Philippa Kaur

Plants increase their freezing resistance upon exposure to low temperatureThe freezing resistance (LT50 values) was found to vary ranging from − 6.9 °C (14-August-2017) to − 31.7 °C (04-November-2018) over the course of study period. The freezing resistance of leaves recorded during the 12 sampling time-points has been provided in Table 1 (also see39). The overlap of confidence intervals around the mean was examined for comparison of LT50 values for the different sampling time-points. Significant differences in freezing resistance were observed across the sampling time-points (Table 1). Leaves of R. anthopogon collected during summer [July and August (Air temperature and photoperiod was about 9.6 °C and 13 h day−1 respectively)] showed marginal resistance to freezing (LT50: − 7 °C) and thus, are more susceptible to freezing damage. Further, as the ambient air temperature and photoperiod decreased towards the end of growing season (i.e., October and November 2017 with air temperature and photoperiod of about − 1.1 °C and 10.5 h day−1 respectively), the plants acquired the highest freezing resistance (LT50: − 30 °C). Interestingly, a sharp increase in freezing resistance (− 29.4 °C) was observed in September 2018, when the daily mean air temperature decreased below 0 °C due to sudden snowfall (Supplementary Fig. S2). Comparison of LT50 values of all the leaf samples of R. anthopogon showed that cold de-acclimation occurred after the snowmelt during early spring in June (LT50: − 13.4 °C) with an increase in air temperature and photoperiod. These results demonstrated that R. anthopogon plants exhibit lowered freezing resistance during the warmer months [hence, these time-periods were referred as non-acclimation (NA)], progressively develop greater freezing resistance during the onset of winter season (hence, referred as cold acclimation) followed by an intermediate level of freezing resistance during the spring [hence, these time-periods were referred as de-acclimation (DA)].Table 1 The estimates of LT50, calculated by fitting sigmoidal curve to electrolyte leakage values of temperature treatments, recorded for leaves collected during the different sampling time-points (from August 22, 2017 to September 18, 2018).Full size tableDuring the acclimation period (i.e., late in the growing season), plants acquired the highest resistance to freezing (Fig. 1). The low electrolyte leakage (= high freezing resistance) observed during this period might be due to changes in cell wall properties (such as increase in lignification and suberization of cell walls), which provide resistance to diffusion of electrolytes from cells of the leaves to the extracellular water47. Moreover, high freezing resistance may also be attributed to high leaf toughness and sclerophyllous habit of this evergreen species48. Further, it was found that freezing resistance was the lowest during mid-summer period. This pattern could be explained by a trade-of between plant growth rates and freezing resistance, where warmer temperatures favour plant allocation to growth49. These observations corroborated well with earlier reports that showed a rapid increase in ‘freezing resistance’ during the transition from summer to early winter and vice versa50.Figure 1LT50 [black point (with solid fill) on the curve] calculated by fitting sigmoidal curve to relative electrolyte leakage (REL %) values recorded during the three different acclimation phases. GOF indicates ‘goodness of fit’ test values for the fitted sigmoidal curves.Full size imagePhotosynthetic rates are higher during non-acclimation and de-acclimation periodIt was found that PN of R. anthopogon varied in the range from 8.336 to 17.64 μmol(CO2)m−2 s−1 and E from 2.281 to 4.912 mol(H2O)m−2 s−1, throughout its growing season. The Gs of leaves was estimated to be in the range from 0.110 to 0.265 mol (H2O) m−2 s−1. WUE, a ratio of PN and E, varied between 52.21 and 87.68 (Table 2). The gas exchange parameters of R. anthopogon varied significantly among the sampling time-points [referred to here as different acclimation phases of the growing period of evergreen shrub (Fig. 2, Table 3)]. In particular, PN was significantly lower on 18-September-2018 (referred as cold acclimation phase), whereas it was higher on 31-August-2018 and 15-June-2018 (referred as NA and DA phases, respectively). Similarly, Gs of leaves was significantly lower during cold acclimation in comparison to the rest of the acclimation phases (i.e., NA and DA). Further, WUE was significantly higher during cold acclimation, while it was lower during both NA and DA (p ≤ 0.05) (Fig. 2).Table 2 Variability in leaf gas exchange parameters of R. anthopogon during the different acclimation phases (NA = Non-acclimation, LA = Late cold acclimation and DA = De-acclimation).Full size tableFigure 2Variability in leaf gas exchange parameters of R. anthopogon during the three acclimation phases [i.e., Non-acclimation (31 August, 2018), Cold acclimation (18 September, 2018) and De-acclimation (15 June, 2018)]. Different alphabets (a, b, c) represent statistically significant values (p  More

