The First Report of a Water Mite Unionicola (Trombidiformes: Unionicolidae) Infection in Filopaludina spp. (Gastropoda: Viviparidae) from Thailand with a Description of a New Species of Unionicola (Polyatax) kasetbangkhenensis sp. nov.

Two species of gastropods, Filopaludina sumatrensis polygramma and F. martensi martensi, were found infested with water mites of the genus Unionicola in Bangkok and Nonthaburi provinces, Thailand. Morphological studies on these water mites, based on the characteristics of their genital acetabular and female genital fields, identified them as a new member of subgenus Polyatax. Unionicola (Polyatax) kasetbangkhenensis sp. nov. was named after the first location where this parasitic water mite was discovered. This species is distinguished from others by the pattern of their female genital field, with their anterior acetabular plates each bearing two acetabula and a short thick spine on the inner margin. Additionally, it differs from other species by the structure of the spines in its pedipalps and legs, as well as in the shapes of its coxal plates.

Blood recovery of wild Mekong snail-eating turtles (Malayemys subtrijuga Schlegel and Müller, 1845) in captivity from leech infestation.

Blood cell counts are valuable diagnostic tools for assessing the health status of chelonians, however, reference standards for healthy blood parameters in various turtle species are lacking. In this study, forty wild female Malayemys subtrijuga were captured from ponds in Kasetsart University, and transported to laboratory for recuperating in captivity. All turtles were infected with a single leech species, Placobdelloides siamensis, with a mean of 513.7 ± 164.9 individuals per turtle, and exhibited penetrating and lesion wounds from leech infestations on both their skin and shell. Subsequently, they were cleaned and treated to eliminate ecto- and endoparasites before the recuperation period began. The turtles did not exhibit significantly differences in weight, carapace length (CL), red blood cell count (RCC), and white blood cell count (WCC) with a mean of 654.2 ± 199.9 g, 15.0 ± 2.5 cm, 327,080 ± 70,156 cells/mm3, and 73,340 ± 15,859 cells/mm3, respectively, during the initial records (week 0). However, after being maintained for 17 weeks, their health significantly improved in term of their blood parameters (RCC and WCC) and weight, except CL which remained unchanged, with a mean of 491,470 ± 16,169 cells/mm3, 18,790 ± 1496 cells/mm3, and 738.9 ± 191.5 g, respectively. Therefore, the health status obtained in this study can be used as a reference for blood parameters, weight, and recuperation period for the treatment of ill wild M. subtrijuga in captivity or as part of conservation management programs for turtles.

Three new species of Pseudorhabdosynochus (Monogenea, Diplectanidae) from several species of Cephalopholis and Epinephelus (Perciformes, Serranidae) from Thailand.

Pseudorhabdosynochus suratthaniensis n. sp. is described from the gills of Cephalopholis argus; P. cephalopholi n. sp., from the gills of C. sonnerati; and P. samaesarnensis n. sp., from the gills of Epinephelus lanceolatus. These fish were all caught in the Gulf of Thailand. Pseudorhabdosynochus suratthaniensis n. sp. is distinguished from congeneric species by the structure of its sclerotized vagina, which has a wide sclerotized trumpet and a single large primary chamber, and by the number of rows of rodlets in each of its squamodiscs. Pseudorhabdosynochus cephalopholi n. sp. is also distinguished by the structure of its sclerotized vagina that, like the P. suratthaniensis n. sp., has a sclerotized trumpet, but it also has a long coiled or curved primary canal near its midlength, and a distal part with a primary chamber and a secondary chamber communicating with the primary chamber through a short secondary canal. In addition, P. cephalopholi n. sp. is distinguished by some sclerotized organs (ventral and dorsal hamuli, ventral bar, and quadriloculate organ) with different lengths, and by the number of rows of rodlets in each of its squamodiscs. Pseudorhabdosynochus samaesarnensis n. sp. is distinguished by its sclerotized vagina that has an anterior cup-shaped trumpet and a short straight or curved primary canal. For Thailand, these are the first species of Pseudorhabdosynochus described from species of Cephalopholis and the second species of Pseudorhabdosynochus described from Epinephelus.

