The striped trumpeter, Latris lineata
(Forster 1801), is distributed in southern hemisphere waters from the Walters Shoals (43°50′E) and Amsterdam Island (77°33′E) in the Indian Ocean through the southern waters of Australia and then to Chatham Island (176°29′W) in the Pacific Ocean (http://www.fishbase.org/summary/FamilySummary.php?id=356
). The overexploitation of this species throughout its range has led to a significant decrease in the wild population, with the total commercial catch decreasing in the last 20 years by almost 100 tonnes to 12.8 tonnes in 2009-2010 (1
). Because of the marked decline in wild stocks, the culture of L. lineata
has been in development at the Tasmania Aquaculture and Fisheries Institute, Hobart, Tasmania, for 16 years. L. lineata
is considered to be a suitable aquaculture candidate; however, issues associated with its complex and lengthy 9-month postlarval stage have been difficult to overcome (1
). The life cycle of L. lineata
has now been successfully closed, and established protocols exist for its reproduction and larval rearing (3
). Despite this, a number of health issues were observed between 1994 and 2010 during the development of L. lineata
for commercial aquaculture. Examples included abnormal swimming behavior, anorexia, swim bladder hyperinflation, skin lesions, and inflammation and swelling of gills in cultured juveniles due to infections from Kudoa neurophila
and chondracanthid copepods (4
). In determining the causes of these health issues, epitheliocystis was also described in these fish (6
). The latter disease is a condition of the skin and gills and is generally associated with infections by Chlamydia
-like organisms (CLOs) (7–10
). These CLOs are Gram-negative, intracellular bacteria that may cause cyst-like lesions in the gill lamellae (9
). The lesions may lead to epithelial hyperplasia and inflammation of the infected tissues, increased mucus production, and respiratory distress, sometimes ending in death (12–15
). Most reported losses in aquaculture attributed to epitheliocystis occur during the larval or juvenile culture stage (16
Little is known about the epidemiology and pathogenesis of epitheliocystis agents. In an effort to understand this, researchers have turned from traditional microbiology methods to molecular techniques in an attempt to understand this condition. This has led to a move toward the fulfillment of Fredericks and Relman's molecular postulates instead of Koch's postulates (17
). As a result, the primary method now used to describe and characterize unknown epitheliocystis agents taxonomically include phylogenetic analysis of DNA sequence data in combination with morphological descriptions. Following this trend, “Candidatus
Parilichlamydia carangidicola” was recently recovered from the yellowtail kingfish in Australia by using molecular techniques and transmission electron microscopy as primary evidence (10
The objective of this study was to identify and characterize the agent causing epitheliocystis in L. lineata, both in cultured individuals and in fish obtained from the wild. Histological examination of epitheliocystis infections in the gill were confirmed by PCR of the 16S rRNA gene and in situ hybridization (ISH). Following this, Bayesian inference and maximum-likelihood phylogenetic analyses were performed by using 16S rRNA sequences to explore the relationships of the striped trumpeter epitheliocystis agent with other epitheliocystis agents in fish and with other members of the order Chlamydiales.
Epitheliocystis is now known to affect >80 different species of fish (7
), and intriguingly, from all of these reports, it appears that the host responses considered to be severe or as having a hyperinfection are all from farmed fish (14–16
). Molecular diagnostics confirmed that this CLO, from both cultured and wild sources, is a member of the order Chlamydiales
, based on the >80% sequence similarity to other species in the order (18
). The nearly identical, nearly full-length 16S rRNA sequences of the CLO obtained over the three sampling periods and from both wild and cultured fish are strong evidence that it is the agent causing epitheliocystis in striped trumpeter fish. The detection of a Chlamydiales
-specific 16S rRNA gene in striped trumpeter epitheliocystis gill inclusions by ISH with DIG-labeled probes provides further evidence that the cysts identified by light microscopy are the source of the amplified novel 16S rRNA gene sequence(s).