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Our model results indicate that in species with a strict social dominance hierarchy where social rank is determined by nepotism, a beneficial mutation occurring in a low-ranking female is not very likely to get established. This outcome emerged despite the immense advantage of the modeled mutation, which doubled its carrier’s survival probability. Moreover, the reproductive skew in our model (see Supplementary Fig. 1) was less radical than the skew reported for the spotted hyena females21, which means that in the model, low-ranking females had a relatively higher reproductive success potential than in reality. In other words, our model may be underestimating the severity of the negative selection a low rank induces.It is reasonable to assume that a low-ranking mutant female in a female dominant society would produce very few surviving offspring due to her low rank and ensuing lack of access to resources. Thus, this female would have only a slight chance to transmit the mutation to the next generation. If this female does reproduce successfully and produces a female which also inherits the mutation, chances of this daughter to pass on the mutation are also slim, as her rank would be even lower than that of her mother. However, if the young produced is a male and has inherited the mutation, chances of transmitting the mutation may increase depending on the male’s reproduction odds. As demonstrated by the four scenarios, the reduction in mutation establishment with decreasing mutant female’s rank became more and more prominent with increasing restrictions on male reproduction. In all four scenarios, the mutation establishment rate median was zero for the lowest ranking mutants, and in all cases but Scenario I, it was 41. Although female dominance hierarchy exists in a few of these species (e.g., Peruvian squirrel monkey41, ring-tailed lemur (Lemur catta)39,42, Verreaux’s sifaka (Propithecus verreauxi))13,25, we did not find any studies indicating female reproductive skew in any of them. Holekamp and Engh25, who reviewed the more classical female dominant species, also reported no evidence for female reproductive skew.This seemingly lack of female reproductive skew among most female dominant species is quite surprising in light of the rather common correlation between social rank and female reproductive success in male dominant species. To mention a few, considerable female reproductive skew is found in baboons (Papio spp), macaques (macaca spp.), feral horses (Equus caballus) and plains zebras (Equus burchelli)8,15,19.Holekamp and Smale28 state that “reproductive skew among female spotted hyenas appears to be greater than that documented among females of male-dominated species characterized by plural breeding”. They suggest that the key determinant of reproductive success among females in this species is rank-related priority of access to food resources. This high priority is reinforced by female dominance over males and is particularly important as this species resides in an environment in which prey availability is seasonal and scarce at times21. Our study suggests that this extreme difference in reproductive success, which, unlike in male-dominated species, is determined by nepotism rather than by physical characters, may induce a handicap on the entire population preventing the establishment of beneficial mutations. This may also hinder adaptation to a changing environment. However, our study results indicate that male equal access to females may, at least partially, counter the inhibition effect on a beneficial mutation establishment. More research is necessary in order to investigate female reproductive skew in species with a social structure similar to that of the spotted hyena, which is characterized by female dominance over males, plural breeding, and a strict female linear social hierarchy determined by nepotism.One intriguing possibility for testing this model’s validity would be an empirical