at E8.25, we observed Venus expression in the neuroepithelium of the forebrain (S2 Fig), consistent with Venus expression in the anterior epiblast at E7.5.
Derivation and characterization of Fgf5-P2A-Venus BAC Tg mEpiSCs mEpiSCs represent a primed pluripotent state that can be utilized as a useful model for studying biological events that take place during the transition from the primed to naïve state and vice versa. To confirm the Fgf5-P2A-Venus BAC Tg expression in vitro, we established mEpiSCs, by culturing the epiblast of E6.5 Fgf5-P2A-Venus BAC Tg embryos in NDiff227 medium supplemented with bFGF and Activin. Immunofluorescence analysis revealed that the Tg mEpiSCs expressed pluripotency markers Oct3/4 and Nanog (Fig 4A). RT-qPCR analysis showed that Oct3/4 expression is significantly higher in the Tg mEpiSCs than in mESCs, while expression levels of Nanog and Sox2 were lower in the Tg mEpiSCs than in mESCs. Notably, Fgf5 and Sox1 expression were detected at much higher levels in the Tg mEpiSCs than in mESCs (Fig 4B). Flow cytometric analysis showed that most Tg mEpiSCs, if not all, expressed Venus, consistent with uniform Venus expression in the epiblast of the E6.5 Tg embryo ( Fig  4C). Taken together, these results indicated that Fgf5-P2A-Venus BAC Tg mEpiSCs have similar properties to bona fide mEpiSCs. Reprogramming of Fgf5-P2A-Venus BAC Tg mEpiSCs into miPSCs Reprogramming of mEpiSCs into miPSCs can be accomplished by ectopic expression of Nanog, Esrrb, Klf2, Klf4 or Klf5 cultured in the presence of 2i/LIF. [12][13][14][15][16]. The reprogramming process upregulates many naïve state-associated markers containing Nanog, Esrrb, Tfcp2l1, Cd31, Rex1, Stella, Nr0b1, Prdm14, Nr5a2, Tbx3, Klf2, Klf4 and Klf5, with a parallel rapid reduction in the expression of primed state-associated markers such as Fgf5. Next, we asked whether Venus fluorescence is suppressed during the reprogramming process toward miPSC state. After ectopic expression of Nanog or Klf5 in the Tg mEpiSCs, the culture medium was changed Higher magnification of optical sections is shown in panels X and Y. Note that Oct3/4 and Venus were coexpressed in the epiblast of the Tg embryo, while T expression was observed in the mesodermal layer of the Tg embryo. Venus expression was also seen in visceral endodermal layer. Open and closed arrowheads indicate mesodermal and epiblast cells, respectively. Ex: extraembryonic region; Em: embryonic region; Epi: epiblast; Me: mesoderm; T: Brachyury/T. Scale bar: 50 μm. All images were captured by a Leica TCS-SP8 confocal microscope using a 20x/0.7 dry objective lens (projection images) and 40x/1. from bFGF and Activin to 2i/LIF (Fig 5A). We found that ES-like colonies emerged in the Klf5and Nanog-overexpressing Tg mEpiSCs within 5-7 days after addition of reprogramming medium. Immunofluorescence analysis revealed that overexpression of Klf5 or Nanog could reactivate the expression of CD31 (also known as PECAM-1: platelet endothelial cell adhesion molecule-1), a useful marker of inner cell mass cells, and increase Nanog expression in the miPSCs (Fig 5B). Importantly, Venus expression in the Tg mEpiSCs was markedly reduced in the miPSCs (Fig 5B). To further validate the characteristics of the miPSCs, several miPSC colonies were randomly picked to generate miPSC lines. These miPSC lines were maintained in 2i/ LIF conditions and used for further experiments. RT-qPCR analysis showed upregulation of pluripotency factors Nanog, Rex1, Esrrb, Tfcp2l1, Klf2, Klf4, and Klf5, and downregulation of lineage commitment factor Fgf5 in miPSCs (Fig 5C). Taken together, these results demonstrated that Venus expression can be used as an indicator when the Tg mEpiSCs are forced to reprogram into miPSCs.
Dynamic heterogeneity of Fgf5 expression in Fgf5-P2A-Venus BAC Tg mEpiSCs mEpiSCs consist of several subpopulations: T-positive and -negative populations, and also Sox1-positive and -negative populations; these positive/negative populations are interconverted [21]. Although we found that Fgf5-P2A-Venus BAC Tg embryo showed uniform Venus expression in the epiblast and most Tg mEpiSCs expressed Venus abundantly, we investigated whether the Tg mEpiSCs contained a Venus-negative population. Careful examination of flow cytometry revealed that a minor fraction of the Tg mEpiSCs was Venus-negative (about 4%) (Fig 4C). To further explore this phenomenon, we purified Venus-positive and -negative mEpiSCs by cell sorting (Fig 6A) and cultured each cell fraction in mEpiSC growth conditions to investigate the ability to re-establish heterogeneity from each subpopulation. Interestingly, Venus-positive cells quickly emerged from the sorted Venus-negative cells, and the sorted Venus-negative cells could re-establish the original cell state within 4 days in culture (Fig 6A). Similarly, the purified Venus-positive cells also generated Venus-negative cells, although the re-establishment of heterogeneity from Venus-positive cells occurred more slowly compared with that from Venus-negative cells (Fig 6A). These results indicated that the Tg mEpiSCs contain at least two subpopulations that can be interconverted. We also examined gene expression patterns in Venus-positive and -negative mEpiSCs by RT-qPCR analysis (Fig 6B). We confirmed that Fgf5 mRNA expression was enriched in Venus-positive cells (Fig 6Ba). Importantly, we found that the expression levels of other epiblast markers, Oct3/4, Nanog, and Sox2, were predominantly detectable in Venus-positive cells (Fig 6Ba). Furthermore, both subpopulations expressed very low levels of naïve pluripotency markers Rex1, Esrrb, Tfcp2l1, and Klf2 compared with mESCs (Fig 6Bd). Interestingly, we found that mesendodermal markers T, Sox17 and Foxa2 were expressed at significantly higher levels in Venus-positive cells than in Venus-negative cells (Fig 6Bb). On the other hand, among the tested ectodermal markers, only Sox1 expression was enriched in Venus-negative cells relative to Venus-positive cells (Fig 6Bc). Taken together, while it was thought that Fgf5 marks mEpiSCs uniformly, our experiments clearly demonstrated that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs.