The prevalence of epitheliocystis and the response of the striped trumpeter to infection reported in this study are in line with those previously reported for this species (6
). The response seen in this study, however, was not as severe as that in previous reports of hyperinfection in the largemouth bass, Micropterus salmoides
, and the Atlantic salmon, Salmo salar
). Epithelial hyperplasia, epithelial lifting along the lamellae, and lamellar fusion in the striped trumpeter were observed, although the filling of the interlamellar spaces was not as severe as that in previous reports. This differs from the nearly complete filling of the interlamellar spaces reported in both the largemouth bass (28
) and the Arctic charr (15
), which resulted in a severely compromised respiratory system.
Striped trumpeter fish were recently reported to be affected by epitheliocystis (6
). While that was the first report of epitheliocystis in the family Latridae, the condition has been reported in other species in the superfamily Cirrhitoidea, including the rock cale, Crinodus lophodon
, and the red morwong, Cheilodactylus fuscus
). Like the striped trumpeter, both of these species originate from Australian waters. In these other species, benign cysts with little or no host response were observed in the gills, which matched that of the wild fish and some of the cultured striped trumpeter fish in this study (33
). Unfortunately, no molecular data on the identity of the epitheliocystis agent from infections in these species are available for comparison, as the molecular techniques used here were not common practice at that time.
Questions remain about the origin of this infection and the potential impact that this novel epitheliocystis agent may have on the health and productivity of the striped trumpeter. In the cultured environment, the water source and treatment and the tank environmental conditions (temperature, salinity, dissolved oxygen, and nitrates) are controlled and monitored daily by staff. However, since infections were found in broodstock from both southwestern and northwestern Tasmanian waters and in fish sampled from the wild, it is reasonable to conclude that epitheliocystis occurs naturally in the environment and was introduced into the culture systems either with the fish or with the seawater supply. Although the bacterial sequences obtained from the different sources were of three distinct genotypes, they are extremely closely related. The development of in vitro methods for culturing these bacteria is needed to help answer questions on how variable these organisms naturally are, how these organisms are transmitted within a population, and what environmental factors, if any, may lead to hyperinfection.
Because of the current inability to culture Chlamydia
-like bacteria in vitro
, an alternative to Koch's postulates must be used. The molecular postulates of Fredericks and Relman were therefore used in this study (17
). The nearly identical sequences identified here from both wild and cultured sources were present in all cases of disease observed in histology. The nature of the CLOs detected here is consistent with the known biological characteristics of Chlamydia
; that is, they are intracellular bacteria requiring a host cell to replicate. The order specificity of the sequences was detected within the epitheliocystis cysts through ISH; and all of these results are repeatable. These results are in line with the molecular postulates of disease causation (17
) and provide strong evidence that the epitheliocystis agent of the striped trumpeter is of Chlamydiales
origin. On the basis of its novel 16S rRNA signature sequence, the sequence divergence from other Chlamydiales
species and the observed phylogenetic relationships of this bacterium to other taxa within the order according to their classification (18
), we propose the name “Candidatus
Similichlamydia latridicola” (gen. nov., sp. nov.) for the Chlamydia
-like epitheliocystis agent infecting the striped trumpeter.
“Candidatus Similichlamydia latridicola” gen. nov., sp. nov., recovered from the striped trumpeter (L. lineata). Similichlamydia gen. nov.; Si.mi.li.chla.my′di.a. L. adj. similis, resembling; N.L. fem. n. Chlamydia, a bacterial genus name; N.L. fem. n. Similichlamydia, resembling Chlamydia. latridicola sp. nov.; la.tri.di.′co.la. N.L. n. Latris -idis, a zoological genus name; L. suff. -cola (from L. n. incola), inhabitant, dweller; N.L. n. latridicola, Latris dweller, isolated from the striped trumpeter (Latris lineata).
Obligate intracellular bacteria infecting fish gills. Membrane-bound inclusions present as granular and tightly packed, staining basophilic under hematoxylin and eosin. Inclusions are found along the gill filament at the base, middle, and tip of the lamellae and incite a host response of cellular hyperplasia. Inclusions react with ISH 16S rRNA probes and stain purple/black. The new genus and species 16S rRNA sequence is 6.0 to 6.3% different from the 16S rRNA of “Candidatus
Parilichlamydiaceae,” placing it within this family, but not as a member of the genus “Candidatus
Parilichlamydia,” according to the classification scheme of Everett (18