study, provided that the value of some adaptive trait can be measured. In the case of the spotted hyena such a trait may refer to hunting success or physical capabilities. It is well established that adult female spotted hyenas are larger and more aggressive than adult males21, but little attention has been allocated to the study of individual physical differences among females of different ranks. Smith et al.43 studied within clan aggression in the context of the fission-fusion behavior characterizing the spotted hyena clans. Their results indicate more frequent aggression and resulting fissions occurring during times of food shortage. Rank was found to be the major correlate of an aggressive incident result. If it is possible to identify low-ranking females with some beneficial trait (independent of rank), it would be interesting to follow such females’ inclusive reproductive success along time, and even more so, the reproductive success of their sons.Another possible path around the conflict this model suggests would be through the selection of male admission into new clans. Male admission into clans is often constrained by severe aggression of resident immigrant males which may prevent or delay male admission21,26. Such behavior may in fact promote mutant male chances, at least in the case of a mutation that improves physical capabilities.One last, though not very likely possible detour around this difficulty is the occurrence of dominance rank exchanges. Such rank improvements are not very common among female dominated societies, except for in the case of aging females who may clear the way for their daughters44. 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Insect rearingThe B. dorsalis strain collected from a carambola (Averrhoa carambola) orchard in Guangzhou, Guangdong Province, was reared under laboratory conditions (27 ± 1 °C, 12:12 h light:dark cycle, 70–80% RH). A maize-based artificial diet containing 150 g of corn flour, 150 g of banana, 0.6 g of sodium benzoate, 30 g of yeast, 30 g of sucrose, 30 g of paper towel, 1.2 mL of hydrochloric acid and 300 mL of water was used to feed the larvae. Adults were fed a solid diet (consisting of 50 g yeast and 50 g sugar) and 50 mL sterile water in a 35 cm × 35 cm × 35 cm wooden cage. For B. dorsalis, the female will start laying eggs once mated and the female will start mating 7 days after emergence. To make sure all females used in our study were gravid females, females were selected 10 day after emergence.Visualization of CF-BD with FISH and PCRFISH was carried out on dissected gut and ovary samples from B. dorsalis. The hybridization protocol for the gut and ovary was performed according to a previously described method32. Briefly, the gut and ovary were collected and immediately soaked in Carnoy’s fixative for 12 h. After sample fixation, proteinase K (2 mg/mL) treatment for 20 min at 37 °C and HCl (0.2 mol/L) treatment for 15 min at room temperature were performed successively. Then, followed by dehydration in ethanol, the samples were incubated in buffer (20 mM Tris-HCl (pH 8.0), 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide) containing 50 nM CF-BD specific probe (5′-AATGGCGTACACAAAGAG-3′) labeled with cy3 at the 5′ end for 90 min. After incubation, the samples were washed with buffer (0.1 M NaCl, 20 mM Tris/HCl (pH 8.0), 5 mM ethylenediaminetetraacetic acid (pH 8.0), 0.01% SDS) and observed under an epifluorescence microscope (Axiophot, Carl Zeiss, Shinjuku-ku, Japan).To further confirm CF-BD in rectum and ovary of mature females, rectums and ovaries of mature females were dissected and fixed in formalin fixation for 24 h. After soaking in graded alcohols and xylene, all samples were embedded in paraffin for section preparation. Samples were sliced into 4 µm each before pasting on the glass slide and then sent for FISH with the same probe (labeled with cy3 at the 5′ end) used above. Moreover, nested PCR was applied to detect CF-BD in 19 ovaries of mature females according to the method of Guo et al., 201733. Briefly, a 1149 bp region of gyrB gene of CF-BD was amplified by the specific outer primer gyrBP1-F (5′-CAGCCCACTCTGAACTGTAT-3′) and gyrBP1-R (5′-TCAGGGCGTTTTCTTCGATA-3′) under a temperature profile of 95 °C for 1 min, which was followed by 25 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 90 s, and 72 °C for 5 min. Then, a 371 bp region of the gyrB gene of CF-BD was amplified by the specific inner primer gyrBP4-F (5′-ACGCTGGCTGAAGACTGCC-3′) and gyrBP4-R (5′-TGGATAGCGAGACCACGACG-3′) under a temperature profile of 95 °C for 2 