Table 1.
List of real-time PCR primers to detect the stomach and midgut bacteria.
Fig 1.
Representative scanning electron microscopy (SEM) micrographs of the inner surface of the digestive of farmed Penaeus monodon.
Inner surface of (A) the dorsal (B) ventral, (C) peritrophic membrane, and (D) fiber seta of the stomach were devoid of bacteria. (E) bacterial cells (arrowed), however, were seen in association with food inside the stomach. (F, G, H, I, J) Healthy midgut have intact microvilli, and a large number of bacteria were observed attached to the peritrophic membrane and food particle in the midgut. (F, I) Massive granules among the epithelial cells can be seen projecting into the midgut lumen between the microvilli. (K, L) only a few bacteria were seen attached to the cuticle lining of the hindgut. Abbreviation: cuticle (cu), spines (Sp), peritrophic membrane (PM), fiber seta (FS), ingested food (IF), bacteria (Ba), microvilli (Mv), granule (Gr), pit (Pi)
Fig 2.
Representative SEM pictures of the inner surface of the digestive tract of a suspected diseased Penaeus monodon from a shrimp farm.
A cluster of unique rod-shaped bacteria attached to (A) fibre setae or (B) to the lining of the stomach. (C) Higher magnification image of the attached bacteria in the stomach exhibiting peritrichous pili-like structures or fimbria., where (D) a few fibres linked to the peritrophic matrix (PM) can be seen (arrowhead). (E) Many holes were created in the PM and a few granules were seen inside the holes. (F) A group of irregular-shaped bacteria were found attached to the PM. (G) A cluster of short-rod shaped bacteria with polar flagella, (h) irregular-shaped, and (I) unidentified particles were seen attached to the hindgut wall. Abbreviation: fiber seta (FS), bacteria (Ba), spines (Sp), fimbria (Fi) granule (Gr), microvilli (Mv), polar flagella (PF), unknown particles (UP), ingested food (IF)
Fig 3.
Representative SEM images of the inner surface of P. monodon infected with Vibrio harveyi.
(A) At 1.5 h post-infection (PI), no bacteria adhering to the surface of the stomach linings were seen, but (B) numerous bacterial cells mixed with ingested food attaching the stomach surface were seen. (C) Numerous rod-shaped bacteria firmly attached to the stomach lining. (D) At 6 h PI, colonizing bacteria cover the epithelium of the anterior midgut. (E, yellow arrow) A higher magnification of the bacteria seen at 6 h show that they possess polar flagella that are linked with each other, and (F) heavy destruction of the epithelial layers by bacteria exposed of the basement membrane underneath. (G) The posterior portion of the midgut showing intact tissue with a thick peritrophic matrix or (H) with a few bacterial cells attached to the microvilli. (I) Scattered clusters of rod-shaped bacteria adhering to the lining of the hindgut. (J-L) At 24 h PI, the numbers of bacteria within the stomach of infected shrimp increased dramatically. Densely packed-bacteria were found covering the epithelium of the anterior midgut. Abbreviation: cuticle (Cu), spines (Sp), peritrophic membrance (PM), ingested food (IF), fiber seta (FS), polar flagella (PF), bacteria (Ba), basement membrane (BM), microvilli (Mv)
Fig 4.
Representative SEM images of the inner surface of the digestive tract of P. monodon infected with Vibrio parahaemolyticus.
(A) At 24 post-infection, numerous straight-shaped bacteria adhering to the fibre seta, to (B) short spines and (C) to the inner surface of the stomach. Some of the spines were broken and had detached from the stomach lining (arrowheads). (D-F) Attached bacteria producing peritrichous pili-like structures. Abbreviation: fiber seta (FS), spines (Sp), cuticle (Cu), fimbria (Fi), peritrophic membrane (PM)
Fig 5.
Representative SEM images of the inner surface of the digestive tract from P. monodon receiving non-pathogenic bacteria (Micrococcus luteus and non-pathogenic Vibrio B4-24).
No observed attachment of bacteria to the stomach (A—M. luteus, B- Vibrio B4-24) or to the midgut (C- Vibrio B4-24). The bacteria found in the hindgut were variable in number where most of the hindgut tissues were intact (D—M. luteus, E- Vibrio B4-24). A high number of pits were found across the surface of the midgut (G, H- Vibrio B4-24), where a large number of cocci- and spindle-shaped granules which resided in the epithelial cells were seen (I- Vibrio B4-24). Abbreviation: spines (Sp), fiber seta (FS), cuticle (Cu), microvilli (Mv), bacteria (Ba), pit (Pi), granule (Gr)
Fig 6.
Real-time PCR presented of the ratio of the gyrB gene of Vibrio harveyi (A) and 16S rDNA bacteria, and the ratio of the gyrB and tlh gene of Vibrio parahaemolyticus (B) and 16S rDNA bacteria in the GI tract of P. monodon.
Error bars were expressed as ±SEM. Asterisk indicates significant differences between the control and challenged group (P<0.05).