Hongwei Ma

Parasitic crustaceans as vectors of viruses, with an emphasis on three penaeid viruses

Parasitic crustaceans serve as both hosts and vectors of viruses as well as of parasites and other microbial pathogenic agents. Few of the presumably numerous associations are known, but many can be anticipated. Recently, branchiurans and gnathiid isopods have been documented to host helminths and blood parasites. Because the agents can be observed readily with a microscope, these are better recognized than are the smaller viral, bacterial, and fungal agents. Some agents are harmful to the host of the crustacean parasite and others are not. Viruses probably fit both these categories, since viruses that do not appear pathogenic are often seen in ultrastructural images from a range of invertebrate hosts, including crustaceans. Some viruses have been implicated in causing disease in the host, at least under appropriate conditions. For example, lymphocystis virus may possibly be transmitted to the dermis of its fish hosts by copepods and to the visceral organs by a cymothoid isopod. Similarly, argulid branchiurans seem to transmit the viral agent of spring viremia of carp as well as carp pox, and copepods have been implicated in transmitting infectious hematopoietic necrosis, infectious salmon anemia, and infectious pancreatic necrosis to salmon. Other viruses can be vectored to their hosts through an additional animal. We exposed three viruses, Taura syndrome virus (TSV), white spot syndrome virus (WSSV), and yellowhead virus (YHV), all of which cause mortalities in wild and cultured penaeid shrimps, to crustacean parasites on fish and crabs. Using real-time polymerase chain reaction analysis, we show that TSV in the cyclopoid copepod Ergasilus manicatus on the gill filaments of the Gulf killifish, Fundulus grandis, the acorn barnacle Chelonibia patula on the carapace of the blue crab, Callinectes sapidus, and gooseneck barnacle Octolasmis muelleri on the gills of C. sapidus, can replicate for at least 2 weeks and establish what should be an infective dose. This result was additionally supported by positive in situ hybridization reactions. All three parasites are the first known non-penaeid hosts in which replication occurs. The mean log copy number of WSSV also suggested that replication occurred in E. manicatus. The mean log copy number of YHV gradually decreased in all three parasites and both hosts over the 2-week period. The vector relationships indicate an additional potential means of transmitting and disseminating the disease-causing agents to the highly susceptible and economically valuable penaeid shrimp hosts.

Phylogeny of Six Genera of the Subclass Haptoria (Ciliophora, Litostomatea) Inferred from Sequences of the Gene Coding for Small Subunit Ribosomal RNA

ABSTRACT. The small subunit ribosomal RNA genes of nine species belonging to six genera of litostome ciliates, namely Amphileptus aeschtae, Chaenea teres, Chaenea vorax, Lacrymaria marina, Litonotus paracygnus, Loxophyllum sp.‐GD‐070419, Loxophyllum jini, Loxophyllum rostratum, and Phialina salinarum, were sequenced for the first time. Phylogenetic trees were constructed using different methods to assess the inter‐ and intra‐generic relationships of haptorians, of which Chaenea, Lacrymaria, Litonotus, and Phialina were analyzed for the first time based on molecular data. Monophyly of the order Pleurostomatida was strongly confirmed, and the two existing families of pleurostomatids, created on the basis of morphology, were confirmed by molecular evidence. Within the Pleurostomatida, Siroloxophyllum utriculariae occupied a well‐supported position basal to the Loxophyllum clade, supporting the separation of these genera from one another. Both the subclass Haptoria and the order Haptorida were partially unresolved, possibly paraphyletic assemblages of taxa in all analyses, creating doubts about the traditional placement of some haptorid taxa. The existing sequence of L. rostratum in GenBank (DQ411864) was conspicuously different from that of the isolate from Qingdao, China sequenced in the present work, indicating that they are different species. The isolate from Qingdao was verified as L. rostratum by morphological analysis, and the published morphology of existing GenBank record of L. rostratum is different from it. Based on both morphological and molecular evidence, the latter may be congeneric with an undescribed species of Loxophyllum from Guangdong Province, China.

Two New Species of Epistylis (Ciliophora: Peritrichida) on the Blue Crab (Callinectes sapidus) in the Gulf of Mexico

ABSTRACT. Two epibiotic peritrichs infested the blue crab, Callinectes sapidus, from the Gulf of Mexico, Mississippi, USA. Epistylis callinectes n. sp. was isolated from the epipods of maxillipeds, bases of gill‐cleaning setae, and gills, and Epistylis clampi n. sp. was isolated from the exterior surfaces of the exoskeleton. Epistylis callinectes has short, symmetrically and dichotomously branched stalks; its zooid is elongate ovoid and conspicuously longer than the individual stalk branches, measuring 40–57 (49) × 18–33 (26) μm in vivo and containing a thick, undivided peristomial lip (PL). It has a single contractile vacuole and a transverse horseshoe‐shaped macronucleus. Its haplokinety (H) and polykinety (Po) complete one and one‐half circuits on the peristome before entering the infundibulum. There is a distal kinetal fragment present at the distal end of both the H and Po. Epistylis callinectes has 48–70 transverse silverlines from the oral area to the trochal band (TB) and 19–26 from the TB to the scopula. Epistylis clampi has long, asymmetrically, and dichotomously branched stalks. Its zooid is elongate vase‐shaped, measuring 35–64 (48) × 21–30 (27) μm in vivo and with a thick, transversely folded PL. The stalks supporting zooids are unequal in length. Its zooid has a single contractile vacuole and a transverse horseshoe‐shaped macronucleus occurs in the upper half of the body. Its H and Po complete approximately one circuit around the peristome before entering the infundibulum. There is a distal kinetal fragment present at the distal end of both the H and Po. This species has 71–112 transverse silverlines from the peristome to the scopula.

Daggerblade grass shrimp (Palaemonetes pugio): A reservoir host for yellow-head virus (YHV)

Yellow-head virus (YHV) is a major pathogen in penaeid shrimps. We surveyed 13 crustacean species in eight families from two orders that are commonly found in the Mississippi coastal area and freshwater environments as potential reservoir or carrier hosts of YHV. Using semi-nested polymerase chain reaction (PCR) on relatively small sample sizes, we did not detect any natural infection. However, when the daggerblade grass shrimp, Palaemonetes pugio, and the blue crab, Callinectes sapidus, were exposed to YHV by injection and per os, YHV was detected in the tissue of P. pugio and in the hemolymph of C. sapidus when tested by semi-nested reverse transcription PCR (RT-PCR) and real time quantitative reverse transcription PCR (qRT-PCR). YHV replicated in P. pugio, causing 8% mortality (9/112) after injection, with the viral titer reaching a peak at 14days post-inoculation (dpi) and remaining detectable at 36dpi. The number of infected animals and viral load, however, were relatively low but the virus still remained infectious to penaeids when administered by feeding. When YHV was injected into P. pugio, in situ hybridization detected a positive response to it at 7dpi in connective tissue of hepatopancreas, muscle, and midgut. Viral RNA in injected C. sapidus remained at a low level for 3days, and it was not detected from 7dpi onwards. In fed C. sapidus, the viral RNA reached a peak at 3dpi and still detectable at 7dpi, but it became undetectable at 14 and 21dpi. These data suggest that P. pugio under some conditions could act as a reservoir host for YHV but that the blue crab could serve as a poor, short term carrier host only.

Stomatogenesis of the marine ciliate Paranophrys magna (Protozoa: Ciliophora: Scuticociliatida) from Qingdao, China

The morphology and stomatogenesis of the marine scuticociliate Paranophrys magna, collected from a crab-culturing pond in Qingdao, China, were studied. It possessed typical characteristics of the genus Paranophrys. The stomatogenesis of this species corresponds basically with the results provided in previous studies for this genus, the main sequence develops can be generalized as follows according to the Qingdao population: (i) in the proter: the remnant of the parental paroral membrane generates the new paroral membrane and the scutica; and (ii) in the opisthe: both the paroral membrane and scutica originate from the proliferation of the anterior part of the secondary primordial field, membranelles 1 & 2 derive from the posterior part of the secondary primordial field, while membranelle 3 from the proliferation of the parental scutica which form the primary primordial field. Some differences between our results and previous descriptions are compared and discussed.

Two New Species of Symbiotic Ciliates from the Respiratory Tract of Cetaceans with Establishment of the New Genus Planilamina n. gen. (Dysteriida, Kyaroikeidae)

ABSTRACT. Examination of mucus discharged from the blowhole of Atlantic bottlenose dolphin (Tursiops truncatus) at Marine Life Oceanarium, Gulfport, Mississippi, and false killer whale (Pseudorca crassidens) and Atlantic bottlenose dolphin at SeaWorld Orlando, Orlando, Florida, using live observations and protargol impregnation revealed mixed infections of Kyaroikeus cetarius and two new species. Planilamina n. gen. is characterized by a C‐shaped argentophilic band located along the laterally flattened margin of cell and extending from the cell apex to subposterior cone‐shaped podite; a deep oral cavity containing one short preoral kinety, two circumoral kineties, seven to 13 infundibular kineties, and a cytostome; a broadly funnel‐shaped cytopharynx reinforced by argentophilic fibers but without nematodesmata; closely packed postoral kinetofragments set in a pocket located anterior left of the podite; and somatic kineties as a right field closely situated at the right surface and a left field bordering the anterior left margin of the oral cavity. The type species for the genus, Planilamina ovata n. sp., is distinguished from its sister species Planilamina magna n. sp. by the following characteristics: body size (28–65 × 20–43 μm vs. 57–90 × 40–63 μm), number of right field kineties (38–55 vs. 79–99), and position of the anterior end of the leftmost kinety in the right somatic field (anterior one‐third vs. mid‐body). The morphogenesis of Planilamina ovata is similar to that of K. cetarius. The diagnosis of Kyaroikeidae is emended to accommodate the new genus.