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Black gill disease of Pacific white leg shrimp (Litopenaeus vannamei) by Aspergillus flavus

Naresh Kumar Dewangan, Ayyaru Gopalakrishnan, Daniel Kannan, Narayanasamy Shettu, Ramakrishna Rajkumar Singh Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608502, Tamil Nadu, India PG & Research Department of Zoology, Pachaiyappa's College, Chennai 600030, Tamil Nadu, India CP Aquaculture India Private Limited, Sambasivanagar, Ongole 523002, Andhra Pradesh, India Journal of Coastal Life Medicine 2015; 3(10): 761-765


Introduction
Aquaculture is playing an important role as a prominent source of nutrition and also presents employment opportunity to millions. This sector is growing as a multi-million dollar industry and the farmed fish contributes 42% of total 158 million tons of fish produced by capture and culture fishery [1]. The share of fisheries sector in the national total export earning is about 6%, out of which approximately 85% is being contributed by shrimp alone [2].
Indian coastal zone has 1.40 million ha brackish water area, out of which 1.19 million ha area has been declared as suitable for shrimp aquaculture. India is the fastest growing nation in shrimp aquaculture with natural resources like brackish water, mud flats, swamps, marshes, lagoons, backwaters and estuaries [3].
The culture of shrimp received maximum importance due to high nutritional value, but the disease outbreak during the culture is a major concern [4]. Some environmental factors, infectious organisms (fungi and viruses, protozoans etc.), and pollutants (chemical and biological) also cause the mass mortalities in the pond [5]. These environmental factors negatively influence the sustainability of shrimp farming and also directly affect to the growth rate and survival in the grow-out culture system [6].
Black gill disease was first reported in Japanese Kuruma prawn, Penaeus japonicas (P. japonicas) by Ishikawa in 1968. Fusarium species was the causative agent for this black gill disease, and later Fusarium species was considered as the most detrimental pathogens for Kuruma prawn in Japan [7]. Chemical contaminants like oil, cadmium, copper, zinc, potassium permanganate, ozone, ammonia,

Objective:
To study the epidemiology of black gill disease in white leg shrimp which is a major problem being faced by the commercial shrimp farmers who are culturing Litopenaeus vannamei (L. vannamei) in India.

Methods:
The normal and infected shrimps were collected from shrimp pond and the gill was preserved in appropriate preservative for histopathological examination and scanning electron microscope analysis. Pathogenic fungus was isolated from black gill of L. vannamei in potato dextrose agar medium. Morphological study and fungal strain identification were done by using light microscopy and scanning electron microscope. Fungal DNA was amplified by ITS4 and ITS5 primers and gene sequencing was done by Macrogen Inc., Korea. Phylogenetic tree was prepared by using MEGA 6 software. Results: Fungal spores and hyphae were observed both in internal and external gill surface of infected shrimps. Fungal spores were round in shape and mature sporangium was observed.
The histopathology study showed clearly that infected gill was damaged by the fungi. Scanning electron microscopic study showed adherence of fungi in infected gill. Internal transcribed spacer gene sequencing revealed that it was caused by Aspergillus flavus.

Conclusions:
The outcome of the present study would help to know the cause of black gill disease and to understand the effect of pathogenic fungi in shrimp culture. This study will initiate researchers for work in field of treatment or prevention of black gill disease in commercial L. vannamei culture.
Although fungi occur widely in marine environment, the distribution of fungi in diseased shrimps has not been studied well as compared to the studies on fungi in freshwater and terrestrial ecosystems [12]. Therefore, the present study was undertaken with the objective to identify the major causative pathogens for the black gill disease in Litopenaeus vannamei (L. vannamei). After separation of gills, wet mount slides were prepared separately for both normal and black gills which were then observed by using the light microscope under different magnifications. Gills were isolated from shrimps and washed thrice in 0.85% NaCl solution.

Materials and methods
After washing, the gills were inoculated in potato dextrose agar (PDA) medium supplemented with ampicillin and streptomycin to inhibit the bacterial growth and then incubated at 25 °C . After gill isolation, lacto phenol cotton blue was used for microscopic observation of fungi. Normal gills were also inoculated in the same medium as control. Experiment was performed in triplicate.
Infected shrimp gills were first preserved in 3% glutaraldehyde, then dehydrated, coated with gold and observed under scanning electron microscope JEOL JSM 5610LV at 15 kV. DNA was amplified by using ITS4 and ITS5 primers [13]. DNA was sequenced by Macrogen Inc., Korea. The sequence similarity was analyzed by using basic local alignment search tool and the phylogenetic tree was constructed by using MEGA 6 software.