Discussion
In this study, we demonstrated that Fgf5-P2A-Venus BAC Tg mice can recapitulate endogenous Fgf5 expression in the postimplantation epiblast and visceral endoderm. To the best of our knowledge, this is the first Tg mouse model allowing for the visualization of endogenous Fgf5 expression during early embryonic development. Initial Tg lines carrying Venus at the first exon failed to drive epiblast Venus expression while Tg lines carrying P2A-Venus in the place of the stop codon drove strong Venus expression in the epiblast of all Tg lines. The exact reason underlying these observations is currently not clear. Because many genes harbor important regulatory elements around the first intron, the insertion of the Venus-pA reporter cassette into the first exon could have potentially abolished transcription by perturbing the epiblast promoter/enhancer elements.
Previous studies showed that primitive streak formation in the posterior portion of epiblast is a crucial event through which the body plan is established during the gastrulation process [44]. More specifically, while epiblast cells that ingress through the primitive streak can form the mesoderm and endoderm, epiblast cells that do not traverse the primitive streak can give rise to the ectoderm [42,43]. Future work will delineate the actual cell fates of Fgf5-positive epiblast cells, which would provide important insights into how the ectoderm lineage is established and regulated in the gastrulating mouse embryo.
Gastrulation begins when a population of epiblast cells is triggered to move to the primitive streak located in the posterior epiblast while the other cells remain in the epiblast. This cell movement leads to the formation of the primary germ layers, namely the ectoderm, mesoderm, and endoderm [43,44]. However, how epiblast cell movement is regulated and which factors stimulate and determine the fates of cell populations at the onset of gastrulation is not fully understood. Therefore, a detailed analysis of Fgf5 and T expression pattern will be required to understand the molecular basis of epiblast cell behavior during gastrulation. mESCs consist of several subpopulations; the subpopulations expressing either PECAM1, Rex1 or Stella efficiently form chimeric animals when injected into blastocysts. mEpiSCs are also heterogeneous in terms of gene expression: T-positive cells are primed to differentiate into mesoderm and endoderm lineages, while T-negative cells are primed to ectoderm [21]. Our data showed that Fgf5 overall uniformly marks mEpiSCs, consistent with the previous report [21]. However, surprisingly, there is a small subpopulation of Fgf5-negative cells in mEpiSCs. Our gene expression analysis revealed that, while Fgf5-positive cells predominantly expressed important mesendodermal markers such as T, Sox17 and Foxa2, Fgf5-negative cells exhibited a high expression level of the ectodermal marker Sox1. Currently, the actual cell type of Fgf5-Venus-negative cells is unknown, but these results suggest the possibility that the heterogeneous expression of Fgf5 observed in our study may be the foundation for the distinct differentiation biases of subpopulations in mEpiSCs. Thus, investigation of the differentiation propensity of Fgf5-negative and -positive mEpiSCs in response to differentiation stimuli will be of a great interest in future studies.
Previous studies indicated potential roles of Fgf5 in the progression of hepatic fibrosis [45], the process of gastrulation [27,28] and hair growth cycle [46], but the molecular basis of how Fgf5 manifests its functions has not been clearly understood. In addition, the impact of biological events on lineage commitment initiated by mEpiSCs is not known. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs established in our study may be used to investigate novel functions of Fgf5 as well as to unravel molecular mechanisms underlying lineage specification in vivo and in vitro. Immunofluorescence staining of the Tg embryo at E8.25 for Venus (green). Note that Venus was expressed in the neuroepithelium of the Tg embryo. Ne: neuroepithelium. Scale bar: 100 μm. (TIF) S1 Table. Primers used for RT-qPCR analysis.


s2fieldsofstudy: Biology Medicine

text:
Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients

Background Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Methods Trypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were incubated with metacyclic forms and the number of internalized parasites was counted. Results All TcI and TcIV T. cruzi strains were poorly infective by the oral route. Parasites were either undetectable or were detected in small numbers in the mouse stomach four days post oral administration. Replicating parasites were found in the stomach and/or in the heart 30 days post-infection. As compared to TcI lineage, the migration capacity of TcIV parasites through the gastric mucin-coated filter was higher but lower than that exhibited by TcVI metacyclic forms previously shown to be highly infective by the oral route. Expression of pepsin-resistant gp90, the surface molecule that downregulates cell invasion, was higher in TcI than in TcIV parasites and, accordingly, the invasion capacity of TcIV metacyclic forms was higher. Gp90 molecules spontaneously released by TcI metacyclic forms inhibited the parasite entry into host cells. TcI parasites exhibited low intracellular replication rate. Conclusions Our findings indicate that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of metacyclic forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1455-z) contains supplementary material, which is available to authorized users.