Title: Trois nouvelles espèces de Pseudorhabdosynochus (Monogenea, Diplectanidae), parasites de plusieurs espèces de Cephalopholis et Epinephelus (Perciformes, Serranidae) de Thaïlande. Abstract: Pseudorhabdosynochus suratthaniensis n. sp. est décrit à partir des branchies de Cephalopholis argus, P. cephalopholi n. sp. des branchies de C. sonnerati, et P. samaesarnensis n. sp. des branchies d'Epinephelus lanceolatus. Ces poissons ont tous été pêchés dans le golfe de Thaïlande. Pseudorhabdosynochus suratthaniensis n. sp. se distingue des espèces congénères par la structure de son vagin sclérifié, qui possède une large trompette sclérifiée et une seule grande chambre primaire, et par le nombre des rangées de bâtonnets dans chacun de ses squamodisques. Pseudorhabdosynochus cephalopholi n. sp. se distingue également par la structure de son vagin sclérifié qui, comme P. suratthaniensis n. sp., a une trompette sclérifiée, mais a également un long canal primaire enroulé ou incurvé près de sa mi-longueur et une partie distale avec une chambre primaire et une chambre secondaire communiquant avec la chambre primaire par un court canal secondaire. De plus, P. cephalopholi n. sp. se distingue par certains organes sclérifiés (hamuli ventraux et dorsaux, barre ventrale et organe quadriloculé) de longueurs différentes, et par le nombre des rangées de bâtonnets dans chacun de ses squamodisques. Pseudorhabdosynochus samaesarnensis n. sp. se distingue par son vagin sclérifié qui a une trompette antérieure en forme de coupe et un court canal primaire droit ou courbe. Pour la Thaïlande, il s'agit de la première espèce de Pseudorhabdosynochus décrite à partir d'espèces de Cephalopholis et de la deuxième espèce de Pseudorhabdosynochus décrite à partir d'Epinephelus.

Placobdelloides tridens sp. n., a new species of glossiphoniid leech (Hirudinea: Rhynchobdellida) found feeding on captive Orlitia borneensis in Thailand, and an update to the host distribution of P. siamensis.

A new glossiphoniid leech species, Placobdelloides tridens sp. n., is discovered on the Malayan Giant Turtle (Orlitia borneensis) at the Nakhon Ratchasima Zoo in Thailand. The morphological study of this new species revealed that it is distinguished from P. siamensis, a turtle leech species that can be found commonly in Thailand. Placobdelloides tridens presented the following diagnostic morphological characteristics: a pear-shaped and triannulate body, well-developed rod-like papillae on the dorsal surface, smooth posterior and anterior suckers with nominal pits inside, a single pair of dark contiguous eyes, light yellow-brown to greenish dorsal color, absence of median line, male and female gonopore separated by a single annulus and a unique trident shape at the tip of the crop ceca. The phylogenetic relationships of P. tridens sp. n., was clarified, and shown to be a sister clade to the P. siamensis and P. sirikanchanae clade. Furthermore, this is a new host record for P. siamensis, which was found on O. borneensis, Batagur affinis and B. borneoensis in the Khao Kheow Open Zoo, Chonburi, Thailand.

Very low prevalence of Opisthorchis viverrini s.l. cercariae in Bithynia siamensis siamensis snails from the canal network system in the Bangkok Metropolitan Region, Thailand.

The liver fluke Opisthorchis viverrini s.l. is associated with a long-term public health problem in Thailand. However, O. viverrini s.l. infection in Bithynia snails in the canal network system (CNS) in the Bangkok Metropolitan Region (BMR) has never been assessed. This study aimed to investigate the occurrence of B. siamensis siamensis and the prevalence of O. viverrini s.l. infection in this snail in the CNS in BMR along with morphological examination and molecular analyses on O. viverrini s.l. cercariae. The snails were randomly sampled from the CNS in all BMR areas from January 2018 to July 2019. Snail specimens were identified and examined for digenean infection by shedding and dissection. The cercariae were identified using morphology and molecular methods, including PCR with a species-specific primer and a Bayesian phylogenetic analysis of ITS2 sequences. Bithynia siamensis siamensis was found in almost all sampling localities, with different quantities and detected frequencies. From a total of 7473 B. s. siamensis specimens, O. viverrini s.l. infections were detected in the Northern Bangkok, Muang Nakhon Pathom, Krathum Baen, and Lam Luk Ka areas with an overall prevalence of 0.05% (4/7473) and prevalence of 0.22% (1/455), 0.21% (1/469), 0.40% (1/253), and 0.16% (1/614) in individual localities with positive snails, respectively. This study is the first investigation of digenean infection in the canal network system-type habitat in Thailand and revealed extremely low O. viverrini s.l. prevalence.