Shrimp sampling
The shrimp farm was 0.66 ha where the dissolved oxygen level

Histopathology
In the case of normal gills, fungi were absent in gill lamella and physiology of gills was not changed (Figure 2A). However, in the case of black gills, the tissue was damaged and fungal hyphae, conidia, and whole sporangium were present inside the gill lamellae

Microscopic observation of shrimp gills
Microscopic study revealed very clear and transparent gills in normal shrimps ( Figure 3A). However, the affected gills were found to be more fuzzy and black in appearance and hyphae strongly attached to central axis of gills ( Figure 3B). Outward growth of the fungi from the gill lamella was very prominent ( Figure 3C). Mature sporangium was present around the gill lamella ( Figure 3D). Fungal hyphae and conidia were also observed inside and in the outer surface of the affected gill lamellae ( Figures 3E and 3F) .

Isolation and colony morphology of fungi
After 48-h incubation, no fungal growth was observed in the case of normal gills ( Figure 4A), but in the affected gills, fungi became clearly visible in the petriplate, and after 72 h, it was transformed into matured colony. The fungus was lime green and yellowish in appearance. However, the reverse side was cream colour. The texture was cottony, granular with spherical shape, flat elevation with entire margin ( Figure 4B).

Microscopic observation of fungi
Hyphae were septate and hyaline, while conidia was smooth, very finely roughened and loosely attached to the vesicles. Conidiophores were found long, coarsely rough and colorless, while vesicles were globose to subglobose. Phialides were present around the vesicle ( Figures 4C and 4D).

Scanning electron microscope (SEM) analysis
SEM analysis revealed the absence of fungi in normal gills ( Figure   5A) and also showed the presence of mature sporangium with long conidiophores in black gills ( Figure 5B). After detachment of spores, clear phialides were visible. Microspores and hyaline mycelium were present in the black gills of L. vannamei. Fungal mat was observed all over the gills ( Figure 5C). Conidia were spared on the gill surface ( Figure 5D).

Phylogenetic analysis
Based on ITS gene sequencing, the pathogenic fungus was found to be A. flavus strain NKD1 (Genbank accession No. KM282572).
In the analysis of basic local alignment search tool, fungi which were isolated from black gills showed 100% similarity to A. flavus (KJ175414) (Figure 6). In phylogenetic tree, it clustered in the same clade of Aspergillus genus with the bootstrap support of 97.
Neighbour joining tree showed the position and similarity of A.
flavus NKD1 to other Aspergillus strains.

Discussion
L. vannamei is one of the important cultivable shrimps all over the world; therefore, the healthy shrimp production is need of the hour.
In the present study, we have isolated A. flavus from black gills of L. vannamei collected from a shrimp pond. Previously, A. flavus was also reported as the predominant species in L. vannamei out of 146 fungal isolates, but it was not isolated from black gills [10].
Apart of A. flavus, Fusarium oxysporium was also reported to be responsible for black gill disease in P. japonicas [14]. Black gill disease in cage-cultured ornate rock lobster Panulirus ornatus in Vietnam by Fusarium species has been observed [15]. Our present study was about L. vannamei in brakish salt water. Recently, a phytopathogenic fungus Gilbertella persicaria has been reported in shrimp Penaeus monodon cultured in brackish water along the east coast of India [16]. However, in the case of fresh water prawn Macrobranchium rosenbergii, the Fusarium infection has also been studied [17]. Histopathological observation in the present study clearly showed that gill tissue was damaged by A. flavus. The pathogenicity of anamorphic fungi in mantis shrimp Oratosquilla oratoria (O. oratoria) has also been reported [18]. In the present study, we have isolated A. flavus fungi from adult shrimps, while the fungal disease in larvae of Penaeus setiferus [19] and egg as well as larvae of Penaeus monodon by Legenidium thermophilus has been reported previously [20]. Fungi can be secondary infection also in other diseased shrimps like loose shell syndrome [21]. Halioticida infection in wild mantis shrimp O. oratoria has also been reported in Japan [22]. Molecular phylogeny and morphology of Fusarium