Background
The outcome of infection by Trypanosoma cruzi, the agent of Chagas disease, may vary greatly: 20-30 % of chronically infected patients develop severe myocarditis, alterations of the digestive tract (megaesophagus and/or megacolon) occur less frequently, whereas the majority remains asymptomatic for life. The genetic background and the immunological status of the host as well as the heterogeneity of the parasite population may contribute to this diversity [1] and possibly the route of infection and the infectious dose. According to the intraspecific nomenclature established in 2009, T. cruzi strains are classified into six discrete typing units (DTUs), TcI-TcVI [2]. In countries North of South America and in the Amazon region, where megaesophagus and megacolon are rare and chagasic cardiomyopathy is commonplace, TcI is the predominant agent of Chagas disease, in contrast to TcII, which has been isolated from most patients in Brazilian central east region where T. cruzi infection is usually associated with mega syndromes [1,3]. TcI has been pointed out as the major cause of resurgent human disease in northern South America, based on genotyping for fine-scale resolution of the geographic distribution, using a large panel of polymorphic microsatellite markers [4]. In recent years, outbreaks of acute Chagas diseases by oral infection have been reported in Venezuela [5,6], Colombia [7,8] and in the Brazilian Amazon [9,10], where TcI is prevalent and a small percentage has been identified as TcIV.
Metacyclic trypomastigotes are implicated in the above referred cases of oral T. cruzi infection. Based on the presence of amastigote nests within sections of gastric mucosa, but no evidence for T. cruzi invasion within the oropharynx or esophagus of animals challenged orally with insect-derived metacyclic forms, the invasion of gastric mucosal epithelium was suggested to be a unique portal of entry for systemic T. cruzi infection [11]. Experiments with TcII and TcVI strains from chagasic patients have revealed that the metacyclic stage-specific surface molecules gp82 and gp90, which act as promoter and inhibitor of target cell invasion, respectively [12,13], play pivotal roles in oral infection [14,15]. Gp82, which selectively binds to gastric mucin [16] is highly conserved between genetically divergent T. cruzi lineages [17] and is resistant to digestion by pepsin at acidic pH, whereas gp90 isoforms with differential susceptibility to peptic digestion may be expressed in different strains so that high or low infectivity by the oral route is associated with the expression of pepsin-susceptible or pepsinresistant gp90 isoform [14,15]. Whether such a diversity in gp90 expression and oral infectivity is also found in TcI and TcIV DTUs remains to be investigated. There is no information on experimental oral infection by these parasites, the available data refer to mice injected with blood trypomastigotes or metacyclic forms by the intraperitoneal route [18] which is an unnatural mode of infection. Here we analyzed metacyclic trypomastigotes of TcI and TcIV strains isolated from chagasic patients in different geographical regions, as regards the gp82 and gp90 expression, the ability to migrate through gastric mucin layer and to invade gastric mucosal epithelium upon oral administration into mice. To clarify the mechanisms of parasite invasion, in vitro cell invasion assays were performed, using human epithelial cells. As surface antigens spontaneously shed from tissue culture-derived trypomastigotes [19] have been reported to play a role in T. cruzi infection [20], we examined whether surface molecules were released by metacyclic forms and influenced host cell invasion.

Oral infection
Five to six week-old female BALB/c mice, bred in the animal facility at Universidade Federal de São Paulo, were used. All procedures and experiments conformed with the regulation of the institutional Ethical Committee for animal experimentation, and the study was approved by the Committee (Protocol No 0234/12). Mice were infected with T. cruzi metacyclic forms by the oral route (5 × 10 7 parasites in 0.1 ml PBS per animal), using 1 ml syringe with gavage needle that was inserted into the animal mouth. For detection of parasites in the gastric mucosal epithelium, the stomach of mice inoculated orally with metacyclic forms was collected 4 days postinfection, fixed with 10 % neutral formaldehyde for 24 h. After processing by gradual dehydration in a graded series of ethanol solution, followed by xylene immersion and embedding in parafin, serial 5 μm tissue sections were cut and stained with hematoxylin and eosin. In another set of experiments, metacyclic forms were given orally into mice (1 × 10 6 parasites in 0.1 ml PBS per animal) and starting on day 10 post-inoculation, parasitemia was monitored twice a week by examining 5 μl blood samples collected from the tail, at the phase contrast microscope. At 30 day post-infection, the stomach, heart and liver were collected and processed for histological preparations, as described above.

Assay of parasite migration through gastric mucin layer
Polycarbonate transwell filters (3 μm pores, 6.5 mm diameter, Costar) were coated with 50 μl of a preparation containing 10 mg/ml gastric mucin in water. Metacyclic forms, suspended in 600 μl PBS, were added to the bottom of 24-well plates (1 × 10 7 parasites/well), the mucin-coated transwell filters were placed onto parasitecontaining wells and 100 μl PBS were added to the filter chamber. After 30 min and/or 1 h incubation at 37°C, 10 μl samples were collected from the filter chamber for parasite counting.