TITLE: Très faible prévalence des cercaires d'Opisthorchis viverrini s.l. chez les mollusques Bithynia siamensis siamensis du réseau de canaux dans la région métropolitaine de Bangkok, Thaïlande. ABSTRACT: La douve du foie Opisthorchis viverrini s.l. cause un problème de santé publique récurrent en Thaïlande. L'infection des Bithynia par O. viverrini s.l. dans le réseau de canaux (RC) dans la région métropolitaine de Bangkok (RMB) n'a jamais été évaluée. Cette étude visait à étudier la présence de B. siamensis siamensis et la prévalence de l'infection par O. viverrini s.l. chez ce mollusque dans le RC de la RMB, avec aussi une étude morphologique et des analyses moléculaires sur les cercaires d' O. viverrini s.l. Les mollusques ont été prélevés au hasard dans le RC dans toutes les zones de la RMB de janvier 2018 à juillet 2019. Les spécimens de mollusques ont été identifiés et l'infection par les Digènes a été étudiée par excrétion et dissection. Les cercaires ont été identifiées à l'aide de méthodes morphologiques et moléculaires, y compris la PCR avec une amorce spécifique à l'espèce et une analyse phylogénétique bayésienne des séquences ITS2. Bithynia siamensis siamensis a été trouvé dans presque toutes les localités d'échantillonnage, avec des quantités et des fréquences de détection différentes. Sur un total de 7473 spécimens de B. s. siamensis, des infections par O. viverrini s.l. ont été détectées dans les régions du nord de Bangkok, Muang Nakhon Pathom, Krathum Baen et Lam Luk Ka avec une prévalence globale de 0,05 % (4/7473) et une prévalence respective de 0,22 % (1/455), 0,21 % (1/469), 0,40 % (1/253) et 0,16 % (1/614) dans les localités avec des mollusques positifs. Cette étude est la première enquête sur l'infection des Digènes dans l'habitat de type réseau de canaux en Thaïlande et a révélé une très faible prévalence d'O. viverrini s.l.

Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite perspective.

We investigated the prevalence, morphological characters and molecular classifications of trematode cercariae in freshwater snails randomly collected from 59 sampling localities in Bangkok from May 2018 to March 2019. We used a crushing technique to observe the cercarial stage inside each snail body and amplified the internal transcribed spacer 2 regions of cercarial DNA using polymerase chain reaction methodology. The associated phylogenetic tree was reconstructed using Bayesian inference analyses. A total of 517 of 15 621 examined snails were infected with trematode cercariae, and the infected snails were classified into 11 species of seven families with a 3.31% overall prevalence of the infection. The Bithynia siamensis siamensis snail displayed the highest prevalence of infection (16.16%), whereas the Physella acuta snail exhibited the lowest prevalence (0.08%) of infection. Eight morphological types of cercariae were observed. The highest prevalence of infection was observed in mutabile cercaria (1.86%). Based on molecular investigations, the phylogram revealed eight cercaria types assigned to at least nine digenean trematode families, of which five belong to groups of human intestinal flukes. Although, with the exception of schistosome cercaria, trematode cercariae are not known to directly damage humans, understanding the general biology of trematode cercariae (including diversity, distribution, infection rates and host range) is important and necessary for the prevention and control of parasitic transmission that impacts aquatic cultivations, livestock farming and human health.

Batracobdelloides bangkhenensis sp. n. (Hirudinea: Rhynchobdellida), a new leech species parasite on freshwater snails from Thailand.

