**6. What is the extent of the geographic spread of the alien pathogen, TSV?**

Diseases are worldwide top issues and challenges in shrimp aquaculture based on the survey of global aquaculture alliance from 2016 to 2017 [60]. Modern shrimp farming is, in a way, shaped by viral disease outbreaks in the nineties and early 2000s [61]. On the other hand, Itsathitphaisarn et al. [62] reiterated that viral pathogens pose a primary threat to global shrimp aquaculture. According to Lightner and Redman [63], there are about 20 viral pathogens that can cause serious epizootics in penaeid shrimp. In the Philippines, major viral pathogens affecting the shrimp aquaculture include white spot disorder infection (WSSV), monodon baculovirus (MBV), irresistible hypodermal and hematopoietic rot infection (IHHNV), hepatopancreatic parvovirus (HPV), yellow head infection (YHV), and Taura disorder infection (TSV) [61].

A disease caused by TSV was first described from Ecuador in the early 1990s. Lightner [64] and Lightner [65] reported that the disease outbreaks caused catastrophic losses with cumulative mortality rates of 60 to >90% in pond-cultured shrimp. The principal host species for TSV are the *P. vannamei* and the *P. stylirostris*, and it has been documented in all life stages (i.e., post-larvae, juvenile, adults) of *P. vannamei* except in eggs, zygote, and larvae [66]. TSV is a particularly virulent pathogen of *P. vannamei*, and it can infect several other

shrimp species including *P. monodon*, *P. aztecus*, *P. duorarum*, *Litopenaeus setiferus*, *L. stylirostris*, *Marsupenaeus jaiponicus*, *Macrobrachium rosenbergii*, *Metapenaeus ensis*, *Fenneropenaeus chinensis*, and *L. schmitti* [61].

According to Wertheim et al. [67], TSV is now widely distributed in the shrimpfarming regions of the Americas, Southeast Asia, and the Middle East. Additionally, evidence showed that TSV is present in natural populations of *P. vannamei* in Central America such as Mexico and Ecuador and may be elsewhere [66, 68]. The international trade of live shrimp resulted in a rapid spread of TSV in the Americas and Asia [69]. TSV was introduced to Asia in 1998 by careless importation of shrimp stocks for aquaculture but has not been reported to cause problems with local crustacean species [70]. Recently, Thitamadee et al. [71] reported TSV has become innocuous due to the widespread use of highly tolerant specific-pathogenfree (SPF) stocks of *P. vannamei* that dominate production.

In the Philippines, there was no documentation on the introduction of *P. vannamei* from Panama into Iloilo in the 1970s and from Hawaii in 1990, and it was not known whether the exotic species introduced any new pathogens [18]. As of 2015, there was no documented report regarding TSV presence in the Philippines [61] and have yet to be detected as stated in the NACA, OIE, and FAO [72] quarterly animal disease report of 2018. However, this will not justify that TSV is not present in the Philippines because there was no study conducted on the detection of the viral disease since the introduction of *P. vannamei* until the importation ban was lifted in 2007 up to 2018. Rosario and Lopez [36] reported that even with the strict implementation of the BFAR formulated FAO 207 series of 2001 which further strengthened FAO 189 series of 1993 which among others prohibit the importation of exotic shrimps and strict surveillance in airports, traders were finding other ways in bringing the illegal shrimp inside the Philippines without passing through the airports. Shrimp Importation, Monitoring, and Surveillance (SIMS) team spearheaded six major confiscations in late 2002 up to 2003 and reported around 700

*Spatial Variability in Environmental Science - Patterns, Processes, and Analyses*

usually takes around 1.5 years to complete the whole life cycle. The matured shrimp females spawn their eggs in the offshore waters [54], while fertilization occurs in the external environment [53]. However, the maturity of the shrimp escaped from farms to natural environments is an important factor in determining their ability to establish a feral population [22]. A study has been conducted to compare the histology of gonads of wild-caught and captive *P. vannamei* of known ages [55]. Captive individuals could develop mature gonads at 11 months after post-larvae 15 (ovaries contained 50% mature oocytes; testes contained 80% mature sperm cells). The result of the study showed that they did not find sexually mature individuals in the wild although some wild-caught males larger than 19 g contained a small percentage of mature sperm cells. However, the authors cannot conclude that escapees can establish a feral population because the study might have under-sampled sexually mature individuals due to inappropriate sampling sites and timing. This issue

