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Toxicity and Potential Pharmacological Activities in the Persian Gulf Venomous Sea Anemone, Stichodactyla haddoni.

Numerous proteins and peptides in venomous marine animals are potentially active molecules with pharmacological properties. Particular condition of the Persian Gulf as a closed ecosystem is a good opportunity to study of biological activities and toxicity of venomous animals. In this study, Stichodactyla haddoni (S. haddoni), a sea anemone, selected to tracing for possible pharmaceutical agents and toxicological characterization. Analgesic, edematogenic, dermonecrotic, LD50, phospholipase, and proteolytic activities of the venom were estimated. LD50 was recorded at 675 μg by intraperitoneal injection. Analgesic activitiy of crude venom on Balb/c mice at both 100 and 150 µg were dose dependent as a linear trend. Three folds increase of activity was seen at both 100 and 150 µg after 240 min comparing to activity of morphine at 200 µg. The crude venom at amount of 0.23 µg produced 50% hemolysis. The highest edematogenic activity was seen on Balb/c mice just two hours after injection for both 168 µg (157%) and 335 µg (247%). The crude venom at 675 µg made 4 mm inflammation area on rabbit skin after 3 h but the amount of 1000 µg induced 8 mm necrosis area. Potent analgesic activity of the venom was seen below its toxic dose that was very greater than the other sea anemones in the other geographical areas. The results indicate that a persistent edematogenic activity could be happened after envenomation. Instant potent edematogenic and rapid dermonecrotic activity were significant phenomena. HD50 at 0.23 µg indicates that a very potent hemolytic agent exists in the venom. The results would also be of high value to better management of envenomation. This study confirmed the great value of further studies on the Persian Gulf S. haddoni venom.

Introduction
Numerous proteins, peptides, and chemical agents in the venom of venomous marine animals are potentially useful biologically active molecules with pharmacological properties. During the past decade, many studies have focused on tracing of novel drugs from marine animals (1-12).
In this regards, venom characterization of marine venomous animals would be led to finding a new potential therapeutic agent (1, 5, 26 and 27).
Among the Persian Gulf venomous animals, S. haddoni, a venomous sea anemone, selected to biochemical characterization and tracing for possible pharmaceutical agents. Stichodactyla haddoni is an ocean dwelling sedentary organism belonging to the family of Stichodactylidae. The venom of Stichodactylidae family could be a potential source of bioactive pharmaceutical compounds (11,(28)(29)(30).
The venom of all sea anemones is produced by specialized cells, known as nematocyst. The most nematocysts are located on the surfaces of tentacles and in lesser density on the body surface. The capsule contains a tightly wrapped and spiralized thread which is extruded under physico-chemical stimuli.
Reference to a survey among many reports, there is no report for an approved drug extracted or derived from the venom of Stichodactylidae family yet (1, 5 and 6).
Venom characterization of clinically important venomous animals concerning geographical distribution is a national task that could be included in the national or regional strategies for antiserum production. Furthermore, the data concluded from simulation of toxicity in animal model may have positive effect on management of envenomation by toxicity national centers and hospitals. Another possible conclusion of venom characterization is induction of an amazing insight to discovery of potential pharmaceutical agents.
Accordingly, the present study was aimed to characterization of the venom of the Persian Gulf S. haddoni from the point of analgesic, phospholipase, proteolytic, hemolytic, coagulation, edematogenic, lethal, and dermonecrotic activities. This study would be assisted in finding potential agents from venom components of the sea anemones and also improving clinical management of S. haddoni envenomation.

Reagents and media
Casein, coomassie brilliant blue R250, lecithin (phosphatidylcholine), and triton purchased from Sigma-Aldrich Co. Thromboplastin-D and activated partial thromboplastin time (APTT) reagents were obtained from Fisher Scientific Co., USA.

Sample collection
One sample of S. haddoni (Figure 1) weighted 9.65 g were collected manually a depth of 20 m from coastal waters of Lark island, the Persian Gulf (the south of Iran) ( Figure 2) in July 2015. The specimens were kept in -20 °C and transferred to venom laboratory at Pasteur Institute of Iran.

Venom extraction and preparation
The frozen specimen thawed in room temperature and surface mucus layer cleaned. Tentacles ( Figure 3) were removed from base by a sterile scalpel, trimmed to small pieces, and divided into two equal parts weighted 4.82 g for venom extraction.

Methanol extraction
Twenty-five mL methanol solution (5%) Toxicity and potential analgesic activities was added to the fragmented tentacles, mixed in a vortex and incubated at 4 °C for 24 h. The solution centrifuged at 10625 g for 10 min (Sigma 1-14, Germany). Supernatant lyophilized in a freeze dryer system (Alpha 1-2 LD plus, Martin Christ Gefriertrocknungsanlagen Co., Germany). The powder resuspended in sterile water for injection and stock solution preserved at -70 °C.

Water extraction
Sterile deionized water (50 mL) added to the fragmented tentacles and extraction performed as detailed above.

Protein Estimation
Concentration of crude venom was determined by bicinchoninic acid (BCA) protein assay method according to manufacturer

Venom extraction and preparation
The frozen specimens thawed in room temperature and surface mucus layer cleaned. Tentacles ( Figure 3) were removed from base by a sterile scalpel, trimmed to small Toxicity and potential analgesic activities 943 instructions (iNtRON Biotechnology Co. South Korea).

SDS-PAGE
To determine the protein profile of extracted venom, Sodium Dodecyl, Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed on 12% polyacrylamide gel based on standard method (36).

Lethal activity
Lethal activity of crude venom was examined in Balb/c mice based on Spearman-Karber's method (37). Specimen conditions for this study were approved by Ethical Committee of the Pasteur Institute of Iran (IR.PII. REC.1394.38) in accordance with EU Directive Guideline (2010/63/EU) for animal experiments.
Briefly, different amounts of venom ranged from 50 to 675 µg were prepared in sterile deionized distilled water (DDW). Toxicity in male Balb/c mice (average weight 20-22 g) was tested by intraperitoneal injection (IP) of 100 µL of crude venom.
Lethality was monitored during 36 h and LD50 calculated based on the following formula:

Analgesic activity
Before the anti-pain assay, each of the Balb/c mice were placed on a hot plate at 55 °C and the numbers of licking were documented during 40 sec by two observers.
The results of this pre-experiment were considered as basic pain signal that was specific for each mouse and used for calculation of analgesic ratio. After 15 min, two doses of crude venom, 100 and 150 µg, were injected intraperitoneally into the abdomen cavity of test groups and anti-pain assay repeated every 30 min for 240 min. This experiment was performed in triplicates. Morphine (200 µg) injected as positive control and its anti-pain activity examined as the same method as test group. At the end of experiment, the percent of analgesic activity in each point of time was calculated according to our novel following math formula:

Methanol extraction
Twenty-five mL methanol solution (5%) added to 6.16 g of fragmented tentacles, was mixed in a vortex and incubated at 4 °C for 24 h. The solution centrifuged at 10625 g for 10 min (Sigma 1-14, Germany). Supernatant lyophilized in a freeze dryer system (Alpha 1-2 LD plus, Martin Christ Gefriertrocknungsanlagen Co., Germany). The powder resuspended in sterile water for injection and stock solution preserved at -70 °C. Sterile deionized water (50 mL) added to 6.16 g of fragmented t extraction performed as detailed above.

Protein Estimation
Concentration of crude venom was determined by bicinchoninic acid (B assay method according to manufacturer instructions (iNtRON Biotec South Korea).

SDS-PAGE
To determine f protein profile of extracted venom Sodium Dodecyl, Po Gel Electrophoresis (SDS-PAGE) was performed on 12% polyacrylam on standard method (36).

Proteolytic activity
Proteolytic activity was based on a new rapid method innovated by Dr. Kamran Pooshang Bagheri (38). Briefly, test solutions (100 µL) containing ascending amounts of venom including 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39 µg were incubated with casein solution (0.5%, containing 0.008 M calcium chloride at pH 7.5) for 2 h at 37 °C. To stopping the reaction and instant staining, coomassie brilliant blue R 250 (50 µL, 1X) was added to the solution. Optical density was read at 595 nm in a microplate spectrophotometer (EPOCH, BioTeK Co., USA) and protein concentration calculated reference to BSA standard curve. Casein (0.5%, containing 0.008 M calcium chloride at pH 7.5) used as control.

Phospholipase activity
Phospholipase activity of the crude venom was determined by colorimetric method (39) with some modifications. Briefly, serial amounts of venom from 0.78 to 100 µg added to a microplate and then, substrate solution (100 µL) was added to each well and incubated at 37 °C for 30 min. Optical density was read at 550 nm in a microplate spectrophotometer (EPOCH, BioTek Co., USA). Macrovipera lebetina (viper snake) and Apis melifera meda (honeybee) venom were used as positive controls. Deionized water was used as negative control.

Edematogenic activity
The ratio of 1/2 and 1/4 of LD50 dose selected to examine edematogenic activity in Balb/c mice (male, 20 g). Based on these ratios, two quantities of S. haddoni venom, 168 and 336 µg, prepared in sterile 0.9% NaCl (W/V), and 30 µL was injected into the subplantar region of the left hind paw. The right hind paw received the equal volume of sterile saline alone and served as the control.
The diameters of left paws were measured using caliper at 0, 0.5, 1, 2, 3, 4, 24, 48, 72 h, 10 days, and 30 days after venom administration. 9 of test groups and anti-pain assay repeated every 30 min for 240 min. This experiment was performed in triplicates. Morphine (200 µg) injected as positive control and its anti-pain activity examined as the same method as test group. At the end of experiment, the percent of analgesic activity in each point of time was calculated according to our novel following math formula: The percentage of edema was calculated based on the following formula:

Hemolytic activity
This assay was performed as described before (38). Fresh human blood was drawn by venous puncture using heparinized tubes. Complete blood count (CBC) assay was performed to verify the health status of the donor. Erythrocytes were washed three times with phosphate-buffered saline (pH 7.4) and a suspension of 2% RBC was prepared in normal saline. Serial amounts of venom prepared in a microplate ranged from 100 to 0.78 µg in 100 µL normal saline and 2% RBC suspension (100 µL) added to each well and incubated for 1 h at 37 °C. The samples were than centrifuged for 10 min at 1664 g (Sigma 3-18k), and the absorbance of the supernatant was determined at 540 nm.
Normal saline and triton X100 were used as negative and positive control respectively. The percent of hemolysis was calculated as follows: 11 from 100 to 0.78 µg in 100 µL normal saline and 2% RBC suspension (100 µL) added to each well and incubated for 1 h at 37 °C. The samples were than centrifuged for 10 min at 1664 g (Sigma 3-18k), and the absorbance of the supernatant was determined at 540 nm.

Dermonecrotic activity
Dermonecrosis activity of the venom was checked on New Zealand rabbits. Different amounts of S. haddoni venom including 200, 675, and 1000 µg prepared in sterile NaCl solution (0.9% W/V). Two-hundred µL of each amount was injected into the shaved areas of back skin intradermally. The equal volume of sterile saline served as negative control. Inflamed and necrotic area was measured using a ruler after 3, 24, and 48 h.

Determination of coagulation activity on human plasma
Effect of venom on extrinsic and intrinsic pathways of coagulation were evaluated in human plasma by prothrombin time (PT) and partial thromboplastin time (PTT) respectively.

Dermonecrotic activity
Dermonecrosis activity of the venom was checked on New Zealand rabbits. Different amounts of S. haddoni venom including 200, 675, and 1000 µg prepared in sterile NaCl solution (0.9% W/V). Two-hundred µL of each amount was injected into the shaved areas of back skin intradermally. The equal volume of sterile saline served as negative control. Inflamed and necrotic area was measured using a ruler after 3, 24, and 48 h.

Determination of coagulation activity on human plasma
Effect of venom on extrinsic and intrinsic pathways of coagulation were evaluated in 10 and Apis melifera meda (honeybee) venom were used as positive controls. Deionized water was used as negative control.

Edematogenic activity
The ratio of 1/2 and 1/4 of LD50 selected to examine edematic activity in Balb/c mice (male, 20 g). Based on these ratios, two quantities of S. haddoni venom, 168 and 336 µg, prepared in sterile 0.9% NaCl (W/V), and 30 µL was injected into the sub plantar region of the left hind paw. The right hind paw received the equal volume of sterile saline alone and served as the control.
The diameters of left paws were measured using caliper at 0, 0.5, 1, 2, 3, 4, 24, 48, 72 h, 10 days, and 30 days after venom administration. The percentage of edema was calculated based on the following formula: � Thickness of the �eft paw � Thickness of the right paw Thicknessof the right paw � � ���

Hemolytic activity
This assay was performed as described before (38) and Apis melifera meda (honeybee) venom were used as positive controls. Deionized water was used as negative control.

Edematogenic activity
The ratio of 1/2 and 1/4 of LD50 selected to examine edematic activity in Balb/c mice (male, 20 g). Based on these ratios, two quantities of S. haddoni venom, 168 and 336 µg, prepared in sterile 0.9% NaCl (W/V), and 30 µL was injected into the sub plantar region of the left hind paw. The right hind paw received the equal volume of sterile saline alone and served as the control.
The diameters of left paws were measured using caliper at 0, 0.5, 1, 2, 3, 4, 24, 48, 72 h, 10 days, and 30 days after venom administration. The percentage of edema was calculated based on the following formula: � Thickness of the �eft paw � Thickness of the right paw Thicknessof the right paw � � ���

Hemolytic activity
This assay was performed as described before (38) and Apis melifera meda (honeybee) venom were used as positive controls. Deionized water was used as negative control.

Edematogenic activity
The ratio of 1/2 and 1/4 of LD50 selected to examine edematic activity in Balb/c mice (male, 20 g). Based on these ratios, two quantities of S. haddoni venom, 168 and 336 µg, prepared in sterile 0.9% NaCl (W/V), and 30 µL was injected into the sub plantar region of the left hind paw. The right hind paw received the equal volume of sterile saline alone and served as the control.
The diameters of left paws were measured using caliper at 0, 0.5, 1, 2, 3, 4, 24, 48, 72 h, 10 days, and 30 days after venom administration. The percentage of edema was calculated based on the following formula: � Thickness of the �eft paw � Thickness of the right paw Thicknessof the right paw � � ���

Hemolytic activity
This assay was performed as described before (38) and Apis melifera meda (honeybee) venom were used as positive controls. Deionized water was used as negative control.

Edematogenic activity
The ratio of 1/2 and 1/4 of LD50 selected to examine edematic activity in Balb/c mice (male, 20 g). Based on these ratios, two quantities of S. haddoni venom, 168 and 336 µg, prepared in sterile 0.9% NaCl (W/V), and 30 µL was injected into the sub plantar region of the left hind paw. The right hind paw received the equal volume of sterile saline alone and served as the control.
The diameters of left paws were measured using caliper at 0, 0.5, 1, 2, 3, 4, 24, 48, 72 h, 10 days, and 30 days after venom administration. The percentage of edema was calculated based on the following formula: � Thickness of the �eft paw � Thickness of the right paw Thicknessof the right paw � � ���

Hemolytic activity
This assay was performed as described before (38). Fresh human blood was drawn by venous puncture using heparinized tubes. Complete blood count (CBC) assay was performed to verify the health status of the donor. Erythrocytes were washed three times with phosphate-buffered saline (pH 7.4) and a suspension of 2% RBC was prepared in normal saline. Serial amounts of venom prepared in a microplate ranged human plasma by prothrombin time (PT) and partial thromboplastin time (PTT) respectively.

Prothrombin time assay
Plasma was collected from a healthy donor. To confirm the health status of the donor, PT and PTT assay were performed and controlled with standard control reagent recommended by manufacturer (Fisher Scientific Co., USA).
Different amounts of S. haddoni venom (7, 14, and 28 µg) was added to citrated plasma (100 μL) and incubated for 5 min at 37 °C in a water bath. Thromboplastin-D (200 μL) was then added and clotting time was recorded by a digital timer. Citrated plasma and PT control reagent were used as normal control.
Partial thromboplastin time assay Different amounts of S. haddoni venom (25, 50, and 100 µg) were added to 100 μL of citrated plasma and incubated for 5 min at 37 °C. One-hundred μL of a PTT reagent was then added and mix together. One-hundred μL of 0.1 M calcium chloride was then added and clotting time was recorded as above. One-hundred µL of a PTT reagent was added to 100 μL of citrated plasma (incubated for 1 min at 37 °C), followed by the addition of 100 µL of calcium chloride (preheated to 37 °C) and the clotting time was measured. Citrated plasma and PTT control reagent used as normal control.

Statistical evaluations
To describe the correlation between the examined doses and percent of activities or time dependency, linear regression test was performed by SPSS software (Ver. 22).

SDS-PAGE
SDS-PAGE results showed 12 separate bands and the molecular weight of observed protein ranged approximately from 8 to 250 kDa ( Figure  4). According to the results, no difference was seen between protein profile of water extract and methanolic extract therefore a mixture of both extracts was used for characterization of the venom.

LD50
Intraperitoneal administration of 675 µg of crude venom was induced 50% lethality in test group. At 450 µg, significant lethargy, weak movement, and tachycardia were seen. At 200 and 300 µg, no signs were seen.

Analgesic activity
Analgesic activity of crude venom at the amounts of 100 and 150 µg was greater than morphine. Anti-pain activities of both amounts of crude venom were dose dependent and slope of activity were highly increased from zero point up to 60 min. Activity of 100 µg crude venom was constant during the time period between 60 and 120 min. This phenomenon was seen for 150 µg crude venom too but from 60 and 90 min. After 90 min of crude venom administration, analgesic behavior of both 100 and 150 µg was similar up to 240 min. From 90 to 150 min for 150 µg and from 120 to 150 min for 100 µg, slope of activity were intensely raised and activity documented as 100% at 150 min. One-hundred percent analgesic activity was constant during 90 min up to 240 min. According to calculated R 2 , both 100 (R 2 = Lane 3: Water extract of S. haddoni crude venom. According to the results, no difference was seen between protein profile of water extract and methanolic extract therefore a mixture of both extracts was used for characterization of the venom.

SDS-PAGE
SDS-PAGE results showed 12 separate bands and molecular weight of observed protein ranged approximately from 8 to 250 kDa (Figure 4). According to the results, no difference was seen between protein profile of water extract and methanol extract therefore a mixture of both extracts was used for characterization of the venom. According to the results, no difference was seen between protein profile of water extract and methanol extract therefore a mixture of both extracts was used for characterization of the venom.
Analgesic activity of morphine was seen faster and greater than both amounts of crude venom during the first 60 min. Activity of morphine was constant from 60 to 120 min during 60 min (91.6%) and slope of activity was rapidly dropped to 33.3% from 120 to 240 min. Based on calculated R 2 for morphine (R 2 = 0.007), the activity was not dose dependent ( Figure 5).

Comparison of analgesic activity of crude venom and morphine
This comparison evaluated generally and also performed at all points of time. According to results, both 150 and 100 µg had lesser activity than morphine from start point up to 120 min. After 128 min, the activity of both doses was significantly raised up to 240 min. Threefold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg ( Figure 6). Based on the regression analysis, both examined doses induced similar trend of activity (R 2 = 0.976) (Figure 7).

Hemolysis assay
The amount of 25 µg crude venom produced 100% hemolysis and HD50 was identified at 0.23 µg. Based on regression analysis, no linearity was seen at examined amounts of venom ranging from 0.023 to 25 µg. This means that hemolysis activity was not seen as dose dependent manner at the examined range (R 2 = 0.299) (Figure 8).
Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this raising trend corresponding to amounts ranged from 0.023 µg up to 0.39 µg. According to calculated R 2 high linearity was seen at this range (R 2 = 0.978) (Figure 9). Slope of hemolysis activity gradually raised from 83.6 to 100% that was corresponding to the amounts ranged from Figure 5. Analgesic activity of S. haddoni crude venom and morphine on Balb/c mice in hot plate assay. Analgesic activity of crude venom at the amounts of 100 and 150 µg was greater than morphine (200 µg). Anti-pain activities of both amounts of crude venom were dose dependent and slope of activity were highly increased from zero point up to 60 min. Activity of 100 µg crude venom was constant during the time period between 60 and 120 min. This phenomenon was seen for 150 µg crude venom too but from 60 and 90 min. After 90 min of crude venom administration, analgesic behavior of both 100 and 150 µg was similar up to 240 min. From 90 to 150 min for 150 µg and from 120 to 150 min for 100 µg, slope of activity were intensely raised and activity documented as 100% at 150 min. Onehundred percent analgesic activity was constant during 90 min up to 240 min. According to calculated R 2 , both 100 (R 2 = 0.744) and 150 µg (R 2 = 0.735) reduced pain as a linear behavior. Analgesic activity of morphine was seen faster and greater than both amounts of crude venom during the first 60 min. Activity of morphine was constant from 60 to 120 min during 60 min (91.6%) and slope of activity was rapidly dropped to 33.3% from 120 to 240 min. Based of calculated R 2 for morphine (R 2 = 0.007), the activity was not dose dependent. Crude venom abbreviated as "Crd". plate assay. Analgesic activity of crude venom at the amounts of 100 and 150 µg was greater than morphine (200 µg). Anti-pain activities of both amounts of crude venom were dose dependent and slope of activity were highly increased from zero point up to 60 min. Activity of 100 µg crude venom was constant during the time between 60 and 120 min. This phenomenon was seen for 150 µg crude venom too but from 60 and 90 min. After 90 min of crude venom administration, analgesic behavior of both 100 and 150 µg was similar up to 240 min. From 90 to 150 min for 150 µg and from 120 to 150 min for 100 µg, slope of activity were intensely raised and activity documented as 100% at 150 min. One-hundred percent analgesic activity was constant during 90 min up to 240 min. According to calculated R 2 , both 100 (R 2 = 0.744) and 150 µg (R 2 = 0.735) reduced pain as a linear behavior. Analgesic activity of morphine was seen faster and greater than both amounts of crude venom during the first 60 min. Activity of morphine was constant from 60 to 120 min during 60 min (91.6%) and slope of activity was rapidly dropped to 33.3% from 120 to 240 min. Based of calculated R 2 for morphine (R 2 = 0.007), the activity was not dose dependent. Crude venom is abbreviated as "Crd". Figure 6. Comparison of analgesic activity of crude venom and morphine. According to results, both 150 and 100 µg had lesser activity than morphine from start point up to 120 min. After 128 min, the activity of both doses was significantly raised up to 240 min. Threefold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg.

16
This comparison evaluated generally and also performed at all point of time.
According to results, both 150 and 100 µg had lesser activity than morphine from start point up to 120 min. After 128 min, the activity was significantly raised up to 240 min. Threefold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg ( Figure 6). Based on the regression analysis, both examined doses induced similar trend of activity (R 2 = 0.976) (Figure 7).

Figure 6.
Comparison of analgesic activity of crude venom and morphine and examined doses. According to results, both 150 and 100 µg had lesser activity than morphine from start point up to 120 min. After 128 min, the activity was significantly raised up to 240 min. Threefold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg. Based on the regression analysis, both examined doses induced similar trend of activity (R 2 = 0.976). Crude venom was abbreviated as "Crd".

Hemolysis assay
The amount of 25 µg crude venom produced 100% hemolysis and HD50 was identified at 0.23 µg. Based on regression analysis, no general linearity was seen at examined amounts of venom ranging from 0.023 to 25 µg. This means that hemolysis activity was not seen in dose dependent manner at examined range (R 2 = 0.299) ( Figure 8).
Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this raising trend corresponding to amounts ranged from 0.023 µg up to 0.39 µg. According to calculated R 2 high linearity was seen at this range (R 2 = 0.978) (Figure 9). Slope of Figure 8. Hemolytic activity of S. haddoni venom. The amount of 25 µg crude venom produced 100% hemolysis and HD50 identified at 0.23 µg. Based on regression analysis, no linearity was seen at examined amounts of venom ranged from 0.023 to 25 µg. This indicates that hemolysis activity was generally not dose dependent (R 2 = 0.299).
hemolysis activity gradually raised from 83.6 to 100% that was corresponding to the amounts ranged from 0.78 to 25 µg. Reference to calculated R 2 , high linearity was seen at this range (R 2 = 0.914) ( Figure 10). 0.78 to 25 µg. Reference to calculated R 2 , high linearity was seen at this range (R 2 = 0.914) ( Figure 10).

Phospholipase activity
The venom did not show phospholipase activity up to 100 µg of the crude venom.

Proteolytic activity
The venom did not induce proteolytic activity up to 50 µg of crude venom.

Edematogenic activity
Edematogenic activity was seen for both examined doses of the crude venom. The crude venom at 335 µg induced greater edematogenic activity than 168 µg. The highest activity was seen just two hours after injection for both 168 µg (157%) and 335 µg (247%).
The slope of activity was rapidly increased at 19 Figure 9. Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this raising trend corresponding to amounts ranged from 0.023 µg (23 ng) up to 0.39 µg (390 ng). According to calculated R 2 high linearity was seen from 0.023 µg up to 0.39 µg (R 2 = 0.978). This result confirmed that hemolytic activity strangely was linear at the range of 0.023-0.39 µg. Figure 9. Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this raising trend corresponding to amounts ranged from 0.023 µg (23 ng) up to 0.39 µg (390 ng). According to calculated R 2 high linearity was seen from 0.023 µg up to 0.39 µg (R 2 = 0.978). This result confirmed that hemolytic activity was linear at the range of 0.023-0.39 µg. Figure 10. Slope of hemolysis activity gradually raised from 83.6 to 100% that was corresponded to the amounts ranged from 0.78 to 25 µg. Reference to calculated R 2 , high linearity was seen at the examined range of doses (R 2 = 0.914). This result demonstrates that hemolytic activity was linear at the range of 0.78 to 25 µg. Figure 10. Slope of hemolysis activity gradually raised from 83.6 to 100% that was corresponded to the amounts ranged from 0.78 to 25 µg. Reference to calculated R 2 , high linearity was seen at examined range of doses (R 2 = 0.914). This result demonstrates that hemolytic activity strangely was linear at the range of 0.78 to 25 µg.

Phospholipase activity
The venom did not show phospholipase activity on human erythrocytes up to 100 µg of the crude venom.

Proteolytic activity
The venom did not induce proteolytic activity up to 50 µg of crude venom.
Toxicity and potential analgesic activities 949 335 µg and showed a linear trend (R 2 = 0.921) in comparison to 168 µg (R 2 = 0.64) after two hours ( Figure 11). Edematogenic activities of 168 and 335 µg of crude venom was slowly reduced to 76.4 and 111.7% after 10 days respectively. Reduction trend for 168 µg was linear and time dependent (R 2 = 0.693) but at 335 µg edema had not time dependent behavior (R 2 = 0.273). The results indicate that a persistent edematogenic activity could be happened after envenomation by the Persian Gulf S. haddoni (Figure 12).
Injection of both amounts of crude venom at 168 and 335 µg induced immediate 61.7 and 74.7% swelling on subplantar region of Balb/c mice respectively as a high linear trend (R 2 = 1). Induction of edema for 335 µg was greater than 168 µg. A linear trend was seen in control group 22 Figure 11. Estimation of edematogenic activity of S. haddoni crude venom on Balb/c mice. The highest activity was seen just two hours after injection for both 168 µg (157%) and 335 µg (247%). The crude venom at 335 µg induced greater edematogenic activity than 168 µg. The slope of activity was more rapidly raised at 335 µg and showed a linear trend (R 2 = 0.921) in comparison to 168 µg (R 2 = 0.64) after two hours. Figure 11. Estimation of edematogenic activity of S. haddoni crude venom on Balb/c mice. The highest activity was seen just two hours after injection for both 168 µg (157%) and 335 µg (247%). The crude venom at 335 µg induced greater edematogenic activity than 168 µg. The slope of activity was rapidly raised at 335 µg and showed a linear trend (R 2 = 0.921) in comparison to 168 µg (R 2 = 0.64) after two hours.  (R 2 = 1) but the amount of swelling was lesser than test groups ( Figure 13).

Coagulation assay
No coagulation activity was seen in PT and PTT assays.

Dermonecrotic activity
A very fast acting dermonecrotic and inflammation activity was seen during 3 h after injection of 675 and 1000 µg of crude venom. Crude venom at 675 µg made 4 mm inflammation area on rabbit skin after 3 h but 1000 µg induced 8 mm necrosis area ( Figure 14).
Infiltration of lymphocytes and necrosis were seen after injection of 1000 µg (A) and 675 µg (B) of venom ( Figures 15A and 15B).

Discussion
During the past decades, many geographical regions have been traced for potential marine pharmaceutical agents while a few studies have been documented about characterization of the Persian Gulf venomous animals. The Persian Gulf is a closed important ecosystem in which many genera have evolved apart from the other similar animals. This particular condition is a good opportunity to study of biological activities in venomous animals concerning the significant value of tracing for potential pharmaceutical agents. From the other point of view, study of the venom toxicity would be assisted in the development of more effective treatment protocols in possible envenomations. During the past decade, many studies have focused on tracing of novel drugs from marine animals. Among many marine derived potential drugs, Ziconotide, a potent anti-pain drug, is a good example that was found in the venom of the fisheating marine snail, Conus magus.
S. haddoni is one of the venomous animals of the Persian Gulf and characterization of its biological activity and toxicity are of great value concerning discovery of new potential pharmaceutical agents as well as study of toxicity of the crude venom.
Hemolysis activity on human erythrocytes Induction of edema at 335 µg was greater than 168 µg. A linear trend was seen in control group (R 2 = 1) but the amount of swelling was lesser than test groups.
23 Figure 12. Edematogenic activity of S. haddoni crude venom during a 10 days period. Edematogenic activities of 168 and 335 µg of crude venom was slowly reduced to 76.4 and 111.7% after 10 days respectively. Reduction trend for 168 µg was linear and time dependent (R 2 = 0.693) but at 335 µg edema had not time dependent behavior. The results indicate that a persistent edematogenic activity could be happened after envenomation by the Persian Gulf S. haddoni.
was not seen in dose dependent manner at estimated doses (R 2 = 0.299) but at two distinct ranges of 0.023-0.39 µg (R 2 = 0.978) and 0.78-25 µg (R 2 = 0.914) the activity was dose dependent and linearity was significant. Similarly, Subraminan et al. (2011) showed the hemolytic activity of Paracondactylis indicus (P. indicus) and Paracondactylis sinensis (P. sinensis) respectively against chicken erythrocytes and also Heteractis magnifica (H. magnifica) and S. hadonii against chicken and goat erythrocytes (33). Sudharsan et al. (2013) reported hemolytic activity of S. mertensii methanolic crude venom extract on chicken and human erythrocytes too (40). One-hundred percent of analgesic activity was seen at both doses after 150 min. This activity was constant up to 240 min while activity of morphine did not reach to 100% and it dropped rapidly after 120 min. Three fold increase of activity was seen at 240 min comparing to activity of morphine, at both 150 and 100 µg. Similar significant linear trend of analgesic activity was seen at both examined doses based on the regression analyses. We successfully measured the percent of analgesic activity by our novel formula and the trend of activity discussed based on this estimation.
As none of documented studies reported based on percent of activity, we could not able to compare our results with the others. Sudharsan et al. (2013) reported the analgesic activity of S. mertensii tentacle methanolic extract (312.5 µg) in tail flick assay (40). Suberamanian et al. (2011) reported analgesic activity of the crude nematocyst extract of different sea anemones including P. indicus, P. sinensis, H. magnifica, and S. haddoni showing analgesic activity at 2 mg/mL (40 µg) by hot plate assay (33) up to 120 sec but the assay time in our study was conducted up to 240 min. The different analgesic activity of the Persian Gulf S. haddoni is an amazing report and demonstrates that different geographical distributions has different effects on biological activity of similar genus and species as Suberamanian et al. collected S. haddoni from Mandapam coast, India. Suganthi et al. (2011) exhibited maximum analgesic activity of the aqueous extract of jelly fish Crambionella stuhalmanni (C. stuhalmanni) and Chrysaora Quinquecirrha (C. Quinquecirrha) (Cnidaria phyllum) at 5 mg/mL (100 µg) by hot plate assay after two hours (43). In comparison, our maximum activity (100%) was obtained after 150 min. This indicates that activity of S. haddoni is slightly slower than C. stuhalmanni and C. Quinquecirrha.
Immediate edema after intradermal injection of venom indicates the existence of a very potent edematogenic molecule in the venom. Maximum thickness was seen after two hours for both 168 and 335 µg of crude venom and edema decreased very slowly during 10 days of follow up measurements to 76.4 and 111.7% respectively. The results indicate that a persistent edematogenic activity could be happened after envenomation by the Persian Gulf S. haddoni. Subramanian et al. in 2011 also showed Figure 14. Dermonecrotic activity of S. haddoni crude venom on rabbit skin. Crude venom at 675 µg made 4 mm inflammation on rabbit skin after 3 h but 1000 µg induced 8 mm necrosis area.

24
A very fast acting dermonecrotic and inflammation activity was seen during 3 h after injection of 675 and 1000 µg of crude venom. Crude venom at 675 µg made 4 mm inflammation area on rabbit skin after 3 h but 1000 µg induced 8 mm necrosis area ( Figure 14).
Infiltration of lymphocytes and necrosis were seen after injection of 1000 µg (A) and 675 µg (B) of venom ( Figures 15A and 15B). edematogenic activity of the crude extract of P. sinensis and S. haddoni collected from east coast of India (33).
No coagulation activity was seen for S. haddoni. Our report is in accordance with Annadurai et al. study in 2012 in which methanolic and aqueous extracts of sea anemones i.e. Heteractis magnifica and S. haddoni had no coagulation activity in both APTT and PT assays (50).
A very fast acting dermonecrotic activity was seen during 3 h after intradermal injection of 675 and 1000 µg of crude venom. This is the first report of a rapid dermonecrolysis by S. haddoni venom. We suggest examining its necrotic activity against skin tumors like melanoma.
Although purification and characterization of venom molecules solely are very useful, since a mixture of many molecules in the venom induce their activity and toxicity beside together, evaluation of crude venom should also be performed and seems that this approach is more real and applicable to determination of toxic activities. This re-evaluation approach would be suggested to characterization of the other marine venomous animals.

Conclusion
Potent analgesic activity of the venom below its toxic dose pointed to existence of a valuable anti-pain molecule in the crude venom. Similar significant linear trend of analgesic activity was seen at both examined doses based on the regression analyses. The results indicate that a persistent edematogenic activity could be happened after envenomation by the Persian Gulf S. haddoni. Instant edema and rapid dermonecrotic activity were the significant phenomena after intradermal injection of the venom. HD50 at 0.23 µg indicates the existence of a very potent hemolytic agent in the venom.
This report, like the history of anti-pain activity in conus venom that led to discovery of ziconotide as a potent drug, can induce an interesting insight in other scientists to discovery of valuable pharmaceutical agents from similar

Discussion
During the past decades, many geographical regions have been traced for potential marine pharmaceutical agents while a few studies have been documented about characterization of the Persian Gulf venomous animals. The Persian Gulf is a closed important ecosystem in which many genera have evolved apart from the other similar animals. This particular condition is a good opportunity to study of biological activities in venomous animals concerning the significant value of tracing for potential pharmaceutical agents. From the other point of view, study of the venom toxicity would be assisted in the development of more effective treatment protocols in possible envenomations. During the past decade, many studies have focused on tracing of novel drugs from marine animals. Among many marine derived potential drugs, Ziconotide, a potent anti-pain drug, is a good example that was found in the venom of the fish-eating marine snail, Conus magus. The data resulting from simulation of S. haddoni envenomation would also be of high value to better management of clinical conditions and following up treatment strategies as well.
According to our results, a high potent peripheral analgesic agent is in the crude venom of the Persian Gulf S. haddoni that its activity was very greater than the other sea anemones in the other geographical areas. It is the first report on instant potent edematogenic, potent hemolytic, and rapid dermonecrotic activities of the Persian Gulf sea anemone venom, Stichodactyla haddoni. This study confirmed the great value of further studies on the Persian Gulf S. haddoni venom.


Delayed aneuploidy stress response of neural stem cells impairs adult lifespan in flies

Summary Studying aneuploidy during organism development has strong limitations, as chronic mitotic perturbations used to generate aneuploidy result in lethality. We developed a genetic tool to induce aneuploidy in an acute and time controlled manner during Drosophila development. This is achieved by reversible depletion of cohesin, a key molecule controlling mitotic fidelity. Larvae challenged with aneuploidy hatch into adults with severe motor defects shortening their lifespan. Neural stem cells, despite being aneuploid, display a delayed stress response and continue proliferating, resulting in the rapid appearance of chromosomal instability, complex array of karyotypes and cellular abnormalities. Notably, when other brain cell-lineages are forced to self-renew, aneuploidy-associated stress response is significantly delayed, indicating that stemness state confers resistance to aneuploidy. Sparing solely the developing brain from induced aneuploidy is sufficient to rescue motor defects and adult lifespan, suggesting that neural tissue is the most ill-equipped to deal with developmental aneuploidy. Highlights Reversible depletion of cohesin results in just a round or two of aberrant cell divisions, generating high levels of aneuploidy. Larvae challenged with aneuploidy during development hatch into impaired adults. Few cell cycles are sufficient for chromosomal instability to emerge from a previously stable aneuploid state. Neural stemness delays aneuploidy stress response. Protecting only the neural tissue from aneuploidy rescues adult abnormalities and lifespan.

19
Larvae challenged with aneuploidy hatch into adults with severe motor defects shortening their 20 lifespan. Neural stem cells, despite being aneuploid, display a delayed stress response and 21 continue proliferating, resulting in the rapid appearance of chromosomal instability, complex 22 array of karyotypes and cellular abnormalities. Notably, when other brain cell-lineages are 23 forced to self-renew, aneuploidy-associated stress response is significantly delayed, indicating 24 that stemness state confers resistance to aneuploidy. Sparing solely the developing brain from 25 induced aneuploidy is sufficient to rescue motor defects and adult lifespan, suggesting that 26 neural tissue is the most ill-equipped to deal with developmental aneuploidy. Aneuploidy, a state of chromosome imbalance, was observed over a century ago by Theodor 42 Boveri. Since then, numerous studies have shown that aneuploidy is largely detrimental both at 43 cellular and organism level. In multicellular organisms chromosome gain or loss results in 44 lethality or developmental defects (1, 2). At the cellular level, studies in yeast and cell culture 45 have demonstrated that aneuploidy has a high fitness cost for the cell, as unbalanced karyotypes 46 lead to activation of multiple stress response pathways, resulting in reduced proliferation, cell 47 cycle arrest, or cell death (Reviewed in (3). The aneuploidy stress response and consequential 48 drop in fitness seems at odds with the hypothesized role of aneuploidy in promoting 49 malignancy, which is usually marked by over-proliferation (4). Ninety percent of solid tumors 50 harbor whole chromosome gains and/or losses (5). Therefore, although usually detrimental to 51 cell fitness, aneuploidy and its effects on cell proliferation can be context dependent, which 52 emphasizes our need for a better understanding of the immediate and ultimate consequences of 53 this abnormal cellular condition in a tissue context and through development.

55
Study of aneuploidy in vivo is challenging since somatic aneuploidy is a rare event and is 56 difficult to capture and trace in real time due to several constraints: i) Cells are equipped with 57 surveillance mechanisms that prevent chromosome mis-segregation (e.g Spindle Assembly 58 Checkpoint (SAC) (Reviewed in (6) making naturally occurring aneuploidy events virtually 59 impossible to evaluate; ii) experimentally-induced aneuploidy, by compromising mitotic 60 fidelity, if often of low prevalence, as it has been demonstrated for several mammalian (7) (8)   61 and Drosophila tissues (9, 10) and iii) induction of somatic or constitutional aneuploidy in 62 metazoans relies on chronic mitotic perturbation (Listed in (11) which usually causes embryonic 63 lethality (Reviewed in(12) as a result of progressive accumulation of damage in the developing 64 organism. Thus, from these studies, it is impossible to disentangle short term and long term 65 consequences of aneuploidy, or to examine kinetics of the response to aneuploid state during 66 development. To circumvent these limitations, we generated a genetic system with the power to 67 induce aneuploidy in an acute and time-controlled manner, in all the dividing tissues of the 68 developing Drosophila. The tool is based on reversible depletion of cohesin, a key molecule 69 regulating mitotic fidelity (13,14). Cohesin is a tripartite ring complex, composed by SMC1, 70 SMC3 and the bridging kleisin subunit RAD21 (15,16). The primary mitotic role of cohesin is 71 to mediate sister chromatid cohesion, by topologically entrapping DNA fibers from neighboring 72 chromatids (17,18). Cells entering mitosis with premature loss of cohesion and sister chromatid 73 separation activate the Spindle Assembly Checkpoint (SAC) resulting in prolonged mitosis (14, 74 19). During this SAC-dependent mitotic delay, chromosomes are shuffled from one cell pole to 75 the other by the mitotic spindle (19). Consequently, chromosome shuffling induces genome 76 randomization and aneuploidy upon mitotic exit with a theoretical rate of nearly 100%. Our 77 engineered system enables a quick restoration of this complex shortly after its inactivation, 78 thereby restricting mitotic abnormalities to a short time-frame, concomitantly with the 79 generation of high levels of aneuploidy. Using such tool, we dissect the kinetics of aneuploidy 80 response across various cell/tissue types and developmental timings. To induce aneuploidy in an acute and time-controlled manner, we developed a genetic system 87 based on rapid removal of cohesin complex, the molecular glue that holds sister chromatids 88 together. To prevent a chronic cohesion depletion state and restrict mitotic failure to a single 89 cell cycle, our genetic system is able to induce cohesin inactivation, followed by subsequent 90 cohesion rescue. The system relies on the artificial cleavage of a modified version of the 91 RAD21 cohesin subunit that contains TEV protease cleavage sites (RAD21-TEV). As 92 previously shown, this system is very efficient at inactivating cohesin upon expression of the 93 exogenous Tobacco Etch Virus (TEV) protease, induced by a heat-shock promoter, resulting in 94 long-term inactivation of this complex (>24h) (20). To restrict cohesin impairment, we modified 95 this system by promptly rescuing cohesin integrity through the expression of TEV-resistant 96 RAD21 protein (RAD21-WT) right after TEV-mediated inactivation. For this purpose, RAD21-97 WT expression is under the control of UAS promoter (UAS-Rad21-wt-myc) that is induced by a 98 Gal4 protein induced concomitantly with the TEV protease (also under a heat-shock promoter, 99 HSprom-GAL4) ( Figure 1A). Given that the TEV protease is under a direct control of heat-100 shock promoter, whereas RAD21-WT relies on a dual expression-system (Gal4-UAS), we 101 anticipated that the temporal delay in RAD21-WT expression relative to the induction of TEV 102 protease would lead to a short time window of cohesin inactivation (RAD21 cleavage) ( Figure   103 1A).

104
To test this, we probed for the kinetics of TEV-mediated cleavage of RAD21-TEV and 105 synthesis of RAD21-WT in different tissues of the developing larvae. After heat shock, both 106 Drosophila larvae brains and wing discs, showed similar kinetics of the TEV-sensitive RAD21 107 disappearance followed by the appearance of RAD21-WT ( Figures 1A´ and 1A´´

114
The cohesive function of cohesin is established in S-phase, concomitantly with DNA 115 replication. Once stabilized on the replicated genome, cohesive cohesin complexes do not turn 116 over (21). As such, loss of cohesin using our system will affect sister chromatid cohesion in all 117 cells that are in S/G2/M phase during the short period between TEV protease expression and 118 synthesis of RAD21-WT ( Figure 1A). In addition to its canonical cohesive function, cohesin 119 has also been recently implicated in other interphase functions, including regulation of gene 120 expression (22). In contrast to the cohesive pool, these cohesin molecules are known to be 121 highly dynamic (21, 23). Moreover, cohesin-mediated loops were recently reported 122 "memorable" and quickly reformed upon cohesin re-establishment (24). We therefore 123 anticipated that this function should not be severely affected by our system. In sharp contrast, 124 mitotic errors induced upon cohesin cleavage are irreversible as there is no way to restore 125 cellular ploidy after a compromised round of mitosis.

127
Whereas canonical chronic mitotic perturbations lead to several rounds of mitotic failures, our 128 novel genetic system should lead to cohesion defects only in the first mitosis following the heat-129 shock, as the expression of RAD21-WT should be able to rescue cohesion in the subsequent cell 130 cycle, if given enough time ( Figure 1A). To confirm that our genetic system works as 131 anticipated, we focused our analysis on two different cycling tissues from the larva: the 132 developing brain and the epithelial wing discs.

133
The developing brain of Drosophila is an excellent model to study the consequences of 134 developmental aneuploidy. The well characterized cell lineages of the tissue in combination 135 with our tractable system to induce mis-segregation of chromosomes offer a unique opportunity 136 to trace the fate of aneuploid cells in real time and analyze their effect on the nervous system 137 development. Through larval development ~100 large neural stem cells called Neuroblasts 138 (Nbs) (25) located in the central brain (CB) region divide asymmetrically to self-renew and 139 generate distinct neuronal lineages via differentiating progeny (26).

140
We evaluated, by live cell imaging, mitotic fidelity in these Nbs using two independent criteria 141 to estimate the state of sister chromatid cohesion: i) the presence of single sisters (a direct 142 consequence of cohesion loss), as opposed to metaphase chromosome alignment and ii) the time 143 cells spend in mitosis, given that premature loss of sister chromatid cohesion is known to 144 activate the SAC and delay mitotic exit (Mirkovic et al., 2015).

145
As expected, the first division after the heat shock results in full cohesin cleavage in Nbs, 146 followed by cohesin rescue in subsequent divisions (Movies S1 and S2). The fast cell cycle of 147 Nbs, coupled with continued proliferation of these cells despite their abnormal genome content 148 (further discussed below), enables analysis of mitotic fidelity throughout several consecutive 149 divisions in great detail. Consistently, in the first mitosis AHS, 95% of Nbs contain single 150 sisters, and exhibit mitotic delay and chromosome shuffling (Figures 2A and 2B Figure S1A to A´´).

156
In contrast to the Nbs, in the epithelial cells of the wing disc, we observe the presence of single 157 sisters and a mitotic delay even at 48hs AHS, despite the presence of high levels RAD21-WT 158 (Figures 1A´´;2D;2E and 2F).These findings are consistent with the long cell cycle of the wing 159 discs cells (27,28). The high incidence of cells affected by reversible-cohesin cleavage is also 160 consistent with a high frequency of cells in S/G2 in this tissue, estimated using the fly FUCCI 161 system (29) ( Figure S3B).To fully demonstrate the ability of our tool to induce aneuploidy in an 162 acute manner in epithelial tissues we tested their regeneration capacity. In Drosophila epithelial 163 cells, multiple cellular insults, including aneuploidy, can activate the Jun N-terminal kinase 164 (JNK) signaling pathway, thus inducing the expression of pro-apoptotic genes and triggering the 165 apoptotic cascade (9, 30). In agreement with these studies, 24hs AHS in the wing disc, Cleaved

219
Premature differentiation and apoptosis were suggested as the main mechanisms of aneuploid 220 Nb elimination, reported in two recent studies (10, 31). However, after acute aneuploidy 221 induction in the entire Nb population, we found a very low frequency of cells undergoing 222 premature differentiation or cell death ( Figure S4).As a proxy for premature differentiation events, we quantified Nb-like cells that had either lost the DPN marker or abnormally exhibit 224 the differentiation marker Prospero (Pros) with or without co-expression of DPN (Figures S4A   225 and S4A´, arrowheads and dashed circles).Pros is the key factor acting as a switch for the 226 transition from stem cell self-renewal to terminal differentiation (33); therefore, this marker 227 should not be present in Nbs. We observed that upon acute aneuploidy induction in the entire 228 Nb population, there is a very low frequency of cells indicative of premature differentiation 229 ( Figure S4). These findings suggest that premature differentiation, although still taking place, is 230 unlikely to be the major form of stem cell elimination. To estimate the levels of apoptosis, we 231 also counted cells positive for cell death markers like CC3 and DCP1. We found a significant

244
To dissect the kinetics of the aneuploid response, we took advantage of the temporal resolution 245 of our system allowing for the tracing of aneuploid fate in real time. We restricted our analysis 246 to 3 rd instar wandering larvae as at this stage no new Nbs are generated from the neuro-247 epithelium (26). Induction of aneuploidy at this developmental stage, therefore, affects the 248 entire Nbs population, which facilitates cell fate analysis. We observed a significant amount of 249 Nbs proliferating for several days and displaying a tendency for chromosome accumulation over Aneuploidy elicits a stress response in the brain tissue.

297
Our findings revealed that aneuploid cells are not promptly eliminated but instead continue to 298 proliferate within certain karyotype restrictions. This should lead not only to the maintenance of 299 aneuploid stem cells (due to Nb self-renewal) but also to the accumulation of differentiated 300 aneuploid progeny (note that each Nb divides every ~2 hours (32)). We therefore tested how 301 such increase in aneuploid cells within the tissue could affect cellular physiology and influence 302 normal tissue development.

303
Several aneuploidy-associated stresses that include oxidative, metabolic, and proteotoxic stress 304 are likely to alter cellular homeostasis (3), which ultimately lead to p53 activation and a p53-305 dependent cell-cycle arrest/senescence (34, 35). Interestingly, elevated levels of p53 have been 306 observed in the Central Nervous System of Down syndrome patients (36). We decided to take 307 advantage of our in vivo system to acutely induce aneuploidy to examine whether abnormal 308 karyotypes trigger a stress response in the developing Drosophila brain and if so, what is the 309 kinetics of such response. We assessed by immunohistochemistry the presence of P53 and the 310 senescence marker Dacapo (DAP, a p21/p27 homologue (37) Figure S5). We concluded that aneuploidy induction has a detectable 320 effect in the entire brain population, triggering stress responses across different cell types.

321
However, despite being promptly affected by aneuploidy induction, Nbs display a delay to this 322 cellular insult and only mount a detectable response ~2 days after becoming aneuploid.

325
The delayed stress response (i.e. ~48hs after induction of aneuploidy) in the neural stem cell 326 pool may imply selective stem cell tolerance for the aneuploid condition when compared to the 327 other cell types of the developing brain. To test this idea we took advantage of the brat mutant 328 condition (38). In brat mutant larvae brains, each Nb divides into two daughter cells grow that 329 retain Nbs properties, leading to the formation of a tumor-like neoplasm (39). We reasoned that 330 cellular stemness confers tolerance to aneuploidy, the complete occupancy of the developing 331 brain by Nbs-like cells observed in the brat mutant phenotype should be sufficient to prevent 332 the stress response observed at 24hs AHS. To test this idea we combined our system for acute 333 induction of aneuploidy with brat mutations to be able to induce aneuploidy in a brat mutant 334 background and analyze the presence of stress markers at 24 and 48hs AHS. As predicted, DAP 335 appearance was significantly delayed in aneuploid brat mutants when compared to aneuploid 336 brains alone ( Figures 6B and 6B´

354
To test this hypothesis, we devised a system to selectively protect only the brain from cohesin 355 removal and consequent aneuploidy. To achieve this, we complemented our reversible cohesin 356 cleavage system with brain-specific expression of RAD21-WT throughout the course of the 357 experiment ( Figure 7A). In this way, TEV expression should lead to cohesion loss in all larval 358 tissues that survive solely on RAD21-TEV at the time of heat shock. In contrast, neural stem 359 cells should be resistant to this challenge, as they express both RAD21-TEV and RAD21-WT 360 ( Figure 7B). Neuroblast-specific expression of RAD21-WT was achieved by the use of 361 inscutable-Gal4 (insc-Gal4) or worniu-Gal4 (wor-Gal4) drivers, to constitutively express UAS-362 Rad21-wt-myc in the developing brain ( Figure 7B). As expected, constitutive presence of TEV-363 resistant RAD21 in the brain prevents any cohesion defects in 3 rd instar larvae Nbs ( Figure 7C).

364
To confirm that the rescue of sister chromatid cohesion occurs exclusively in the brain, we 365 performed parallel characterization of the first mitotic division after the heat shock in the wing, 366 derived from the same larvae. As anticipated, full cohesin cleavage was observed in all the 367 dividing epithelial cells from the wing discs ( Figure 7D). Notably, protecting only the brain 368 from developmental aneuploidy fully rescued the severe motor defects of the ecloded flies from 369 the 72hs AEL heat-shock, as demonstrated by mobility essays (Figures 7E´ and Movie S6).
Even more surprisingly, the brain protection was enough to rescue the lifespan of ~70% of the 371 adult flies affected by organism-wide aneuploidy during development, demonstrating that the 372 brain is indeed the most sensitive tissue when challenged with aneuploidy ( Figure 7E).

403
Neuroblasts have been used as a system to study aneuploidy response in previous studies (10, 404 31). These studies postulate two different but not mutually exclusive mechanisms of response to 405 induced aneuploidy: premature differentiation (31) and cell death by apoptosis (10). We 406 reasoned that if these are the major mechanisms of response to aneuploidy in neural stem cells, 407 they should be detectable in high frequency after the aneuploidy induction by our acute 408 approach. Contrary to that notion, after examined in detail the kinetics of the response, both 409 premature differentiation and cell death were detected at low frequency even days after cells 410 became aneuploid. It is important to note that the degree of aneuploidy in the Nbs upon cohesin 411 loss should be around 98% due to the extensive genome shuffling prior to mitotic exit.

412
Therefore, the finding that aneuploidy does not eliminate the entire Nb population, strongly 413 argues against the existence of specific, active mechanisms controlling the integrity of the 414 neural stem cell genome. The more plausible explanation is that the Nb elimination due to 415 aneuploidy stems from a wide spectrum of abnormalities due to a randomized genome.  Interestingly, our results highlight that cell identity determines the kinetics of this stress 445 response. Aneuploidy response is specifically delayed in the neural stem cell pool (displayed 446 mainly at ~48hs AHS) compared to the rest of the tissue, which exhibits it considerably earlier.

447
Forcing self-renewal is sufficient to delay stress response in the entire tissue, suggesting that 448 cellular stemness alone makes cells less sensitive to aneuploidy-induced stresses. Accordingly, 449 unusual resistance to altered ploidy was observed in human and mouse embryonic stem cells 450 (ESCs), mostly achieved by relaxing the cell cycle control and uncoupling the spindle 451 checkpoint from apoptosis (49). The ability of neural stem cells to continue dividing despite the 452 aneuploid karyotype dubbed them as aneuploidy "tolerant" (10). Yet, based on our findings it is 453 clear that keeping these aneuploid cells is catastrophic for normal tissue architecture and 454 development. Thus, aneuploidy may be "tolerated" better in Nbs, but the tissue as a whole is 455 unable to be functional. In contrast, the "sensitivity" of epithelial cells enables the tissue to 456 clean up and regrow properly.

458
The developing brain restricts organism recovery after induced aneuploidy.

459
Chromosomal aberrations have been long associated with neurological disorders (50). However, 460 their impact on brain development and function remains complex and poorly understood, 461 partially due to limitations of available experimental approaches. In almost all animal model 462 systems used to study aneuploidy and its consequences until now, the organisms die 463 prematurely due to the chronic disruption of mitotic fidelity to generate chromosome imbalance. 464 Therefore, it is only possible to address the short term effect of aneuploidy in nervous system 465 development, but not to understand the ultimate consequences for brain function. Our acute 466 system reversibly affects chromosome segregation to induce just a pulse of aneuploidy, 467 enabling the organism to recover from the insult and complete its development. The most 468 noticeable phenotype observed in the adult was the severe motor and behavioral defects that 469 clearly affect the lifespan of the flies, evidencing the sensitiveness of the nervous system to 470 aneuploidy. Previous studies in Drosophila have shown that the mitotic disruption in larvae Nbs 471 generates a reduction of their brain size (10, 31) reinforcing the idea about a link between 472 aneuploidy and microcephaly. However, our results showed that induced acute aneuploidy has 473 no significant impact in the size of the adult brain. These findings suggest that the continued 474 proliferation of neural stem cells, caused by incomplete cell elimination and delayed 475 aneuploidy-stress response, is sufficient to support the development of an apparently normal-476 sized organ. It is conceivable that the observed normal size reflects a sample selection, as this 477 analysis was restricted to flies that survived the aneuploid challenge (~70%). Supporting this 478 possibility, a screening performed to isolate anatomical brain mutants of Drosophila have 479 shown that mutant strains showing altered brain shape and particularly small brains are very 480 weak being mostly lethal at pupa stage (51). Despite unaltered shape and size of the adult brains, we reasoned that the neural circuits are likely impaired in those brains giving rise to the 482 adult phenotype observed in all the surviving flies.

483
In accordance with the notion of the brain as the tissue most sensitive to aneuploidy, we show 484 that preventing aneuploidy exclusively in the brain is sufficient to rescue all the behavioral 485 defects previously observed. This brain protection not only rescued motor defects but also the 486 lifespan of the flies ecloded upon 72hs AEL heat-shock, suggesting that neural tissue is the most

508
RFP, respectively. The progeny was then heat shocked once at 37°C for 45min at the desired 509 developmental stage. The correct genotype larvae were selected based on the absence of the 510 "tubby" phenotype; the heat shocked "tubby" larvae were used as negative controls (control 511 HS). As genetic control we used the same genotypes for the induction of aneuploidy but without 512 performing the heat-shock.

513
To determine the proportion of adult eclosion, the crosses mentioned were raised in cages to 514 monitor the time of egg collection. After 6hs collection, the plates were removed from the 515 cages, the number of eggs counted and the plates were kept until larvae hatched. The plates 516 were then heat-shocked at 37°C for 45min at different larvae developmental time (~48hs AEL, 517 ~72hs AEL, ~96hs AEL and ~120hs AEL (± 6hs)) and placed in a new clean plastic cage. Once 518 they reached pupae stage ("yellow body") the pupae were gently removed with a wet brush and 519 separated in "tubby" (control HS) and "no tubby" phenotype (condition). The different batches 520 of pupae were placed over agar plates covered with two layers of absorbent paper to maintain 521 the humidity and counted. The plates with the pupae were kept at room temperature until flies 522 ecloded and the proportion of eclosion calculated.

547
Lifespan was measured at room temperature according to standard protocols. In brief, newly 548 ecloded animals (0 to 3 days) were collected (50 per genotype: "control", "Aneuploidy" and 549 "Aneuploidy + brain rescue"), and then placed in vials (up to 10 per vial), and transferred to

932
C-Quantification of Cohesive states of 3 rd instar larvae Nbs and epithelial cells from wing discs 933 following heat-shock. Insc-Gal4 protects the brain from cohesin loss, but has no effect in the 934 wing disc.