A new snail-eating leech, Batracobdelloides bangkhenensis sp. n., was discovered at Kasetsart University, Bangkhen Campus, Bangkok, Thailand. This species is found free living in the benthic zone of ponds; feeds on freshwater snails, including Bithynia siamensis siamensis, Indoplanorbis exustus, Radix rubiginosa, Physella acuta, and Pomacea canaliculata; and uses a shell as a shelter during the parental care period, with a colony of 7-15 juvenile individuals held on the venter inside the shell of host. Batracobdelloides bangkhenensis displays distinct morphological characters, including a rice-shaped body showing transparency, cephalization, two eye pairs merged on somite III, an anterior sucker twice as large as the cephalic region, a central mouth in the anterior sucker, seven light brown transverse rows in the neck region, absent dorsal papillae, rich green pigments on the dorsum, a male gonopore on XIIa2/XIIa3 (27-28), a female gonopore on XIIIa1/XIIIa2 (29-30), and diffuse aggregations of minute, spherical salivary glands in the neck region. Comparisons of the COI and COI-ND1 genes showed a monophyletic clade for Batracobdelloides, and the phylogenetic tree of the COI gene also indicated that B. bangkhenensis is distinct from other species in the genus, with strong support values.

New Records of Fish Parasitic Isopods (Crustacea: Isopoda) from the Gulf of Thailand.

From a total of 4140 marine fishes examined, eight species of parasitic isopods were reported from marine fishes in the Gulf of Thailand. These isopods were identified in two families, Corallanidae (Argathona macronema and Argathona rhinoceros) and Cymothoidae (Cymothoa eremita, Cymothoa elegans, Smenispa irregularis, Nerocila sundaica, Norileca indica and Norileca triangulata). Most of these parasitic isopods were found in the buccal cavity of their fish hosts with one host recorded as follows: C. eremita was found from Nemipterus hexodon, C. elegans was found from Scatophagus argus, N. sundaica was found from Saurida tumbil. The majority of the isopod specimens recorded in this study was S. irregularis, which was found in the buccal cavities of five host fish, Pampus argentius, Alepes melanoptera, Caranx hippos, Parastromateus niger and Terapon jarbua, with a prevalence of 11.67%, 10.43%, 9.78%, 6.10% and 4.21%, respectively. Argathona rhinoceros was found in the nasal cavity and branchial cavity of Epinephelus coioides, whereas A. macronema and N. triangulata were found on the skin of Epinephelus coioides and Seriolina nigrofasciata, respectively. The highest species diversity was found in E. coioides, which harbored two species of parasitic isopods, A. macronema and A. rhinoceros. Cymothoa eremita, C. elegans, S. irregularis and N. triangulata were recorded for the first time in the Gulf of Thailand. The reported discovery of C. eremita, S. irregularis, N. sundaica and N. triangulata in their fish hosts were new recorded hosts. Moreover, A. macronema and N. triangulata were found for the first time in the central Indo-Pacific region.

The life cycle of the Siamese shield leech, Placobdelloides siamensis Oka, 1917.

Placobdelloides siamensis is a glossiphoniid leech with a short life cycle. In a laboratory setting, ten mature P. siamensis left their host (a turtle) after feeding for approximately three days and initiated copulation. The adults spent 3-4 days gestating before depositing eggs (272.8±62.9 eggs/clutch; range: 186-359 eggs/clutch). The eggs then changed from a creamy white to a creamy brownish color before hatching. Hatching occurred after incubation on the parent's ventral surface for 5-7 days. The transparent brood, with a single pair of red eyes, spent a couple of weeks under the venters of their parents. After this period, they left their parents and grew to maturity in 10-15 days; leeches were considered mature when their color was similar to that of their parents and they performed their first copulation. In addition, the mature leeches survived for 163 days on one feeding.

Parasitism of Placobdelloides siamensis (Oka, 1917) (Glossiphoniidae: Hirudinea) in Snail-eating Turtles, Malayemys spp., and the effects of host and aquatic environmental factors.

