Two eyestalk ablation protocols (i.e., cauterization and surgery approaches) were performed on anesthetized female crabs. The eyestalk ablation of mud crabs hastened the ripening of ovaries without decreasing the survival rate.
Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the induction of ovarian maturation is important to meet the consumer demand for mature mud crabs and hasten seed production. Eyestalk ablation is an effective tool to enhance ovarian maturation in mud crabs. However, there is no standard protocol for the eyestalk ablation of mud crabs. In this study, two eyestalk ablation techniques are described: cauterization (the use of hot metal to ablate the eyestalk of an anesthetized crab) and surgery (the removal of the eyestalk using surgical scissors). Before eyestalk ablation, sexually mature females (CW > 86 mm) were anesthetized using an ice bag (−20 °C) with seawater. When the water temperature reached 4 °C, the ice bag was removed from the water. Flowing seawater (ambient temperature: 28 °C) was used for recovery from the anesthesia immediately after eyestalk ablation. Mortality did not occur during or after the process of eyestalk ablation. The eyestalk ablation protocol presented here accelerated the ovarian maturation of the mud crabs.
All four mud crab species belonging to the genus Scylla are commercially important crustacean species in aquaculture1,2. The growth of crustaceans, including mud crabs, and their transformation from the pre-mature (sub-adult or pubertal) phase to the sexually mature (adult) phase occur through a molting process that involves the periodic shedding of older and smaller exoskeletons. Carapace width (CW), chelipeds, and abdominal flap morphologies are widely used to determine the sexual maturity of Scylla spp.3,4,5. The process of molting is regulated by the action of various hormones and requires a huge amount of energy6. In addition to the normal molting process, the loss of limbs, either voluntarily or induced by external factors, expedites the molting of crabs without affecting their survival rate7,8,9. Therefore, limb autotomy is commonly used for molt induction in the soft-shell mud crab farming industry7,9.
Unilateral or bilateral eyestalk ablation is mostly popular in freshwater prawn and marine shrimp for gonad maturation and seed production10,11,12,13. Common eyestalk ablation techniques in crustaceans include the following: (i) ligation at the base of the eyestalk using a string14,15; (ii) cauterization of the eyestalk using hot forceps or electrocautery devices16; (iii) removal or direct pinching of the eyestalk to leave an open wound12; and (iv) removal of the eyestalk contents through incision after slicing the distal portion of the eye with a razor17. The eyestalk X-organs are important endocrine organs in crustaceans as they regulate crustacean hyperglycemic hormones (CHH), molt-inhibiting hormones (MIH), and vitellogenesis-inhibiting hormones (VIH)6,18,19,20,21,22. Eyestalk X-organs (or the sinus gland complex) synthesize and release gonad-inhibiting hormones (GIH), also known as vitellogenesis-inhibiting hormones (VIH), belonging to the neuropeptide hormone family6. Unilateral or bilateral eyestalk ablation reduces GIH synthesis, resulting in the dominance of stimulating hormones (i.e., gonad stimulating hormones, GSH) and the acceleration of the ovarian maturation process in crustaceans23,24,25,26. Without the influence of GIH after eyestalk ablation, female crustaceans devote their energy to ovary development27. It has been found that unilateral eyestalk ablation is sufficient for the induction of ovarian maturation in crustaceans11 and that the ablated eyestalk of shrimps and crabs can regenerate after several moltings28. There are four ovarian development stages recorded in Scylla spp.: i) immature (stage-1), ii) early maturing (stage-2), iii) pre-maturing (stage-3), and iv) fully mature (stage-4)29,30. The immature ovarian stage is found in immature females. After pubertal molting and mating, the immature ovary starts developing and finally matures (stage-4) before spawning31.
An eyestalk ablation protocol is essential for mud crab broodstock development and seed production. In the global food market, mature mud crabs with fully mature ovaries (stage-4) rather than crabs with higher muscle content are preferred by consumers and, thus, have a higher commercial value, even higher than large males. There is no complete protocol for the eyestalk ablation of mud crabs. The eyestalk ablation protocol in this work minimizes stress by using fully anesthetized crabs and minimizes physical injury to personnel from crab bites. This protocol is easy and cost-effective. Here, we present a protocol for the eyestalk ablation of Scylla spp. that can induce the maturation of the gonad. Two techniques of eyestalk ablation (cauterization and surgery) were tested and their efficiencies compared based on the gonadal development rate of female mud crabs.
This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia. The sacrifice of the experimental samples was done according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Sexually pre-mature female mud crabs (orange mud crab S. olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.
1. Sample collection and disinfection
Figure 1: Abdominal morphology of female mud crabs used to identify the sexual maturation stages. Please click here to view a larger version of this figure.
2. Acclimatization
3. Induced molting for sexual maturity
4. Anesthesia
5. Eyestalk ablation
6. Post-anesthesia care
7. Observation of ovarian maturation
Gonad maturation
Creamy white ovarian tissues (immature ovaries, stage-1) were found in 100% of the dissected females (n = 6) before performing the eyestalk ablation (Figure 2). The gonad maturation rate of the eyestalk-ablated female crabs (n = 63; 31 females with the cauterization technique and 32 females with the surgery technique) was higher compared to female crabs that were not subjected to eyestalk ablation (n = 31) after 30 days of individual rearing (Figure 3). The highest percentages of pre-maturing ovaries (stage-3) were found in the eyestalk-ablated female crabs (Figure 3; both cauterization and surgery techniques), and a one-way analysis of variance (ANOVA) showed significant differences (p < 0.05) among the experimental female crabs' ovarian maturation stages (Table 1). The control group had a higher prevalence of immature female crabs compared to the cauterization and surgery treatment groups (Tukey's HSD test, p < 0.001). The cauterization and surgery treatments did not show any significant differences in terms of the percentage of female crabs across all maturation stages (Tukey's HSD test, all p > 0.1). Both the cauterization (Tukey's HSD test, p = 0.004) and surgery (Tukey's HSD test, p = 0.006) treatments had significantly higher percentages of pre-maturing stage 3 female crabs than the control treatment, and only cauterization and surgery treatments were able to produce stage-4 female crabs from an immature stage within 30 days after treatment (Table 2).
Figure 2: Four ovarian maturation stages of female mud crabs. Differences in the coloration and volume of the ovaries between the stages are clearly pointed out by the black arrows. Please click here to view a larger version of this figure.
Figure 3: Ovarian maturation stages of female crabs subjected to eyestalk ablation (surgery and cauterization) and control after the 30 days rearing period (n = 94). The error bar represents the standard deviation. The superscript letters indicate significant differences between treatments at each maturation stage at p < 0.05. Please click here to view a larger version of this figure.
Maturation stage | Sum of Squares | df | Mean Square | F | P | |
Immature (stage-1) | Between Groups | 3755.556 | 2 | 1877.778 | 169 | <0.001 |
Within Groups | 66.667 | 6 | 11.111 | |||
Total | 3822.222 | 8 | ||||
Early maturing (stage-2) | Between Groups | 1355.556 | 2 | 677.778 | 8.714 | 0.017 |
Within Groups | 466.667 | 6 | 77.778 | |||
Total | 1822.222 | 8 | ||||
Pre-maturing (stage-3) | Between Groups | 4688.889 | 2 | 2344.444 | 17.58 | 0.003 |
Within Groups | 800 | 6 | 133.333 | |||
Total | 5488.889 | 8 | ||||
Fully matured (stage-4) | Between Groups | 822.222 | 2 | 411.111 | 9.25 | 0.015 |
Within Groups | 266.667 | 6 | 44.444 | |||
Total | 1088.889 | 8 | ||||
Note: The mean difference is significant at p = 0.05 level. |
Table 1: Comparison of eyestalk-ablated (cauterization and surgery) and control female crabs'gonadal maturation stages following a one-way ANOVA test. The mean difference was significant at p = 0.05.
Maturation stage | Treatment | Comparison treatment | P |
Immature (stage-1) | Cauterisation | Surgery | 1 |
Cauterisation | Control | <0.001 | |
Surgery | Control | <0.001 | |
Early maturing (stage-2) | Cauterisation | Surgery | 0.129 |
Cauterisation | Control | 0.014 | |
Surgery | Control | 0.232 | |
Pre-maturing (stage-3) | Cauterisation | Surgery | 0.934 |
Cauterisation | Control | 0.004 | |
Surgery | Control | 0.006 | |
Fully matured (stage-4) | Cauterisation | Surgery | 0.109 |
Cauterisation | Control | 0.012 | |
Surgery | Control | 0.237 | |
Note: The mean difference is significant at p = 0.05 level. |
Table 2: Post-hoc Tukey's HSD test of the differences between eyestalk-ablated (cauterization and surgical) and control female crabs' gonadal maturation stages. The mean difference was significant at p = 0.05.
Survival rate
The average survival rate of the eyestalk-ablated female crabs was 95.45% ± 4.98% (mean ± standard deviation) in the 30 days rearing period. No mortality occurred within the first 7 days after eyestalk ablation and handling. During the 30 days rearing period after eyestalk ablation, the mortality rate was not significantly different (Kruskal-Wallis test, p = 0.67) between the treatments. The molting success rate of the limb-autotomized female crabs was 80% ± 2.86% (n = 115).
This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.
The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Anesthesia via immersion in 2-phenoxyethanol was used as it is comparable to the use of tricaine methanesulfonate (MS-222) in arthopods but cheaper and does not require the use of additional buffer40. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).
The gonad maturation of the mud crab is regulated by hormones21,41,42. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).
The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,43. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,43,44. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,45. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique takes time for the wound to heal and this would allow for chance of infection. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,46,47.
Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.
Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.
Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,48. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,49,50. In addition, future research on pain assessment following eyestalk ablation on mud crabs is recommended to highlight the change in behaviours associated with pain and stress, as evident in freshwater prawn Macrobrachium americanum51.
Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.
Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,52,53. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,53. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.
The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.
The authors have nothing to disclose.
This study was supported by the Ministry of Education, Malaysia, under the Higher Institution Centre of Excellence (HICoE) program, Malaysia, accredited to the Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu (Vot No. 63933 & Vot No. 56048). We acknowledge the support of Universiti Malaysia Terengganu and Sayap Jaya Sdn. Bhd. via the Private Partnership Research Grant (Vot. No. 55377). An adjunct Academic Fellow position from Universiti Sains Malaysia to Khor Waiho and Hanafiah Fazhan is also acknowledged.
Aeration tube | Ming Yu Three | N/A | aquarium and pet shop |
Airstone | Ming Yu Three | N/A | aquarium and pet shop |
Autoclave machine | HIRAYAMA MANUFACTURING CORPORATION | N/A | MADE IN JAPAN |
Bleaching powder (Hi-Chlon 70%) | Nippon Soda Co.Ltd,Japan | N/A | N/A |
Blow torch | MR D.I.Y. Group Berhad | N/A | N/A |
Circular tank (32L) | BEST PLASTIC INDUSTRY SDN. BHD. | N/A | N/A |
Cotton hand gloves (thick) | MR D.I.Y. Group Berhad | N/A | N/A |
Cotton towel | MR D.I.Y. Group Berhad | N/A | N/A |
Digital thermometer | Hanna Instrument | HI9814 | Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814 |
Digital Vernier Caliper | INSIZE Co., Ltd. | N/A | |
Dissecting tray | Hatcheri AKUATROP | N/A | Research Center of Universiti Malaysia Terengganu |
Dropper bottle/Plastic Pipettes Dropper | Shopee Malaysia | N/A | N/A |
Ethanol 70% | Thermo Scientific Chemicals | 033361.M1 | Diluted to 70% using double distilled water |
Fiberglass tank (1 ton) | Hatcheri AKUATROP | N/A | Research Center of Universiti Malaysia Terengganu |
Fine sand | N/A | N/A | collected from Sea beach of Universiti Malaysia Terengganu |
First Aid Kits | Watsons Malaysia | N/A | N/A |
Flat head nickel steel metal rod (Screw driver) | MR D.I.Y. Group Berhad | N/A | N/A |
Formaldehyde | Thermo Scientific Chemicals | 119690010 | |
Gas cylinder (butane gas) for blow torch | MR D.I.Y. Group Berhad | N/A | N/A |
Gas lighter gun (long head) | MR D.I.Y. Group Berhad | N/A | N/A |
Glass beaker (100 mL)) | Corning Life Sciences | 1000-100 | |
Ice bag | Watsons Malaysia | N/A | N/A |
Perforated plastic baskets | Eco-Shop Marketing Sdn. Bhd. | N/A | N/A |
PVC pipe 15mm | Bina Plastic Industries Sdn Bhd (HQ) | N/A | N/A |
Refractometer | ATAGO CO.,LTD. | ||
Refrigerator | Sharp Corporation Japan | N/A | Chest Freezer SHARP 110L – SJC 118 |
Scoop net | MR D.I.Y. Group Berhad | N/A | |
Seawater | Hatcheri AKUATROP | N/A | Research Center of Universiti Malaysia Terengganu |
Siphoning pipe | MR D.I.Y. Group Berhad | N/A | N/A |
Spray bottle | Mr. DIY Sdn Bhd | N/A | N/A |
Stainless surgical forceps | N/A | N/A | N/A |
Stainless surgical scissors | N/A | N/A | N/A |
Submersible water pump | AS | N/A | model: Astro 4000 |
Tincture of iodine solution (Povidone Iodine) | Farmasi Fajr Sdn Bhd | N/A | N/A |
Tissue paper | N/A | N/A | |
Transparent plastic aquarium | Ming Yu Three | N/A | aquarium and pet shop |
Waterproof table | Hatcheri AKUATROP | N/A | Research Center of Universiti Malaysia Terengganu |