remains important for further investigation. A monitoring program in offshore areas may provide opportunities for us to obtain sexually mature individuals. Likewise, Panutrakul et al. [44] found no evidence that the shrimp present in the wild could reach maturation in the Bangpakong River although gonadal development has been observed. Moreover, Wakida-Kusunoki et al. [35] argued that it was not possible to find evidence of *P. vannamei* becoming established in the zone of the Mexican coast of the Gulf of Mexico. The low frequency of *P. vannamei* encounters in the monitoring program of artisanal shrimp fishing in lagoon system and the negative presence of the shrimp in surveys of the commercial shrimp catches of coastal waters near to the mouth of this lagoon indicate the absence of an established population. They suggested further sampling and monitoring are required to find evidence that confirms the establishment of a population of the shrimp in the Southern Gulf of Mexico. According to Panutrakul et al. [44], natural reproduction would require released adults and a high probability that the mature adults could find mates. In the Philippines, the population of *P. vannamei* in the wild therefore already exists, although it is still uncertain if this population is now breeding [29] until this time.

**5. Can** *P. vannamei* **potentially compete with native shrimp species?**

Studies have shown that there is a potential risk of a negative impact of the introduced Pacific whiteleg shrimp *P. vannamei* on native species and the invaded ecosystems [21, 30, 44]. An alien species like *P. vannamei* could potentially interact with local species through food competition, either by exploitative or interference

Recently, Chavanich et al. [56] conducted a laboratory assessment of feedingbehavior interactions between the introduced *P. vannamei* and five native shrimps plus a crab species in Thailand. Results showed that the shrimp was nonselective with respect to the palatability of the five native shrimps as food. The shrimp was behaviorally dominant when competing for food one-on-one with the native shrimp species. According to Gamboa-Delgado et al. [57], the shrimp is an opportunistic feeder that can adapt well to changes in diet composition. Though laboratory studies could not represent the feeding interactions under field conditions, the non-native shrimp could become a serious threat to native shrimps when the frequency of escapes is increasing and when they begin to reproduce successfully. One of the key factors influencing the success of invading species is propagule pressure or total quantity [58, 59]. Increasing the propagule pressure may enhance the foundation of an invasive population [58]. In Bangpakong estuary, increased frequency of encountering the shrimp is reflecting an increase in propagule pressure because the frequency of escapes is increasing [30]. However, Chavanich et al. [56] suggested

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competition [21].

have illegal *P. vannamei* farms operating in Luzon. Moreover, despite all the efforts of the BFAR, the culture industry for the shrimp in the Philippines has grown and may produce as much as 5000 metric tons in 2003 [29]. In fact, Philippines was one of the main producer countries of *P. vannamei* based on FAO fishery statistics in 2006 [73]. According to de la Peña [74], there is always the possibility of contamination with TSV if the illegal shipments of the shrimp remain uncontrolled, and this proves recently as the study of Vergel et al. [75] reported for the first time the presence of TSV in *P. vannamei* in the Philippines using morphological and molecular techniques. BLASTn search results showed that the TSV sequences have very high sequence similarity at 86–100% with TSV viral isolates from other countries (Taiwan, Thailand, Venezuela, the USA, Colombia, and Belize). The detected prevalence rates of the study comprise a small sample population with limited areas in the Philippines, namely, Bulacan (33%), Batangas (47%), Bohol (7%), and Cebu (13%). The authors suggested further testing in other sites in the country and implementation of mitigation methods and policies to prevent further spread of the viral disease. Likewise, detection of TSV in the wild is also important to be conducted. In the study of Barnette et al. [76] using PCR and immunological analyses, results suggested that TSV has already spread into the Bangpakong River and the Gulf of Thailand. The viral disease appeared to be more widespread in dry seasons than wet seasons. The presence of TSV has been detected in *P. monodon* adults; local shrimp species of the Bangpakong River such as *P. monodon*, *P. semisulcatus*, *P. merguiensis*, *M. brevicornis*, *M. affinis*, *M. tenuipes*, *Parapenopsis hungerfordi*, and *M. rosenbergii*; two other species belonging to the Family Caridea; and wild-caught *P. vannamei* including green mussel *Perna viridis*, blue swimming crab *Portunus pelagicus*, and Asian sea bass *Lates calcarifer*. TSV was detected in *L. setiferus* and *Farfantepenaeus aztecus* in Laguna Madre [77] and *L. schmitti* in Maracaibo lagoon, Venezuela [78].