The Siam Shield Leech, Placobdelloides siamensis, is a common leech found on Malayemys turtles in Thailand. Sixty Snail-eating Turtles (29 Malayemys macrocephala and 31 M. subtrijuga) were caught over twelve months (February 2017 - January 2018) to determine host characteristics (body size, weight and sex), parasitism (prevalence, intensity and density) and seasonal aquatic environmental factors (conductivity, nitrate nitrogen, dissolved oxygen, pH, salinity and total dissolved solids). There was no significant difference of infection rate between species and sex in both turtle species. Leech prevalence indicated that all turtle individuals were infected throughout year, while the infection rate was significantly higher in larger and heavier turtles mainly on the carapace with an average number of leech approximately 474.80 ± 331.38 individuals for individual host infection and 76.53 ± 20.27 individuals for infection per 100 g body weight. The high level of leech parasitism also caused a rot wound and shell hole which caused the host to die. Aquatic environmental factors did not influence the infection of leeches in both turtle species. Therefore, the factors that influenced the infection rate of P. siamensis were based on only host body size and weight without effect from season. In addition, this study also showed two new hosts, including Cyclemys oldhamii and Heosemys grandis and the widespread distribution from northern, north-eastern, western, central and southern Thailand were reported.

Infections of Digenetic Trematode Metacercariae in Wrestling Halfbeak, Dermogenys pusilla from Bangkok Metropolitan Region in Thailand.

This study aimed to investigate metacercarial infections in the wrestling halfbeak, Dermogenys pusilla, collected from Bangkok metropolitan region of Thailand. A total of 4,501 fish from 78 study sites were commonly examined with muscle compression and digestion methods (only head part of fish) during September 2017 to July 2018. The overall prevalence of metacercarial infection was 86.1% (3,876/4,501 individuals), and the mean intensity was 48.9 metacercariae per fish infected. Four species, i.e., Posthodiplostomum sp., Stellantchasmus falcatus, Cyathocotylidae fam. sp., and Centrocestus formosanus, of digenetic trematode metacercariae (DTM) were detected. The prevalences were 65.8%, 52.0%, 2.1%, and 1.2%, respectively and their mean intensities were 23.1, 51.6, 1.4, and 3.2 per fish infected, respectively. The seasonal prevalences were 81.0% in winter, 87.8% in summer and 87.4% in rainy, and the mean intensities were 38.9, 46.6, and 55.2 metacercariae per fish infected, respectively. Conclusively, it was confirmed that the wrestling halfbeak play the role of second intermediate hosts of 4 species of digenetic trematodes including S. falcatus and Posthodiplostomum sp. in Bangkok metropolitan region. And then the metacercariae of C. formosanus and Cyathocotylidae fam. sp. are to be first found in the wrestling halfbeak by this study.

Pseudorhabdosynochus kasetsartensis n. sp. (Monogenea: Diplectanidae) from the cloudy grouper Epinephelus erythrurus (Valenciennes) (Perciformes: Serranidae) in the lower Gulf of Thailand.

Pseudorhabdosynochus kasetsartensis n. sp. is described from the gills of the cloudy grouper Epinephelus erythrurus (Valenciennes) caught in the lower Gulf of Thailand. The new species is distinguished from other species assigned to the genus by the structure of its sclerotised vagina which has a wide and prominent sclerotised trumpet, long, thin, coiled or curved primary canal, short secondary canal, and primary and secondary chambers that are blind extremities of the primary and secondary canals, respectively. This is the first species of Pseudorhabdosynochus Yamaguti, 1958 described from E. erythrurus and the first record of a species of Pseudorhabdosynochus in Thailand.

Placobdelloides sirikanchanae sp. nov., a new species of glossiphoniid leech and a parasite of turtles from lower southern Thailand (Hirudinea, Rhynchobdellida).

Abstract A new species of glossiphoniid leech, Placobdelloides sirikanchanae sp. nov., is reported in the Asian leaf turtle (Cyclemys dentata) and the dark-bellied leaf turtle (C. enigmatica) from Songkhla Province, southern Thailand. The examination of morphological characters revealed that this new species is similar to P. siamensis (Oka, 1917), a common turtle leech species found in Thailand. Placobdelloides sirikanchanae sp. nov. demonstrates distinct morphological characters, with an elongated, narrow body, 13-17 well-developed knob papillae on each annulus, dark brown to greenish dorsal color with a crimson median line, the absence of a scarlet dot, different male and female gonopore distributions, a rough posterior sucker with a random pit distribution, and 104-115 eggs per clutch. The phylogenetic relationships of COI-ND1 genes were clarified and shown to be distinct from those of P. siamensis. Additionally, habitat preferences tended toward low oxygen conditions such as puddles or water patches on rubber plantations.