Flow cytometry and indirect immunofluorescence assays
Live metacyclic trypomastigotes (1 × 10 7 ) were incubated on ice for 1 h with monoclonal antibody 3 F6 or 1G7, directed respectively to metacyclic stage-specific surface molecule gp82 or gp90. Thereafter, the parasites were fixed with 4 % para-formaldehyde for 20 min. Following washings in PBS, the parasites were incubated with Alexa Fluor 488-conjugated anti-IgG for 1 h at room temperature and the number of fluorescent parasites was estimated with a BD AccuriTM C6 Flow Cytometer. Control parasites were incubated with the secondary antibody only. To visualize HeLa cell lysosomes, coverslips with adherent cells were incubated for 1 h at 37°C in D10 or in PBS ++ , or were incubated with parasites in D10 for 1 h. After fixation with 4 % p-formaldehyde in PBS for 30 min, the cells were treated with 50 mM NH 4 Cl in PBS for 30 min and washed in PBS. The cells were then incubated for 1 h at room temperature with mouse anti-human LAMP2 diluted 1:8 (v/v) in a PBS solution containing 0.15 % gelatin, 0.1 % sodium azide and 1 % saponin (PGN-Saponin). After washings in PBS, the coverslips were incubated for 1 h with Alexa Fluor 568-conjugated antimouse IgG (Invitrogen), diluted 1:300 in PGN-Saponin containing 500 ng/ml phalloidin-FITC and 10 μg/m DAPI (4′,6′-1-diamino-2-phenylindole dihydrochloride), followed by washes in PBS and subsequent mounting of coverslips in ProLong Gold (Invitrogen). Confocal images were acquired in a Leica TCS SP8 laser-scanning microscope (Leica, Germany) using an oil immersion Plan-Apochromat 63X objective (numerical aperture 1.4). The series of images obtained from confocal z-stacks were processed and analyzed using Leica LAS AF (Leica, 2012, Germany) and Imaris (Bitplane) software.

Preparation of parasite conditioned medium
Metacyclic forms (10 8 ) were incubated at 37°C for 1 h in 100 μl complete D10 medium, nutrient-deprived PBS ++ or PBS. After centrifugation, the pellet was discarded and the supernatant (conditioned medium) was collected. For use in cell invasion assays the conditioned medium was diluted 1:100 in D10 or PBS ++ and for Western blot 10 μl was loaded.

Production and purification of recombinant gp82 protein
The recombinant protein containing the full-length T. cruzi gp82 sequence (GenBank TM data base, accession number L14824) in frame with gluthatione S-transferase (GST), was produced in E. coli DH5-α and purified as detailed elsewhere [23].

Statistical analysis
The Student's t test, as implemented in GraphPad software (Version 6.01), was employed.

Oral infection of mice with T. cruzi metacyclic forms
The ability of TcI parasites to infect mice by the oral route was examined in a set of experiments. Forty mice were separated in 8 groups of five animals. In one set of experiments, aimed at checking the invasion of gastric epithelium by metacyclic trypomastigotes, 4 groups were used and each group of five mice received a different parasite strain (5 × 10 7 parasites per animal). Four days after oral administration, the stomach of mice was collected and processed for histological preparations. A high number of parasites was used in this case because in previous studies with TcI strain G, from the wild transmission cycle, we had found that even with a high inoculum very few amastigote nests, which correspond to intracellulary replicating amastigotes, were detectable at 4 day post oral infection. Despite the large inoculum, we could not detect amastigote nests by microscopic analysis of histological sections of the stomach (Table 1). Another set of experiments was performed to determine whether there was any difference in tissue tropism between T. cruzi strains. For that purpose, the remaining 4 groups were used and each group of five mice received a different parasite strain (1 × 10 6 parasites per animal) and the course of infection was followed for 30 days. Starting on day 10, the parasitemia levels were monitored twice a week, and at day 30 the stomach, heart and liver were collected for histological analysis. In this case, the inoculum was smaller because we reasoned that, after several rounds of cell invasion and intracellular replication, amastigote nests would be detectable.
Parasitemia was undetectable throughout the period of observation but infection was confirmed in all groups by detection of amastigote nests in the stomach and/or in the heart of mice (Table 1), but not in the liver. Of note was that in the stomach of mice infected with strain 1522, which derived from a chronic chagasic patient with end-stage heart failure [24], parasites were found in the heart but not in the stomach whereas the opposite applied to infection by strain 28 (Table 1). Amastigote nests were present at high numbers in the stomach of some mice infected with strain 28 or 588 (Table 1), presumably resulting from multiple rounds of invasion and intracellular replication in the gastric epithelium. The fact that parasites were not detected in the mouse stomach at 4 day after oral administration, but only at a later time point, after intracellular replication cycles, suggested either an inefficient migration of metacyclic forms  Metacyclic forms of the indicated parasite strains were given orally into mice. In one experiment, mice received 5 × 10 7 parasites and 4 days later the stomach was collected for histological preparations. In another experiment, mice received 1 × 10 6 parasites and 30 days later, the stomach and the heart were collected for histological preparations b ND not determined through the mucus layer, inefficient invasion of target epithelial cells and/or intracellular parasite multiplication. In addition to TcI strains, we examined one TcIV strain, 1434, derived from an orally infected patient. Amastigote nests, in small numbers, were detected in 3 mice at 4th day post-infection in the histological sections of the stomach, but parasites were not found in the stomach or in the heart 30 days after infection (Table 1). Parasitemia was negative throughout the course of 30 day infection. Positive hemoculture confirmed infection by all strains. Histological preparations were also examined for the presence of inflammatory processes. Regardless of the parasite strain, inflammatory foci were barely detectable in the stomach at day 4 or 30 postinfection, or in the heart at day 30.