min, which was followed by 35 cycles of 95 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min.Influence of CF-BD on B. dorsalis ovary developmentTo evaluate the effect of CF-BD on ovary development, newly emerged B. dorsalis females were injected with streptomycin and CF-BD suspension (both dilute in sterile water). Specifically, 10 µL 25% glycerol solution containing CF-BD was added into 100 mL Luria-Bertani (LB) liquid medium and culturing for 1 day by shaking (180 rpm) in 30 °C incubator. After culturing, CF-BD was collected by centrifuging (3000 rpm, 15 min) the medium in a 50 mL centrifuge tube. Then collected CF-BD was re-suspended with 5 mL sterile water. CF-BD concentration was measured on a hemocytometer and CF-BD concentrations used in the following assays were prepared by diluting the original concentration with sterile water. A 0.5 mm inside diameter capillary needle with 1 μL streptomycin or CF-BD suspension was used for injection. The injection operation was carried out on a microinjector (Eppendorf FemtoJet), and every female was injected in the abdomen near the ovipositor. The concentrations of streptomycin used were 20 mg/mL, 10 mg/mL and 5 mg/mL, respectively. And CF-BD suspension concentrations were 3 × 107 cfu/mL, 1.5 × 107 cfu/mL and 7.5 × 106 cfu/mL, respectively. For control, the female fly was injected with 1 μL sterile water in the abdomen near the ovipositor. Then the development level of the ovary was assessed by comparing the width and length of ovary between streptomycin (or CF-BD suspension) injection flies and control. For CF-BD injected flies, developmental facilitation was observed for ovaries 2 days before the flies reached sexual maturity (flies will reach sexual maturity after 7 days). For antibiotic injected flies, ovaries were dissected after 7 days.Oviposition assaysThe method reported in previous studies was followed for the oviposition experiments17. Briefly, a 2-choice apparatus was assembled in a cage made up of wood and wire gauze (length: width: height = 60 cm: 60 cm: 60 cm) with two petri dishes (diameter: 3 cm) at the bottom of the cage (Fig. 2a). All devices were sterilized before each experiment. Fresh fruits of guava (Psidium guajava Linn.) and mango (Mangifera indica L.) were sourced from the local market in Guangzhou, China. These fruits were sterilized on the surface with ethanol and ground into puree with a sterilized grinder, and puree (2 g) was added to the sterilized Petri dishes of the cages (one dish with puree containing 100 μL CF-BD (0.8*108 cfu/mL) in sterile water, and one dish with puree containing 100 μL sterile water). Then the prepared cages were divided into two groups for different assays. Group 1: At 0 h, 50 gravid females of B. dorsalis were placed in the cages and egg numbers in the petri dishes were recorded after 2 h. Group 2: At 4 h, 50 gravid females of B. dorsalis were placed in the cages and egg numbers in the petri dishes were recorded after 2 h.To test the oviposition attraction of 3-HA, a 4-choice apparatus was assembled in a cage made up of wood and wire gauze (length: width: height = 60 cm: 60 cm: 60 cm) with four petri dishes (diameter: 3 cm) at the bottom of the cage. In the Petri dishes, 2 g puree, 2 g puree + 0.2 mg 3-HA, 2 g puree + 2 mg 3-HA and 2 g puree + 20 mg 3-HA were added. Then, the egg-laying behavior was observed31.To test the oviposition attraction of 3-HA to flies with genes knocked down, 20 females injected with dsRNA were placed into the above cage with two Petri dishes. In the Petri dishes, 2 g guava puree and 2 g guava puree + 20 mg 3-HA were added. Then, the egg-laying behavior was observed using the above method. Oviposition of normally reared females was performed as a control. The oviposition index was calculated using the following formula:Oviposition index = (O − C)/(O + C), where O is the number of eggs in the treatment and C is the number of eggs in the control.Volatile analysisThe volatile compounds in guava and mango purees were analyzed by GC–MS according to the method described in a previous study17. Briefly, 2 g puree mixed with sterile water or CF-BD was added into a 20 ml bottle, and then a 100-μm polydimethylsiloxane (PDMS) SPME fiber (Supelco) was used to extract the headspace volatiles for 30 min. GC–MS was performed with an Agilent 7890B Series GC system coupled to a quadruple-type-mass-selective detector (Agilent 5977B; transfer line 250 °C, source 230 °C, ionization potential 70 eV). The 3-HA concentrations in puree mixed with sterile water and CF-BD were measured with the standard curve