New record of Trichodina unionis (Ciliophora, Trichodinidae) from freshwater gastropods in Bangkok, Thailand.

Trichodinids, which are ciliate protists, are causative agents of an aquatic animal disease, trichodiniasis, especially among both captive and wild fish. This disease can adversely affect aquaculture and have economic impacts. The objectives of this study were to evaluate the prevalence and mean intensity of Trichodina unionis infection, describe qualitative and quantitative morphological characters, and perform a molecular phylogenetic analysis. The gastropod samples were randomly collected by hand-picking and a hand net. Trichodina unionis was collected by the crushing method under a stereomicroscope. Among all 4977 examined gastropods, 55 individuals of two gastropod species, Gyraulus siamensis and Physella acuta, were found to be infected by T. unionis, with overall prevalence and mean intensity of infection of 1.11% and 16.65, respectively. The characteristics of the denticles indicated T. unionis as having moderately wide blades and moderately curved blade margins, with distinctive bend angles near the distal end. The quantitative characters showed some variations, which could be due to food availability. Molecular phylogenetic analysis conducted with 18S rRNA provided a monophyletic tree of our specimens and previously identified T. unionis, confirming species identification. This study represents the first record of T. unionis in Thailand.

Redescription of the Siamese shield leech Placobdelloides siamensis with new host species and geographic range.

The Siamese shield leech Placobdelloides siamensis (Oka, 1917) Sawyer, 1986 (Euhirudinea: Glossiphoniidae) was collected from five new host species, Southeastern Asian Box Turtle (Cuora amboinensis), Yellow-headed Temple Turtle (Heosemys annandalii), Malayan Snail-eating Turtle (Malayemys macrocephala), Mekong Snail-eating Turtle (M. subtrijuga), and Khorat Snail-eating Turtle (M. khoratensis) and was found for the first time in Udon Thani, Thailand. Examination of live leeches provided, for the first time, data on coloration and the combination of parental care behavior, both carrying cocoons and attaching cocoons to the substrate. This species was separated from its congeners based on the following characters: one pair of eyes; spines at proboscis subterminal; mouth terminal on oral sucker; absent plaque in neck region; gonopores located in furrow and separated by two annuli; distinctly triannulated mid-body segments; crop with seven pairs and branched caeca; caudal sucker slightly over half of maximum body width; and strongly dorsal papillae. Phylogenetic relationships based on the COI and ND1 genes were clarified and demonstrated that the species is distinct from others. The original description was amended and the taxonomic history is discussed.

A new species of Tetraphyllidean (Onchobothriidae) cestode from the brown-banded bambooshark Chiloscyllium punctatum (Elasmobranchii: Hemiscylliidae).

Yorkeria chonburiensis n. sp. (Tetraphyllidea: Onchobothriidae) is described from the spiral intestine of a specimen of the brownbanded bamboo shark, Chiloscyllium punctatum, collected from the Gulf of Thailand, Chon Buri Province, Thailand. Yorkeria chonburiensis n. sp. is distinguished from all other species of Yorkeria by the pattern of proglottids (craspedote as opposed to acraspedote) and the proglottid number (more than 168). The new species is larger than all other members of Yorkeria, and the medial hooks are slightly larger than the lateral hooks with a ratio of 1:1.87-1:1.96. The position of the genital pore is 29-34% from the anterior margin of proglottid, greater than all other members of Yorkeria.

Potential application of extracts from Indian almond (Terminalia catappa Linn.) leaves in Siamese fighting fish (Betta splendens Regan) culture.