Migration of T. cruzi metacyclic forms through the gastric mucin layer
An assay to mimic the parasite translocation through the mucus layer in the stomach was performed using transwell filters coated with gastric mucin, which is the main macromolecular component of the mucus. Previous studies had shown that TcVI metacyclic forms, which efficiently invade gastric epithelium when administered orally into mice, traverse the gastric mucincoated transwell filter as effectively as the empty filter and this property is associated with the expression of pepsin-resistant g82 molecule that selectively binds to gastric mucin [16,25]. Gastric mucin-coated transwell filters were placed on top of wells containing metacyclic forms. After 30 and 60 min incubation at 37°C, the number of parasites that reached the upper chamber was counted. TcI parasites displayed reduced capacity to migrate through the gastric mucin layer, as compared to strain CL (TcVI) that served as control (Fig. 1a), confirming that gastric mucin acted as a barrier for migration. Metacyclic forms of TcIV strain 1434 traversed the gastric mucin-coated filters more efficiently than TcI parasites, but still at lower rates than CL strain (Fig. 1a).
We examined the expression of gp82 and its resistance to pepsin in TcI and TcIV strains. Metacyclic forms were treated for 1 h with 2 mg/ml pepsin in citrate solution at pH 3.5, a condition that extensively degraded BSA (Fig. 1b), and the detergent soluble extracts were analyzed by Western blot, along with untreated parasites, using monoclonal antibody (mAb) 3 F6 directed to gp82. Before reaction with mAb 3 F6, equal loading of the parasite samples was checked by Ponceau-S staining (Additional file 1: Figure S1A). Expression of gp82, which was preserved intact after pepsin treatment, was similar in all strains, apparently at somewhat higher levels in strain 1434 than in TcI strains (Fig. 1b). As this could explain the higher migration capacity of strain 1434, we compared the gp82 expression of this strain to that of CL strain that efficiently traversed the gastric mucin-coated filter (Fig. 1a). Gp82 expression was comparable in 1434 and CL strains (Additional file 1: Figure  S1B upper panel). The presence of gp82 molecules on the parasite surface was confirmed by flow cytometry analysis (Fig. 1c). To further examine whether strain 1434 expressed higher gp82 levels than TcI strains, the Western blot analysis was repeated by using strain 515 as a representative of TcI lineage. MT samples of the two strains, prepared in the same day and electrophoresed in the same SDS-PAGE gel, showed similar profile (Additional file 1: Figure S1C). From all these data and additional FACs analysis of the two strains, showing slightly higher gp82 levels in strain 1434 (Additional file 1: Figure S1D), it is not possible to conclude that the relative efficiency of strain 1434 metacyclic forms in migrating through the gastric mucin layer is due to the differential expression of gp82. As T. cruzi is known to spontaneously release surface molecules [19,20,26], parasite-shed molecules could also be responsible for the observed effects. We checked whether gp82 was shed into medium during 1 h incubation in PBS, condition used in gastric mucin migration assay. Western blot analysis of the supernatant after parasite removal, obtained as described in the methods section, revealed that strains 515 and 1434 shed considerable amounts of gp82, in contrast to CL strain that released gp82 at barely detectable levels, whereas gp90 was shed at high levels by strain 515 and at very low levels by strains 1434 and CL (Fig. 1d). If the molecules differentially released by strains 515, 1434 and CL in fact interfere with the parasite migration through the gastric mucin coat, that would explain the high and low efficiencies of strains CL and 515, respectively, as well as the intermediate ability exhibited by strain 1434 (Fig. 1a). An experiment to demonstrate the influence of released gp90 on strain 515 migration was performed by placing gastric mucin-coated transwell filters on top of wells containing metacyclic forms alone, or mixed with anti-gp90 mAb 5E7 that does not recognize live parasites or mixed with unrelated mAb 2C2 directed to T. cruzi amastigote molecule [27]. After 30 and 60 min incubation, samples from the filter chamber were taken for parasite counting. Parasites mixed with anti-gp90 mAb 5E7, but not those mixed with unrelated mAb 2C2, traversed the gastric mucin coat at higher numbers than control parasites (Fig. 1e), indicating that shed gp90 interferes with parasite migration through unknown mechanism that does not involve binding, provided that gp90 does not bind to gastric mucin [28]. Interference by gp82, which could not be demonstrated because anti-gp82 mAb 3 F6 binds to live parasites, would be understandable because it binds to gastric mucin. We also examined whether gp90 and gp82 were differentially released in neutral and acidic pH. Conditioned medium obtained from metacyclic forms of strain 515 incubated in PBS, pH 7.2, or in citrate buffer, pH 3.5, was analyzed by Western blot. Release of gp82 was similar at both pH, whereas gp90 shedding was lower at acidic pH (Additional file 1: Figure S1B lower panel).