drawn by the authentic standards of 3-HA. And 3-HA concentration in puree mixed with sterile water and CF-BD was compared with a paired sample Student’s t-test.Olfactometer bioassaysAn olfactometer consisting of a Y-shaped glass tube with a main arm (20 cm length*5 cm diameter) and two lateral arms (20 cm length, 5 cm diameter) was used. The lateral arms were connected to glass chambers (20 cm diameter, 45 cm height) in which the odor sources were placed. To ensure a supply of odor-free air, both arms of the olfactometer received charcoal-purified and humidified air at a rate of 1.3 L/min.To test the attraction effect of puree supplemented with CF-BD or 3-HA for females, puree mixed with CF-BD was prepared and placed in one odor glass chamber. In the control odor glass chamber, puree mixed with sterile water was placed. After 4 h, gravid females were individually released at the base of the olfactometer and allowed 5 min to show a selective response. The response was recorded when a female moved >3 cm into one arm and stayed for >1 min. Females that did not leave the base of the olfactometer were recorded as nonresponders. Only females that responded were included in the data analysis. Odor sources were randomly placed in one arm or the other at the beginning of the bioassay, and the experiment was repeated ten times. The system was washed with ethanol after every experiment. More than 100 females were selected for testing, and each female was used only once for each odor. A chi-square test was performed to compare the attraction difference between puree mixed with sterile water and CF-BD.Olfactory trap assaysThe attraction of purees supplemented with CF-BD to mature females was also tested. The test chamber was assembled with a plastic cylinder (120 × 30 cm) covered by a ventilated lid. The test chamber contained an odor-baited trap (2 g puree + 100 μL CF-BD (0.8*108 cfu/mL)) and a control trap (2 g puree + 100 μL sterile water). The traps were made of transparent plastic vials (20 × 6 cm) and were sealed with a yellow lid on which small entrances were present to let the flies in (Fig. 3a). After 0 h or 4 h of fermentation, 100 gravid females were released in the cage. The fly number in each trap bottle was recorded after 2 h. The number of flies was compared with a paired sample Student’s t-test.The attraction effect of puree supplemented with 3-HA on mature females was tested by placing four traps (2 g puree, 2 g puree + 0.2 mg 3-HA, 2 g puree + 2 mg 3-HA and 2 g puree + 20 mg 3-HA) in the test chamber. Then, the attraction effect was observed31.Video observation of egg-laying behaviorEgg-laying behavior was observed in a Petri dish. Briefly, guava puree was added to a centrifuge tube on which a hole was made. Then, one gravid female was placed into the petri dish, and the lid was closed. Above the petri dish, a camera was placed to record the behavior of the female before laying eggs.EAG analysisEAG analysis was performed to determine whether 3-HA could elicit electrogram responses in the ovipositors of gravid females and Obps knocked down gravid females. For EAG preparations, the ovipositor of a gravid female was cut off and mounted between two glass electrodes (one electrode connected with the ovipositor tip). The ovipositor tip was cut slightly to facilitate electrical contact. Dilution of 3-HA in ethanol (0.1, 1 and 10 mg/mL) was used as a stimulant. Ethanol was used as control. For each ovipositor, ethanol and 3-HA diluted in ethanol were used as stimulants. The signals from the ovipositors were analyzed with GC-EAD 2014 software (version 4.6, Syntech).Transcriptome sequencing and gene identificationTo identify the olfactory genes that contribute to B. dorsalis oviposition preference, the transcriptome sequencing results of the female ovipositors at different developmental times (0 day, 3 days, 6 days, 9 days and 12 days) were compared. For each time, 5 ovipositors were dissected for RNA extraction. In addition, five replicates were included for each time. In the next step, paired-end RNA-seq libraries were prepared by following Illumina’s library construction protocol. The libraries were sequenced on an Illumina HiSeq2000 platform (Illumina, USA). FASTQ files of raw reads were produced and sorted by barcodes for further analysis. Prior to assembly, paired-end raw reads (uploaded to National Genomics Data Center, Accession number: PRJCA004790) from each cDNA library were processed to remove adapters, low-quality sequences (Q  More