Indian almond (Terminalia catappa Linn.) leaves with green and red coloration were tested for bactericidal activity with pathogenic bacteria and their acute toxicity to Siamese fighting fish (Betta splendens Regan). Powder of Indian almond leaves was extracted with water at the ratio of 1:10 (w/v) and then freeze-dried to a dry powder. Bactericidal efficacy was tested against 28 isolates of pathogenic bacteria (Aeromonas hydrophila, Edwardsiella tarda, Enterobacter spp., Plesiomonas shigelloides, Pseudomonas spp., Shewanella putrefaciens, Staphylococcus sp. and Streptococcus sp.) isolated from Siamese fighting fish. A paper disc diffusion method was tested on Mueller Hinton Agar (MHA) by inoculating 1 x 106 CFU/mL of each bacterial suspension. Paper disks (5 mm) were impregnated with either 10 microl of green or red Indian almond leaf extract at a concentration of 12,000 ppm, then laid on the surface of the MHA. The results revealed that aqueous extract of red Indian almond leaves could inhibit the growth of tested bacteria better than the green extract. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined. Extracts of green and red leaves were diluted in Mueller Hinton Broth (MHB) to obtain a starting extract concentration of 6,000 ppm then twofold serially diluted in a 96-well microtitre plate. The pathogenic bacteria were inoculated into each well at a density of 1 x 105 CFU/mL and incubated at 35 degrees C for 24 h. The growth of bacteria was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliium bromide. The MIC of the red leaf extract ranged from 187.5 to 375 ppm which was lower than for green leaf extract (375-750 ppm). The MBC range of the red leaf extract was 375-750 ppm which was lower than for the green leaf extract (750-1,500 ppm). Acute toxicity tests (96-h LC50) of Indian almond green and red leaf water extracts in Siamese fighting fish were conducted. A logarithmic-spaced series of Indian almond leaf extract concentrations (6 concentrations) between the highest concentration that did not kill fish and the lowest concentration that killed all fish were used. Each concentration had three replicates (20 fish/replication). Mortality was observed after 96 hours. The LC50 value was calculated using probit analysis. The 96-h LC50 value for green and red leaf extracts was 1,765.69 and 1,651.21 ppm, respectively. When Siamese fighting fish were cultured in water added with Indian almond water extract at 5 concentrations for 5 days and then challenged with Aeromonas hydrophila at a concentration of 1 x 106 CFU/mL, the survival rate of the Siamese fighting fish infected by A. hydrophila could be decreased by bathing with Indian almond red leaf extract at 750 ppm while green leaf Indian almond extract was effective for Aeromonad septicemia therapy in Siamese fighting fish when used at a concentration of 1,000 ppm. In conclusion, red leaf Indian almond aqueous extract had high potential for the control of pathogenic bacteria at a concentration of 750 ppm which should be safe for Siamese fighting fish taking into consideration the toxic level of the extract.

Growth, nonspecific immune characteristics, and survival upon challenge with Vibrio harveyi in Pacific white shrimp (Litopenaeus vannamei) raised on diets containing algal meal.

A 70-day growth trial was conducted with postlarvae 12 (PL12) Pacific white shrimp (Litopenaeus vannamei) to study the suitability of soybean meal and oil originating from a single-celled microorganism (thraustochytrid) as fishmeal and fish oil substitutes in practical diets for L. vannamei. The growth, survival rate and immune characteristics were evaluated. Seven experimental diets were designed with soybean meal used as the primary protein source; each formulation contained 33% crude protein and 8% lipid. Fish oil was completely substituted with 3% soybean oil and meals originating from single-celled heterotrophs rich in docosahexaenoic acid (DHA) and arachidonic acid (ARA) were added at different concentrations. A commercial shrimp feed was used as the control diet. The final weights and survival rates of the shrimp were not significantly different among all treatments. However, shrimp raised on diets supplemented with marine algal meals rich in DHA and ARA showed significant improvement in immune parameters, such as total hemocyte count, phenoloxidase activity, superoxide dismutase activity, and bactericidal activity. Additionally, the survival rate after challenge with Vibrio harveyi was increased. These findings demonstrated that substitution of thraustochytrid-derived meals as an alternative to fish-based ingredients in shrimp diets provided similar growth rates while increasing the immune parameters and providing vibriosis resistance.