Host cell invasion by T. cruzi metacyclic forms
Reduced capacity of metacyclic forms in invading gastric epithelium upon oral administration into mice has been associated with expression of pepsin-resistant gp90 at high levels, and correlates with poor infectivity toward cultured human epithelial cells [14,15]. We examined the ability of TcI and TcIV metacyclic forms to enter host cells. Parasites were incubated with HeLa cells for 1 h in full nutrient DMEM and the number of intracellular parasites was counted. Low invasion capacity was a common feature of all strains, TcIV strain 1434 strain exhibiting higher efficiency than TcI strains (Fig. 2a). Western blot analysis, using monoclonal antibody directed to gp90, revealed that all parasites expressed Fig. 1 Migration of gp82-expressing T. cruzi metacyclic forms through the gastric mucin layer. a Transwell filters coated with gastric mucin were placed onto wells containing the indicated parasite strains. Samples were collected from the filter chamber at 30 min and 60 min for parasite counting. Values are the means ± SD of three independent experiments. As compared to CL strain, a significant lower migration of TcI strains (**P < 0.0005) and TcIV strain 1434 (*P < 0.005) was observed at 60 min. b Metacyclic forms, untreated (−) or treated (+) with 2 mg/ml pepsin, at pH 3.5, were analyzed by Western blot using mAb 3 F6 directed to gp82. As control of pepsin activity, BSA stained by Coomassie blue is shown. c Live parasites were incubated on ice for 1 h, in absence or in the presence of mAb 3 F6. After fixation, the parasites were incubated with Alexa Fluor 488-conjugated anti-IgG and the number of fluorescent parasites was estimated. d Parasites were incubated for 1 h in PBS. After centrifugation to remove parasites, the conditioned PBS was analyzed by Western blotting using mAb 3 F6 and mAb 5E7, directed to gp82 and gp90 respectively. e Transwell filters coated with gastric mucin were placed onto wells containing metacyclic forms of strain 515 alone, or in the presence of anti-gp90 mAb 5E7 that does not recognize live parasites or unrelated mAb 2C2. After 30 and 60 min incubation, samples from the filter chamber were taken for parasite counting. Values are the means ± variation of duplicates pepsin-resistant gp90 (Fig. 2b). The presence of gp90 on parasite surface was checked by flow cytometry analysis in strains 515 and 1434. In repeated assays using different parasite samples, gp90 was found at much lower levels in strain 1434 (Fig. 2c, Additional file 1: Figure  S1E). We expected a higher cell invasion capacity of strain 1434 because the observed profile of gp90 is similar to that of CL strain metacyclic forms (Additional file 1: Figure S1B upper panel), which are not recognized by anti-gp90 monoclonal antibodies (mAbs), gp90 being detectable in detergent extract by Western blot.

Effect of surface molecules released by T. cruzi metacyclic forms in host cell invasion
We determined whether gp82 and gp90 were shed into medium during 1 h incubation in complete D10, i.e., under condition used in the cell invasion assay. Western blot analysis of the conditioned medium, prepared as described in the methods section, revealed that gp90 was shed at the highest levels by strain 515 and at the lowest levels by strain CL, whereas gp82 release by strains 515 and 1434 was clearly visualized but was barely detectable in CL strain supernatant (Fig. 3a). Next, we tested the possibility that the invasive capacity of metacyclic forms was influenced by gp90 released into the medium. CL strain metacyclic forms were incubated for 1 h with HeLa cells in D10 medium alone or in D10 plus 1 % of conditioned medium of 515 strain, preincubated or not with anti-gp90 mAb 1G7 or with unrelated mAb 2C2. CL strain was used because of its cell higher invasion capacity and lack of reaction with mAb 1G7. As shown in Fig. 3b, conditioned medium significantly reduced parasite invasion, its inhibitory activity was mostly neutralized by anti-gp90 mAb 1G7 but not by unrelated mAb 2C2. Presumably, the gp82 present in conditioned medium also contributed to the observed inhibitory effect, but this was difficult to demonstrate because anti-gp82 mAb that does not recognize live parasites is not available. In HeLa cells incubated in D10 for 30 min with metacyclic forms of strain 515 or 1434, there was some spreading of lysosomes (Fig. 3c) that may result from interaction with gp82 released to medium. The recombinant gp82 protein has been shown to induce lysosome scattering to the cell periphery [29], and event that culminates in exocytosis and contributes for parasitophorous vacuole formation required for T. cruzi invasion [30][31][32].
In another set of experiments, we examined whether molecules released by metacyclic forms of strains 515 and 1434 inhibited the host cell invasion by these parasites. As the invasion capacity of these strains is low, the assays were performed in PBS ++ , a nutrientdeprived medium that induces lysosome scattering and exocytosis [30]. Lysosomes, which are predominantly perinuclear in HeLa cells incubated in full nutrient D10, are mobilized toward cell periphery upon 30 min incubation in PBS ++ (Fig. 3d). Invasion of metacyclic forms of strains 515 and 1434 was significantly higher in PBS ++ than in D10 (Fig. 3e). To determine whether there was differential release of gp82 and gp90 in D10 and PBS ++ , the conditioned medium of parasites was analyzed by Western blot. Regardless of the strain, higher levels of gp82 and gp90 were released in D10 than in PBS ++ , the difference being particularly remarkable for gp82 (upper part of Fig. 3f and g). To check the effect of conditioned medium and the of gp90 on parasite invasion, HeLa cells were incubated for 1 h with metacyclic forms of strain 515 or 1434 in PBS ++ , PBS ++ plus 1 % conditioned medium of respective strain prepared in D10, preincubated or not with anti-gp90 mAb 5EG7 or unrelated mAb 2C2. In this case we used mAb 5E7 that reacts with a cryptic epitope of gp90 and does not recognize live parasites. Significant inhibition of cell invasion of strains 515 and 1434 by respective conditioned media was observed (Figs. 3f and g). The capacity of mAb 5E7 in reverting the a HeLa cells were incubated for 1 h with the indicated parasite strains in full nutrient DMEM. After fixation and Giemsa staining, the number of intracellular parasites was counted in a total of 250 cells. Values are the means ± SD of four independent assays performed in duplicate. As compared to CL strain, a significant lower invasion of strains 28, 588 and 1522 (***P < 0.0005), strain 515 (**P < 0.001) and strain 1434 (*P < 0.005) was detected by Student t test. b Metacyclic forms, untreated (−) or treated (+) with 2 mg/ml pepsin, at pH 3.5, were analyzed by Western blot using anti-gp90 mAb 1G7. As control of pepsin activity, BSA stained by Coomassie blue is shown. c Live parasites were incubated on ice for 1 h, in absence or in the presence of mAb 1G7. After fixation, the parasites were incubated with Alexa Fluor 488-conjugated anti-IgG and the number of fluorescent parasites was estimated inhibitory effect of conditioned medium was more pronounced for strain 515 whereas unrelated mAb 2C2 had no significant effect.