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Sewage and TSE quantity characteristicsThe WWT facilities have been implemented for Zabol with a capacity of 39,000 m3/day. Table 1 shows the volume of water consumption and sewage production based on the sewage coefficient in urban communities of the study area.Table 1 Water consumption, TSE volume and receiving resources in the study area—2019.Full size tableAs shown in Table 1, the total water consumption in the study area is 22.538 mcm/year while based on the development conditions. Afterward, the sewage volume was calculated to 16.194 mcm/year, considering the sewage coefficient and water consumption.Continuously, the sewage data obtained from the Water and Wastewater Organization of Zabol city, Iran, showed that the sewage entrance to the treatment plants of the study area is about 19,000 m3/day and 137 working days. Therefore, the TSE volume of the WWT plant was calculated based on the following scenarios of (1) data obtained from the Water and Wastewater Organization, Iran, and (2) based on the capacity of WWT plant. Note that the working days for both scenarios will be 137. The calculation is based on Eq. (1). The total TSE volume for scenarios 1 and 2 is 2.8 and 5.1 mcm/year, respectively.The difference between the calculation based on capacity and the existing data is due to the removal of raw sewage before entering the treatment plant, which has caused health and environmental problems in the region. Data obtained from Iran Department of Environment34 showed that 1.68 mcm/y of sewage were extracted for the farms. Previous studies in the same study area also reported the significant (P  5. Note that typical abundance of total and fecal coliforms (FC) in raw sewage are 107–109 and 106–108 100/mL, respectively, and were reduced by 1–5 orders of magnitude in treated TSE, depending on the type of treatment39,40. Classical treatments, which do not include any specific disinfection step, reduce fecal micro-organisms densities by 1–3 orders of magnitude40, but because of their high abundance in raw sewage, they are still discharged in large numbers with treated TSEs in the environment.Figure 6The results of the abundance of total coliforms (TC) and fecal coliforms (FC).Full size imageAdditionally, the results of yearly values of physicochemical factors of Zabol TSE (mg/L) including BOD5, COD, TDS, TH, and EC in the period of 2017–2019, showed in Fig. 7. The yearly results suggested that the values through the years of investigation did not show significant changes. In the following parts, the possibility of TSE evaluated considering various standards.Figure 7The results of yearly values of physicochemical factors of Zabol TSE.Full size imagePotential application of TSEComparing the quality of the TSE and sewage are based on various regulations showed in Table 3. It includes the food and agriculture organization (FAO), US environmental protection agency (USEPA), the Canadian water quality index (CWQI), and Iran’s national standards (INS), considering the irrigation and recreational application.Table 3 Guidelines for interpretations of water quality of sewage and TSE of Zabol WWT plants (average in the period of 2017–2019) compared to the standards of regulations.Full size tableAccording to the FAO Guide41 for Classifying Agricultural Water Quality, as shown in Table 3, the most crucial parameters for the application of TSE in irrigation include electrical conductivity (EC), sodium uptake ratio (SAR), chlorine, BOD, COD, and FC. However, three out of seven parameters namely BOD, COD, and FC in the TSE are largely erratic with the limits recommended in the standards.Based on USEPA42, the value of total suspended solids in TSE of Zabol WWT plant largely inconsistent with the limits recommended in the standards for TSE reuse. However, TDS, EC, and pH, met the criteria. Moreover, except TSS and pH, the other chemical parameters of sewage also meet the criteria. It is worth mentioning that EPA does not require or restrict any types of water reuse. Generally, states maintain primary regulatory authority (i.e., primacy) in allocating and developing water resources. Some US states have established programs to specifically address reuse, and some have incorporated water reuse into their existing programs. EPA, states, tribes, and local governments implement programs under the Safe Drinking Water Act and the Clean Water Act to protect the quality of drinking water source waters, community drinking water, and waterbodies like rivers and lakes.According to INS regulations for irrigation and recreation reuse of TSE33, the value parameters tested for the TSE of the Zabol WWT plant are following the limits recommended in the standards for consumption as irrigation (except chlorine) and recreation projects.Finally, the CWQI is a means to provide consistent procedures for Canadian jurisdictions to report water quality information to both management and the public. The CWQI value ranges between 1 and 100, and the result is further simplified by assigning it to a descriptive category in Table 4.Table 4 The CWQI value and descriptive.Full size tableThe results of CWQI software for analyzing the TSE of the WWT plant in the study area, as shown in Table 5 and Fig. 8, indicated its poor quality for drinking, and aquatic. While it is fair for livestock and marginal for irrigation. However, considering the purpose of this study for irrigation of the native plants, it met the criteria. Note that the input data set is based on the period of 2017–2019.Table 5 The results of TSE in various applications assessed by CWQI.Full size tableFigure 8CWQI tets results for TSE of WWT plant in the study area.Full size imageThe results of this section indicated the consideration of various parameters due to various regulations and demonstrated that the treatment technology upgrade was significantly better than those of urban miscellaneous water and agriculture water standards, indicating this system can be widely used for urban landscape hydration. Moreover, squeezing the sewage treatment process for being cost effective could be recommended considering the measurements of FC, BOD, and COD.Optimal area suggestion for project executionConsidering three steps of wind erosion which are detachment, transportation, and deposition, the sand fixation methods have to be done in the detachment area to be more effective. Hence, the most advantageous regions for project execution were selected based on the factors of (a) discovering the dust origins, and (b) vegetation cover. Regarding the first concern, it was shown that the dry sediments of the Farah river43, and the presence of dunes between the two sand movements corridors in Sistan, namely Jazinak (near Zabol city) and Tasuki corridors (shown in Fig. 9), was increased the dust concentration in Zabol city37,44 while the agricultural lands, and other infrastructures such as roads, and irrigation canals developed in the area between Zahedan and Zabol city.Figure 9Locations and names of Hamuns lake and sand movement corridors in the study area © 2022 by Springer Nature Limited is licensed under Attribution 4.0 International (created by ArcMap 10.5).Full size imageSubsequently, based on a guide that 30% of vegetation cover has a significant effect on the process of soil detachment45,46, and soil protection in the desert areas47, the regions with less than 30% vegetation cover in the study area based on field observation was investigated and showed in Fig. 10. Field observation demonstrated that most areas along with the Jazinak sand corridor and Zabol city have 1–15% and 15–30%36, which are in the priority for stabilization.Figure 10The critical dust hotspot and dust origins in the study area © 2022 by Springer Nature Limited is licensed under Attribution 4.0 International (created by ArcMap 10.5).Full size imageThe results are consistent with Abbasi et al.37, reported that the Hamun Baringak Lake plays a crucial role in the aeolian mobilization of sediments in the Sistan region because of the hydrological droughts that led to the gradual decline of the wetland vegetation cover. Notably, Jahantigh48, in the same study area, reported that the average forage yield of Aeluropus lagopoides in Hamun Hirmand lake in the condition of the water inflow and during drought, was estimated to be 8869 and 173 kg/ha, respectively. It can be explained by the effect of water presence on plant production and cover. However, the average of bare soil of Hamun lake was estimated to be 7.5% and 84.2% in the two periods of water inflow and drought, respectively48. It indicated the impact of dusty days. Therefore, the mentioned areas with the vegetation cover below 30% prioritized for stabilization techniques to dust reduction or mitigation.The detailed field investigation of the land use and vegetation cover, as shown in Fig. 12, indicated the presence of native plants such as A. lagopoides and Tamarix spp. Based on Fig. 11, among the Tamarix genus, the three species of T. aphylla, T. stricta, and T.hispida were observed in the study area. T. stricta is a native species to Iran with benefits including, traditional therapeutic uses in Persian Medicine49,50. Also, the