To gain more scientific understanding regarding Crayfish in a tropical climate since its first introduction in 1987, this research focused on perceptions of non-native species trading and invasive species as it relates to management. Pet store owners and farmers were interviewed. There are two Crayfish species in Thailand—Procambarus clarkii and Cherax quadricarinatus and the Crayfish trade is expanding due to high demand. The results demonstrated that respondents had a basic knowledge of Crayfish biology, primarily related to farming production. However, most respondents lacked understanding of the negative impact and invasiveness of Crayfish and, therefore, the potential risk of invasion. Respondents suggested: (1) controlling invasive species by the government using stringent law enforcement and prohibition of the release of nonnative species into the wild and harsher legal penalties for those who do not comply with the law; and (2) an educational campaign, especially on the negative impacts of Crayfish, should be publicized targeting both traders and pet owners. We further examined Crayfish biology and behavior using a laboratory experiment. The study considered the impact of different densities of Crayfish in captivity on survival rates and tolerance of Crayfish to different environmental conditions. The potential effects of native freshwater species as control agents for Crayfish were also determined. Results revealed that stocking Crayfish at a high density tended to achieve higher growth and survival rates than at a low density, as Crayfish reared at a high density were unable to exhibit agonistic (fighting) behavior in a very confined area. It was also revealed that Crayfish are sensitive to polluted water conditions (dissolved oxygen 2.55 ± 0.25 mg l−1; biological oxygen demand 8.45 ± 0.04 mg l−1) and could not survive there very long. This intolerance limits the potential of Crayfish to spread further to other water bodies in Thailand. Biological control agents demonstrated that Anabus testudineus, or the Climbing Perch, appeared to be the most effective predator and may help resist invasion of exotic Crayfish in the wild.
Thailand is home to thousands of nonnative plants and animals that are imported into the country for different purposes such as food and decorative pets. Crayfish are not native to Thailand and are among the species introduced there since 1987 (Kannasuit, 2006). Crayfish, such as Procambarus clarkii, are popular compared to other ornamental fauna, such as fishes and crabs, because their exoskeletons possess beautiful, bright colors that attract pet lovers. Crayfish are also cultured in Thailand for food production (Cherax quadricarinatus) in the north where the temperature is similar to their native habitats (Gherardi, 2006). However, continuous introductions of Crayfish and increasing demand from buyers has led to concerns that the aquarium trade can be a source of aquatic invasive species (Padilla and Williams, 2004; Reed, 2005; Chucholl, 2013a; Patoka et al., 2014; Faulkes, 2015). In addition, studies in many countries have indicated that Crayfish are a main cause of ecological problems such as Crayfish plague and biodiversity reduction (Alderman et al., 1990; Lynne et al., 2006; Gherardi and Acquistapace, 2007; Shane and Yeo, 2007; Jose et al., 2009; Francesco et al., 2009; Christina and Yeo, 2010).
Understanding species invasiveness and its behavior is crucial for predicting the possible impacts of invasive species and determining the best management practices. Improved understanding of the behavior of invasive species is important as it contributes to understanding their competitive ability and spread (Holway and Suarez, 1999). Crayfish behavior that may alter habitats and influence other species in shared habitats, includes interactive, agonistic (fighting), foraging, burrowing, and maternal-offspring (Mason, 1970; Stein and Magnuson, 1976; Correia and Ferreira, 1995). Another key factor in invasion success is the ability of species to reside in a wide range of habitats (Williamson, 1996; Chaichana and Sumpan, 2015). Crayfish can live under different environmental conditions (Distefano et al., 1991; Firkins, 1993) and, thus, making them successful invaders. For example, three common Crayfish (Orconectes rusticus, O. propinquus and O. robustus) in Canada have variable levels of tolerance to low pH conditions (pH 5.5–6.1) (Berrill et al., 1985) and three European Crayfish species (A. pallipes, A. italicus and A. torrentium) can tolerate a very low oxygen concentration for an extended period of time (Demers et al., 2006). There is also an expression in invasion biology when predators are absent in an invaded area and as a result, nonnative species can become dominant in a new infested area (Chaichana and Jongphadungkiet, 2012). Eiswerth and Johnson (2002) reported that the introduced species are more competitive than native species in their new settings, as the lack of predators allows them to colonize and alter ecosystems swiftly. Therefore, knowing the ability of native predators to control the spread of invasive species is essential.
Most studies of Crayfish invasion have been done in temperate countries and Crayfish are the largest and amongst the longest-lived invertebrate organisms in temperature environment (Alderman et al., 1990; Vorburger and Ribi, 1999; Lynne et al., 2006; Gheradi, 2007; Tetzlaff et al., 2011). In Thailand, although Crayfish were introduced to the country almost 30 years ago, scientific investigation regarding their behavior and other related information is still limited or unknown. Therefore, the objectives of this study were to evaluate the current Crayfish trade situation in Thailand and the views of traders on Crayfish and invasive species. Traders and buyers are important components of the invasion process and are engaged in the introduction phase of the process. In fact, Crayfish in Thailand have been intentionally released into the wild. We also examined the behavioral traits of the Crayfish (Procambarus clarkii) when reared at different densities. This study will lead to a better insight of the likely impact that Crayfish may pose to tropical freshwater resources. In addition, we investigated the response of Crayfish to a range of environmental conditions (dissolved oxygen and biological oxygen demand; BOD) to predict the potential of Crayfish to spread further to other areas. Lastly, biological control agents were studied using native predators to test whether native species can eradicate introduced Crayfish in the wild.
Crayfish trade in Thailand
In this study, we used questionnaires and interviews to obtain data from respondents (pet shop owners and farmers) regarding the Crayfish trade in Thailand and their general knowledge concerning Crayfish and invasive species. Questions regarding attitudes of respondents toward import of nonnative species as well as management and control of introduced species were also included in questionnaires. We randomly interviewed 10 owners/farmers (30% of 30 owners) who sell Crayfish as pets and food in and around Bangkok. Closed- and open-ended questionnaires were employed and divided into two parts (Part I: Demographic information of pet shop/farm and Part II: Knowledge of Crayfish and invasive species) (Tables 1 and 2).
Effects of different stocking densities on growth and survival of Crayfish
This study investigated whether different stocking densities increase or decrease the growth and survival of Crayfish. Juvenile P. clarkii (approx. 2.78 ± 0.75 g and 4.78 ± 0.42 cm) of similar size were raised in glass aquaria (12.5 × 25 × 17.5 cm with 75% clean water and no shelter provided) at three individuals per 5,470 cm2 (or one individual per 1,823 cm2) and at five individuals per 5,470 cm2 (or one individual per 1,094 cm2). Densities were chosen based on space availability in glass aquaria. Each treatment was in triplicate. Crayfish were fed by Hydrilla verticillata daily. Amounts of food given depended on the food requirement of Crayfish in each treatment. The measurement of the food consumption, Crayfish weight (on a digital balance with ±0.01 g precision), feed-conversion ratio (FCR), and Crayfish survival was carried out every week for seven weeks (end of the trial). Any interactive, agonistic, or aggressive behavior of Crayfish was also observed after food feeding.
Tolerance of Crayfish under different conditions of water quality
We determined the tolerance of Crayfish cultured in different types of water quality to test our assumption that being classified as an invasive species, Crayfish are adaptive and tolerant of a broad range of environmental conditions. We used P. clarkii with an average total length of 2.78 ± 0.7 cm and weight of 4.78 ± 0.42 g and raised them in glass aquaria (12.5× 25× 17.5 cm) at a density of one Crayfish per aquarium and it was run was in triplicate (nine Crayfish in total). Clean, moderately polluted, and polluted water were used in this experiment and data (survival rate) were collected for four weeks. Tap water was used as clean water (initial dissolved oxygen 4.80 ± 0.47 mg l−1 and biological oxygen demand 1.15 ± 0.02 mg l−1) and moderately polluted (initial dissolved oxygen 2.55 ± 0.25 mg l−1 and biological oxygen demand 8.45 ± 0.04 mg l−1) and polluted water (initial dissolved oxygen 0.59 ± 0.02 mg l−1 and biological oxygen demand 14.15 ± 0.03 mg l−1) was collected from ponds around Kasetsart University (Table 3). We used a multi-parameter analyzer (Consort 933) to measure the temperature, pH, dissolved oxygen (DO), total dissolved solid (TDS) and conductivity. Concentrations of ammonium, soluble reactive phosphorus (SRP) and BOD5 were also analyzed weekly based on standard methods of the examination of water and wastewater until the end of experiment. No oxygen supply was added to any treatment. The initial water quality conditions were compared with water quality conditions at the end of experiment.
Biological control alternatives
We investigated the biological control of Crayfish (P. clarkii) by using native predatory fishes and the giant Malaysian prawn (Macrobrachium rosenbergii), the largest freshwater prawn species in Thailand. The predatory fish species selected in this experiment were Channa striata (common snakehead), Pangasianodon hypophthalmus (iridescent shark), Osphronemus goramy (giant gourami) and Anabas testudineus (climbing perch). We used various native predators to determine whether different fauna had a different degree of impact on the survival of the Crayfish. In each treatment, three Crayfish (each weighing 5 g) were released into each experimental aquarium (12.5× 25× 17.5 cm) as prey and predator species was subsequently put in the aquarium. All trials were repeated in triplicate for each species of predators and lasted for seven weeks. The percentage destructive rate of Crayfish by predator was calculated based on the remaining numbers of Crayfish.
For statistical analysis, we used Excel 2007 and SPSS Statistic 16.0 software packages. Descriptive statistics (percentage) were used for the demographic information and questionnaire answers of respondents. One-way ANOVA was used to determine the differences between the growth rates and food consumption of Crayfish reared in different densities. We used Student's t test to evaluate the differences of Crayfish survival in biological control experiment and Tukey HSD was used for multiple comparisons of water quality among treatments. These studies were conducted during 2013–2014.
Crayfish trade in Thailand
Eighty percent of respondents were male and 20% were female. The majority (60%) of respondents held a bachelor degree and the remainder finished high school. Most respondents (90%) were trading in Crayfish as an additional source of income; whereas, the remainder (10%) relied mainly on the Crayfish trade. Some 60% of the respondents had been in this business less than five years and 40% had owned shops/farms for more than five years. Furthermore, 100% of the Crayfish available in the market were cultured in Thailand – Procambarus clarkii for the pet trade and Cherax quadricarinatus for consumption. The best-selling Crayfish in the market was P. clarkii followed by C. quadricarinatus. The average age range of regular customers was between 21 and 30 years old.
The result of interviews regarding the general Crayfish knowledge of respondents (Part II) is presented in Table 1. Most respondents had a basic understanding of Crayfish as they answered most questions correctly. However, when respondents were asked whether Crayfish could cause Crayfish plaque, no one answered this question correctly (100%). In addition, nine out of 10 people did not know that Crayfish in Thailand are regarded as a potential invasive species by the Office of Natural Resources and Environmental Policy and Planning (Table 2).
In Part II of the questionnaire, five open-ended questions were used to appraise respondents' attitudes (pet-shop owners) toward the introduction of invasive species to Thailand. Most respondents tended to have similar answers regarding introduction of nonnative species in Thailand that it was due to the demand from a buyer either as pets or food consumption. Respondents also believed that the main cause of the presence of exotic species in the wild was the result of intentional release when the raised species become too big or produce too many offspring. To control and prevent impacts posed by invasive species, most respondents answered that it was unlikely as long as there are people who want to buy and raise exotic species. Detailed answers are presented in Table 2.
Effects of stocking densities on growth and survival rates of Crayfish
It was revealed that Crayfish raised at a high density tended to consume more food than at a low density. The mean food consumption of Crayfish at low (28.76 ± 12.48 g) and high (41.28 ± 2.57 g) densities was not significantly different (F1, 4 = 2.88, P = 0.164). Furthermore, stocking Crayfish at a high density resulted in a relatively higher growth (average final weight 4.56 ± 1.66 g) compared with stocking Crayfish at a low density (average final weight 3.35 ± 1.66 g) (F1, 4 = 1.560, P = 0.280). The FCR of Crayfish stocking also corresponded well with the food consumption of Crayfish. The FCR of Crayfish at low and high densities was 21.09 ± 5.47 and 16.26 ± 1.47, respectively (F1, 4 = 2.179, P = 0.214). The results of the survival rate showed that high stocking densities of Crayfish had a higher survival rate (80%) than at lower stocking densities (66.67%).
Tolerance of Crayfish in different conditions of water quality
At the end of the experiment, Crayfish did not survive in moderately polluted and polluted water treatments (Table 4). In contrast, Crayfish reared in clean water could survive up to three weeks without any oxygen supply. The result also indicated that most water quality conditions especially DO and BOD5 were more deteriorated after the experiment. Statistical analysis was showed in Table 4.
Biological control alternatives
The most effective predatory fish of Crayfish was Anabas testudineus as indicated by statistical difference between survival percentages between Anabas testudineus and Crayfish (P = 0.006) (Figure 1). Osphronemus goramy could also reduce a number of Crayfish but no statistical difference was found (P = 0.11). Other predators—Osphronemus goramy, Pangasianodon hypophthalmus, Macrobrachium rosenbergii and Chana striata—had relatively lower impacts on Crayfish survival, respectively. In fact, C. striata (P < 0.01), P. hypophthalmus (P = 0.084) and M. rosenbergii (P = <0.01) had lower survival rates than that of Crayfish. Interactive and agonistic (fighting) behavior of Crayfish was detected. In addition, we observed that both prey and predators had wounds as a result of fighting. Fish were attacked by Crayfish at their fins (tail, dorsal and pectoral fin) and some wounds appeared on the bodies of fish. Predator fish also attacked Crayfish at their eyes, antenna, chelipeds and legs.
Crayfish trade in Thailand
The Crayfish trade in Thailand has steadily expanded due to the high demand in both the ornamental and food sectors especially during the past five years. Two main species (Procambarus clarkii and Cherax quadricarinatus) are cultured in many parts of Thailand such as central, eastern and northern regions and then distributed to markets in Bangkok. The central Crayfish market for pets is Chatuchak ornamental fish market. According to our interviews, respondents have a basic knowledge of Crayfish biology and culturing as these are crucial for farming production. However, when we asked respondents deeper questions regarding the negative impact of Crayfish, such as Crayfish plaque or the invasiveness of Crayfish, most respondents did not know and, therefore, this could lead to a potential risk of invasion. This risk comes from the fact that nonnative species are released into the wild because people are unaware or may think that nonnative species cause no harm to ecosystems. This finding is consistent with Padilla and Williams (2004), Patoka et al. (2014) and Nunes et al. (2015) who stated that the aquarium trade is an important source for species to invade aquatic habitats and can pose a dangerous threat. Respondents did not anticipate the consequences of introduced species and some important knowledge in relation to negative impacts of Crayfish was still limited. Therefore enhancing awareness and education on the problem of invasive species is necessary (Yan et al., 2001; Reed, 2005) especially to farmers, traders, pet owners, and the general public.
The study also disclosed that most respondents agreed in general with the control of non-indigenous species using measures such as stringent law enforcement and prohibition of releasing nonnative species in the wild (Kerr et al., 2011; Ameden et al., 2007; Smith et al., 2009). In Thailand, farmers have to register with Department of Fishery for Crayfish farming and have to follow farming regulations under the new law released in 2016. However, there is still a limitation for officers to monitor whether all Crayfish farmers had registered and followed regulations strictly. Respondents also supported the idea of a legal penalty for those who do not comply with the law. Most respondents proposed that buyers should know species that they want to purchase, including life history and potential risk to ecosystems. In practice, when exotic plants and animals are no longer wanted (too big, eat too much, produce too many offspring), pet owners are unaware of the impact on ecosystems when they release them into nature (Kerr et al., 2011). In fact, the Department of Fishery has a campaign that pet owners can return unwanted exotic freshwater species at Provincial Fishery Stations and get a native species in return. This campaign reduces introduction of invasive species into natural habitats. Similarly, there is one successful event in Florida called “Nonnative Amnesty Day Event” that is organized annually by the Florida Fish and Wildlife Conservation Commission (FWC) in cooperation with local zoo. The main purpose of this event is to allow pet owners to surrender their exotic pets and animals will then be examined and given to qualified adopters. This event is also a platform to educate children and pet owners about exotic pets.
It is also interesting that, from the respondent's point of view, the control of the introduction of invasive species is almost impossible as long as the species is still wanted by buyers. As traders, they have to provide species to meet the demand of buyers. Therefore, this loop between trader and buyer seems never ending and, consequently, results in the continuous import of nonnative species into the country. The solution proposed by respondents was that government agencies must strictly control the import of invasive non-native species at borders. Public policy especially pre and post-border inspection is important since it has to deal with invasive species and issues such as disease control and eradication as well as restricting the movements of invasive species (Sumner et al., 2005). In fact, by law exotic animals that are imported to Thailand via airport, port and ground transportation have to be monitored for diseases at Fish Inspection Office and Animal Quarantine Station and they must not be in the prohibited species list. The Royal Thai government has a strict law that any species listed on a prohibited aquatic species list in the Royal Decree (applies to 93 animals and plants) cannot be imported into Thailand (Department of Fisheries, 1990).
Effects of stocking densities on growth and survival rates of Crayfish
Different stocking densities (low and high) tended to have biological effects on the food consumption, growth and survival of Crayfish. At a high density, Crayfish appeared to consume more food (.89 times more) than at a low density, although no significant difference was found. This can be attributed to the fact that the more Crayfish, the more food consumed and, thus, leading to a gain in weight. From direct observation, interactive, agonistic and aggressive behavior of Crayfish raised at low and high densities was also revealed. At a high density, Crayfish did not show strong agonistic (fighting) behavior because of their inability to fight in a very confined area. Generally, spread of Crayfish occurs when it advances toward another individual and raises its claws ready to fight or attack (Garvey et al., 1994). In contrast, at a low density or in a more open area, Crayfish exhibited more and intensely aggressive fighting and spent more energy in agonistic behaviors possibly as a result of protecting their territory against other Crayfish intruders or competition for food. Therefore, this led to the lower survival of Crayfish at a low density. However, our results were different from a previous study that as density of Crayfish increased, mean final length and weight decreased (Ramalho et al., 2008). It could be stated that Crayfish used in this study were larger in size (2.78 g) than in the previous study that used very small juvenile (0.037 g). Accordingly, larger Crayfish show clearer agonistic behavior for food competition and territory.
Tolerance of Crayfish in different conditions of water quality
One of the important traits of an invasive species is being adaptive or highly tolerant in a harsh environment (Davidson et al., 2011). We tested whether Crayfish were tolerant to deteriorated water quality and, if so, there is a high chance of Crayfish to establish and spread further. In this study, when raised in moderately polluted and polluted water, we found that Crayfish died almost immediately due to insufficient oxygen concentrations and increased BOD5. Deterioration of water quality could be the result of leftover food and waste produced by Crayfish (during experiment). In contrast, Crayfish survived only in the clean-water condition. However, other European Crayfish species, such as Austropotamobius pallipes and A. torrentium, are more tolerant to very low levels of oxygen concentrations (Demers et al., 2006). Thus, different species may have a different degree of tolerance to harsh environmental conditions. In Thailand, Crayfish have already been present in the Pra Prong reservoir, Sra Keaow province (Wanjit and Chaichana, 2013) as a result of intentional introduction. Since Crayfish are found in a good environmental habitat mentioned above and are sought for food consumption, their spread could then be controlled by fishermen. In contrast, other nonnative species, such as Amazon sailfin catfish (Pterygoplichthys pardalis), have spread very quickly across the country since they are not consumed by the Thai people (Chaichana and Jongphadungkiet, 2012) and they have broader environmental tolerances.
Biological control alternatives
Alternative control of Crayfish using local fauna was determined. Anabas testudineus was the most effective control agent and reduced Crayfish numbers significantly. A. testudineus attacked the eyes of Crayfish from the back first and then removed walking legs until the Crayfish could not defend against predators. This attacking behavior of A. testudineus was also observed in Osphronemus goramy. However, O. goramy moved more slowly than A. testudineus and, therefore, was a less effective predator on Crayfish. In addition, Pangasianodon hypophthalmus and Channa striata were less effective than other predators. They were finally attacked by Crayfish, especially their caudal fins and bodies, until they could not swim away and were eaten. Macrobrachium rosenbergii was also attacked by Crayfish because they possess smaller claws and thinner shells. Crayfish are aggressive, territorial animals and attacked M. rosenbergii by destroying claws and walking legs until the M. rosenbergii could not move. Thus, it could be stated that smaller predators are likely to be attacked by Crayfish. This is consistent with reports that Crayfish having larger chelae were better able to defend themselves against predators (Stein and Magnuson, 1976). In other countries, several studies have also showed that native predators can impact Crayfish. For example, in Italy, the native European Eel, Anguilla anguilla, is an effective predator of P. clarkii because of its benthic feeding preference and ability to tolerate partially deoxygenated waters, properties that match the lifestyle of Crayfish and the typical habitats they occupy (Nystrom and Perez, 1998). A study in a northern Wisconsin lake also showed that largemouth bass (Micropterus salmoides) can cause high mortality of small invading Crayfish (Aquiloni et al., 2010). For practical implications in Thailand, natural communities with abundant native predators such as A. testudineus and O. goramy may resist invasion of exotic Crayfish as biotic resistance can play a major role to keep out invasive species (deRivera et al., 2005; Carlsson et al., 2011). An example of biotic resistance showed that dense populations of native sunfishes (Lepomis) were capable of eliminating juvenile Crayfish population in Wisconsin lakes (Tetzlaff et al., 2011).
In conclusion, this study revealed the perception of farmers and pet shop owners regarding Crayfish and the impact of invasive species in general. The results showed that most respondents were knowledgeable about Crayfish, except for the impact on ecosystems. Respondents also proposed, that to control and eradicate invasive species, the government agency should promote an educational campaign to the general public and should be strict in applying the law. The results also demonstrated that different Crayfish densities affected the amount of food consumption, growth and survival of Crayfish. Crayfish were not tolerant to polluted water and therefore were confined to clean water. Lastly, biological control agents proved that the use of A. testudineus to control introduced Crayfish was promising.
We would like to thank the Department of Environmental Technology and Management, Faculty of Environment, Kasetsart University, Thailand for partial research fund.