Intracellular replication of parasites
In addition to cell invasion assays, experiments were performed to examine the intracellular replication of Fig. 3 Effect of gp90 released by T. cruzi metacyclic forms in host cell invasion. a Conditioned medium of the indicated strains, prepared in D10 medium, was analyzed by Western blot. b HeLa cells were incubated for 1 h with CL strain metacyclic forms in D10, D10 plus conditioned medium (CM) of strain 515, prepared in D10, preincubated or not with anti-gp90 mAb 1G7 or unrelated mAb 2C2. Values are the means ± SD of three independent assays performed in duplicate. Reversal of the inhibitory effect of CM by mAb 1G7 was significant (*P < 0.05). c Metacyclic forms of the indicated strains were incubated for 1 with HeLa cells and then processed for confocal immunofluorescence to visualize lysosomes (red), actin cytoskeleton (green) and nucleus (blue). The image showed a representative microscopic field of experiment under 60X objective. Scale bar = 15 μm. d HeLa cells were incubated for 30 min in D10 or in PBS ++ and processed for confocal fluorescence analysis as in (c). Note the lysosome scattering in cells incubated in PBS ++ . Scale bar = 20 μm. e Metacyclic forms of the strains 515 and 1434 were incubated for 1 h with HeLa cells in D10 or in PBS ++ , and then processed for Giemsa staining. Values are the means ± SD of three independent assays performed in duplicate. Cell invasion was significantly higher in PBS ++ than in D10 for strain 515 (**P < 0.005) and strain 1434 (*P < 0.01). f, g HeLa cells were incubated for 1 h with metacyclic forms of strain 515 (f) or strain 1434 (g) in PBS ++ , PBS ++ plus conditioned medium (CM) of respective strain prepared in D10, preincubated or not with anti-gp90 mAb 5EG7 or unrelated mAb 2C2. Values are the means ± SD of three independent assays performed in duplicate. Significant inhibition by CM was observed in invasion by strain 515 (*P < 0.05) and by strain 1434 (**P < 0.005), which could be reverted by mAb 5E7 but not by mAb 2C2. Detection of gp90 released by each strain in D10 and in PBS ++ is shown in the upper part of the corresponding figure parasites, using strain 515 as representative of TcI lineage and the highly infective CL strain. Hela cells were incubated for 1 h with 515 strain MT in PBS ++ or with CL strain MT in D10. At 1 h, the number of strain 515 MT that invaded cells in PBS ++ was comparable to that of CL strain MT in D10 (Fig. 4a), the great majority of infected cells containing one parasite. After 1 h incubation and washings with PBS to remove non internalized parasites, the cells were maintained in DMEM containing 1 % FBS for 96 hs before processing for intracellular parasite counting. We chose 96 hs to monitor the parasite multiplication because that was the time for checking the presence of amastigote nests in the gastric epithelium after oral infection of mice. The number of intracellular parasites in cells infected with CL strain was about 4 fold higher as compared to infection by strain 515 and trypomastigotes differentiated from amastigotes could be visualized in some cells (Fig. 4b). In contrast to cells infected with CL strain, with cytoplasm full of amastigotes, most cells infected with 515 strain contained a few amastigotes (Fig. 4c).

Involvement of gp82 and protein kinase C (PKC) in host cell invasion by T. cruzi metacyclic forms
In PBS ++ , the invasion capacity of strains 515 and 1434 reached levels comparable to that of CL strain metacyclic forms. We ascertained that other TcI strains exhibited the increased invasion capacity in PBS ++ (Fig. 5a), suggesting that their entry into host cells is influenced by expression of surface molecules as well as by their shedding. We presumed that cell invasion by metacyclic forms of different strains in PBS ++ is gp82-mediated. To validate the assumption, metacyclic trypomastigotes were incubated for 1 h with HeLa cells in PBS ++ , in absence or in the presence of 40 μg/ml GST or the recombinant protein containing the full-length gp82 sequence fused to GST, and the number of internalized parasite was counted. Invasion was significantly inhibited by the recombinant gp82 but not by GST (Fig. 5b). In CL strain, the gp82-mediated invasion has been associated with activation of target cell signaling pathways involving protein kinase C [30,33]. We examined if this applied to T. cruzi strains analyzed here. HeLa cells in D10 were pretreated with 100 nM phorbol myristate acetate (PMA), a drug that inhibits diverse cell processes by downregulating PKC [34,35], before incubation with parasites. As shown in Fig. 5c, pretreatment of cells with PMA significantly diminished invasion by all strains. Treatment of cells with PMA has been shown to inhibit PBS ++ -induced lysosome scattering to the cell periphery [30]. We confirmed that in cells pretreated with PMA and incubated in PBS ++ the lysosomes remained in the perinuclear region (Fig. 5d).