soil EC in the habitat of T. aphylla (15.70 mhos/cm) is almost the same as the control area (15.80 mhos/cm) in the depth of 0–30 cm; while the available potassium in T. aphylla habitat (460 mg/l) was also more than the control area (180 mg/l)51. Hence, the afforestation of Tamarix spp. has caused the addition of soil amendments and increased the clods.Figure 11The most land use/cover in the study area.Full size imageConsequently, the water requirement of the plants in the desert area consisting of T.aphylla, is reported in Table 6. The water requirement of T. stricta was estimated based on Table 6 to be 580 m3/ha for 500 plants no./ha with a vegetation cover of 10–30%.Table 6 Annual water requirement of the T. aphylla for irrigation in the early stages of establishment in terms of planting density (Rad, 2018).Full size tableMoreover, Fig. 12 shows the vast (50% more) soil coverage of T. stricta in the collar area compared to T. aphylla. Therefore, it is more appropriate to cultivate T. stricta than T. aphylla for the biological restoration of the region. Note that the introduced dust mitigation technique using TSE of Zabol WWT can play a specific role in the rehabilitation of soil cover in the mentioned area due to the low water need of native plants. Consequently, it has a significant impact on dust reduction in Zabol city.Figure 12The picture of (a) T. stricta and (b) T. aphylla in the study area.Full size imageHence, based on the hotspots of dust origins in the study area, the most appropriate sites for the project executions of TSE were selected, as shown in Fig. 13. Investigations indicated that a total of 27,500 ha are suitable for the project excision. Hence, considering the water requirement of 500 m3/ha/year, TSE volume of 5.1 mcm/year, vegetation cover of below 30%, and other observations such as the soil coverage in the collar area, the native plant of T. stricta selected for the afforestation of 10,000 ha on the west part of Zabol. This region has the priority in stabilization due to companionship to the corridors with a vegetation cover of 16–30%.Figure 13Area suggested for the dust mitigation project execution by the application of TSE © 2022 by Springer Nature Limited is licensed under Attribution 4.0 International (created by ArcMap 10.5).Full size imageCost analysisFinally, due to the vast area of TSE application, the total of 27,500 ha, with the puprose of dust mitigation, the project execution costs must have been addressed. Hence, Fig. 13 shows the distance of Zabol city to Hamun Hirmand and Baringak lake for transportation calculation. Accordingly, the distance from Zabol to Hamun Hirmand and Baringak lake is 14 and 33 km, respectively. The whole area around Zabol city to Hammon Hirmand lake is cultivated lands; hence, the existing roads reduced construction costs.The two main modes of transportation are trucks and pipelines. There are various pros and cons to both methods. Truck transportation is favored for low volume and short distances, while its costs rapidly increase for large-scale transportation. On the other hand, pipeline transportation is appropriate for large volumes, and long travel distances as it has a positive impact on reducing greenhouse gas emissions. Using pipelines also reduces noise, reduces highway traffic, and improves highway safety.Based on the literature, the variable and fixed transportation cost components depend on the type of product shipped, design requirements, and other decisions related to facility planning. For the sewage sludge with a pH level of 7.0 ± 0.1; hence, a low-cost PVC pipe suggested. Moreover, for cost optimization, as the WWT facilities in the study area do not generate enough volume daily, it makes economical sense to store sewage for a few days to increase the shipped volume. However, reducing the storage to a single day condenses these investment costs drastically52.It was estimated that the total costs for a facility-owned and rented single trailer truck with a capacity of 30 m3 to be $5.6/m3 and 7.4/m3/km, respectively53. Hence, the variable unit transportation cost along a pipeline with a capacity of 480 m3/day is estimated to be $0.144/m3/km. In despite of previous studies mentioning that it is more economical to use a pipeline rather than a rented single trailer truck if the volume shipped is greater than 700 m3/day, in the study area, it is more economical to use a facility-owned single trailer truck, while the shipped volume is 1200 m3/day due to the low cost of petroleum and very close distance of the suggested area. More