Discussion
Our previous studies had indicated that cell invasion capacity of T. cruzi metacyclic trypomastigotes is dependent on expression of surface molecules gp82 and gp90, which trigger opposite effects upon interaction with host cells, gp82 inducing signaling cascades leading to an increase in cytosolic Ca 2+ concentration and lysosome scattering followed by exocytosis [29]. Metacyclic forms of T. cruzi strains, such as CL and Y82, which express gp82 and low gp90 levels, efficiently enter host cells in vitro and, when administered orally into mice, effectively invade the target gastric epithelial cells [16,25]. The present study has revealed that gp82 and gp90 molecules that are differentially released into medium by different T. cruzi strains influence the cell invasion process.
We have found that during 1 h incubation in full nutrient D10 medium, condition used in our cell invasion assays, CL metacyclic forms release gp82 molecule at barely detectable levels and gp90 in small amounts, in sharp contrast with strain 515 parasites that release high amounts of gp82 and gp90. During incubation in D10 medium, gp82 and gp90 released by metacyclic forms of strain 515 presumably interfere with parasite-host cell interaction, gp82 competing with the molecule present on parasite surface for the host cell receptor, and gp90 further contributing to down modulate signaling events required for invasion. This is consistent with the wide difference in the invasion capacity of strains CL and 515. Metacyclic forms of strain 1434 expressed and released surface gp90 at levels comparable to CL strain, but their invasive capacity was much lower, which may be due to high amounts of gp82 released into medium. In contrast to CL strain, when given orally into mice, metacyclic forms of strain 515 and other TcI strains that have similar surface profile, as well as TcIV strain 1434, were poorly infective, parasites being either undetectable or Values are the means ± SD of three independent assays performed in duplicate. The difference in the invasion rate in D10 and PBS ++ was significant for strains 515, 588 and 1522 (***P < 0.005), 28 (**P < 0.01), 1434 (*P < 0.05). b Metacyclic forms were incubated with HeLa cells in PBS ++ , in absence or in the presence of rGP82, the recombinant protein based on gp82, or GST as control. After 1 h, the cells were processed as in (a) for intracellular parasite counting. Values are the means ± SD of three independent assays performed in duplicate. The recombinant gp82 significantly inhibited invasion by strains 28 (*P < 0.05), 515, 588, 1522 and 1434 (**P < 0.005). c HeLa cells were pretreated for 30 min with PMA and then incubated with metacyclic forms in PBS ++ . After 1 h incubation, the cells were processed as in (a) for intracellular parasite counting. Values are the means ± SD of three independent assays performed in duplicate. Pretreatment with PMA significantly inhibited invasion of strains 28, 515, 1522 (**P < 0.0001), 588 and 1434 (*P < 0.001). d Hela cells, untreated or pretreated for 30 min with PMA in D10, were incubated in PBS ++ for 30 min and processed for confocal immunofluorescence for visualization of lysosomes (red) and nucleus (blue) under 60X 0bjetive. Note the perinuclear accumulation of lysosomes in PMA-treated cells. Scale bar = 20 μm detectable in small numbers in histological sections of the stomach collected 4 days post infection. The low infectivity by oral route of T. cruzi strains that shed gp82 and/or gp90 could be due to the inhibitory effect of these molecules on parasite invasion of gastric epithelial cells. Based on findings that the recombinant gp82 inhibits invasion by TcI/TcIV strains, we presume that gp82 released by these strains may reduce the parasite infectivity by competing for target cell binding. The premise is compatible with the finding that gp82 is released by highly invasive TcVI strains in minimal amounts, and at high levels by poorly invasive TcI strains. Why parasites of TcI/TcIV lineage release molecules that reduce their own infectivity? One idea that comes to mind is that T. cruzi lineage TcI/TcIV has been associated with the wild transmission cycle, and the infection of animals in nature since ancient times may have induced parasite adaptations that favored the survival of the host. Strains of T. cruzi lineage TcII/TcVI, associated with the domestic transmission cycle, have been found to be highly invasive and may kill the host [14,25], what is disadvantageous to the parasite. Another important factor that could account for the gastric epithelium parasitism by TcI strains, which was undetectable at day 4 post oral infection, is their low intracellular multiplication rate. Cell invasion assays with TcI strain 515 showed that, at day 4 post infection of HeLa cells, a few amastigotes per cell was detected, as opposed to four-fold higher numbers in cells infected with CL strain. An additional factor that may be contributing to the observed low infective capacity is the parasite ability to traverse the mucus layer that protects the gastric mucosa. In vitro, the ability to migrate through gastric mucin-coat was very low in TcI strains and to a lesser degree in strain 1434. Gp82 and gp90 molecules released by parasites appear to affect translocation through gastric mucin layer. As binding of metacyclic forms is mediated by gp82, there would be a competition between surface and released gp82 molecules. The inhibitory effect of shed gp90 cannot be explained on the same basis because this molecule does not bind to gastric mucin [28].
Further support to the hypothesis that surface molecules shed into medium interfere with parasite invasion was provided by experiments with strains 515 and 1434 in nutrient-depleted PBS ++ medium. During 1 h incubation in PBS ++ medium, the release of gp90 and of gp82 in particular was greatly reduced, what could explain the increased cell invasion as compared to D10 medium. As a short time incubation of cells in PBS ++ results in lysosome scattering, a condition that facilitates the parasite entry into cells, and the surface molecules are released only in small amounts, the rate of invasion reaches levels similar to those of CL strain. We found that in PBS ++ cell invasion by all strains examined was gp82mediated and involved the participation of protein kinase C, similarly to what was previously observed with CL strain [30].
From our findings we infer that the differential capacity of T. cruzi strains in invading gastric epithelium upon oral administration is associated with the differential expression and/or release of gp82 and gp90 molecules. Four TcI strains from different geographical regions were analysed and exhibited similar characteristics. As we had access to only one TcIV strain, we do not know whether the observed features apply to this DTU in general.

Conclusions
Our findings have indicated that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of metacyclic forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly.