MFR PAPER 1148

CLYDE J. UMPHLETT and E.M. McCRAY, JR.

A Brief Review of the Involvementsof Lagenidium, an AquaticFungus Parasite, with Arthropods

ABSTRACT-Several species of the genus Lagenidium, an aquaticplanomycetous fungus, have been reported as parasites of arthropods inclu~­

ing crabs, barnacles, and mosquito larvae. Lagenidium callinectes,.a superfi­cial parasite of egg masses of the blue crab, has on some occasIOns beenfound on as many as 40 percent of the egg masses collected from ChesapeakeBay waters. The same fungus species has been found as a parasite in the ova ofthe barnacle Chelonibia patula in waters off the North Carolina coast. Asecond species, Lagenidium chthamalophilum, has been observed in 34 per­cent of the gil/lamellae of the barnacle Chthamalus fragilis. A Lagenidium sp.has been observed in laboratory-reared brown shrimp, Penaeus aztecus, andwhite shrimp, Penaeus setiferus. Lagenidium giganteum has been shown tobe a virulent pathogen of larvae of several species of culicine mosquitoesincluding Aedes aegypti, with over 90 percent of test larvae in laboratoryexperiments killed consistently. Lagenidium giganteum has been shown to bean effective larval pathogen under field conditions also, but does not appear tobe as effective against anophelines as against cUlicines.

INFECTIONS INOTHER ARTHROPODS

Couch (1935) described in NorthCarolina the only species of

three millimeters. Development of theeggs at the interior of the sponge was notretarded by the infection. Heavily dis­eased sponges were infected to the ex­tent that about 25 percent of the eggs inthe mass contained the fungus, and in agiven sample of experimental crabssome 80 to 90 percent exhibited somedegree of infection. Development of thefungus was rapid at salinities between 5and 30 ppt, but abnormal developmentwas noted in fresh pond water.Rogers-Talbert observed also that eggsof the oyster and mud crab were at­tacked in the laboratory under condi­tions favoring very rapid transmissionof the infection. Scott (1962), in a surveyof the phycomycetous fungi of marineand brackish waters in the vicinity ofGloucester Point, Va., reported that 40percent of the blue crab egg masses col­lected were infected with Lagenidiumcallinectes. Bland and Amerson (1973)surveyed over 2,000 ovigerous crabsduring the summer of 1971 and obtainedisolates of L. callinectes with whichthey performed a detailed morphologi­cal study, but did not report the extentof the fungus in the crab population.

Another marine species has been de­scribed by Johnson (1958). L. chlha­malophilum in the barnacle Chthama­Ius jragilis was reported in 34 percentof all host lamellae inspected. Thispercentage of infection was based onhosts collected from piling and moor­ing stakes, since 86 barnacles of thesame species collected from salt marshcord grass exhibited only three infec­tions with L. chthamalophilum. At­tempts to infect the barnacle, Bala­nus amphitrite, with fungus materialfrom C. jragilis were unsuccessful.

Lightner and Fontaine (1973) re­cently observed that a Lagenidium sp.was infective to larval white shrimp,Penaeus seti/erus, and a brown shrimp,Penaeus aztecus, reared under labora­tory conditions. Natural mortality oc­curred in 12.4 percent of the shrimpafter the fungal mycelium had invadedand replaced nearly all the internal tis­sues, while 20.0 percent of the larvalshrimp died after experimental expo­sure to the fungus.

Lagenidium callinectes was de­scribed by Couch (1942) as being para­sitic in ova of the blue crab, Callinectessapidus. Johnson and Bonner (1960) re­ported the occurrence of the same fun­gus on lamellae of the barnacle,Chelonibia patula. Couch describedthe fungus mycelium as being intracellu­lar in the crab ova, whereas Johnsonand Bonner found that the preponder­ance of fungal hyphae in the barnaclewas extramatrical. They concluded,however, since the fungus transferredreadily from barnacle to blue crab eggsin cross inoculation experiments thatthe fungus on the barnacle was indeedLagenidium callinectes. In a com­prehensive study of the disease causedin blue crabs by L. callinectes,Rogers-Talbert (1948) described thefungus as a peripheral parasite of the eggmasses. She noted that the eggs weresusceptible to infection in all stages ofdevelopment. The spread of the fungusover the sponge was rapid, but it usuallyappeared to penetrate no deeper than

61

CRUSTACEAN INFECTIONS

Clyde J. Umphlett is with the Bot­any Department, Clemson Uni­versity, Clemson, SC 29631 andE.M. McCray, Jr. is with TechnicalDevelopment Laboratories, Centerfor Disease Control, Savannah,GA 31402.

Interest in environmental conserva­tion in recent years has prompted asearch for means of controlling popula­tions of noxious organisms with otherthan chemical pesticides, and there isinterest of long standing regarding dis­eases occurring in populations of desir­able and profitable organisms such ascertain crustaceans. Out of these twointerest areas has arisen a small litera­ture dealing with certain aquatic fungiamong which is the genus Lagenidium.This aquatic fungus has a long history ofparasitism, and several species havebeen recorded from a variety of hostsincluding algae, other fungi, certain in­sects, and some of the lower and highercrustaceans (Sparrow, 1960). Species ofLagenidium have been found in hostsfrom both freshwater and marinehabitats.

Figure 1.-iLagenld/um g/ganleum parasitizing Culexresluans): Non-septate hyphae growing In abdominalhemicoele.125x.

~.~

Figure 2.-(Lagenidlum giganleum paraslllzing Culexresluans): Septate hyphae dissected from host; each cell Ispotentially a sporangium, 125x.

Figure 3.-iLagenldlum glganfeum para­slllzing Culex resluans): Discharge tubeforming from sporangium and penetratingexoskeleton of host cadaver. 600 x.

Figure 4.-(Lagenldlum glganleum Figure 5.-iLagenldlum glganleum paraslllzing Culex resluans): Clusterparasitizing Culex resluans): 811lagel- of encysted zoospores on anal segment; Infeclion inlflated here. 600x.late zoospores swarming in vesicle justprior to release. 600x.

LaRenidium reported thus far to occuras a parasite in mosquito larvae. L.giganleum was described as a sap­rophyte which could function as a weak,facultative parasite of culicine mos­quitoes. Willoughby (1969) reported onthe nutrition of a saprophytic strain of

this species which he had isolated ontermite wings from a stream in England,but he did not cite any parasitic relation­ship of his isolate with mosquito larvae.One of the authors (CJU) isolated intopure culture two strains of what wasapparently L. giganleum in 1963. One

62

of the strains originated from aparasitized culicine larva, the otherfrom a parasitized anopheline larva.Both were from Orange County, N.C.A very brief and unreported test of thefungus strain from the culicine larva atthat time indicated that the fungus could

age (Fig. 7). In a short preliminary fieldtest, Umphlett and Huang (1972) re­ported that 43 percent of the larvae of C.restuans collected from the experimen­tal pool three days after the introductionof inoculum were infected, 8 percent ofthe Anopheles sp. larvae in the samecollection were infected, and 4 percentof the larvae of Psorophora sp. wereinfected with the test fungus. Larvae ofAnopheles sp. occurred in three subse­quent collections, but C. restuanslarvae did not appear in any sample afterthe first. Umphlett and Huang (1972)suggested that L. giganteum was notstrikingly effective against Anophelesspp. McCray, Umphlett, and Fay,(1973) subsequently corroborated thisby reporting no mortality in Anophelesspp. tested. However, Giebel andDomnas (In press) reported that theywere able to obtain up to 85 percentmortality of Anopheles quadrimacu­latus in some tests, but remarked thatin some experimental series no infec­tion or only a low rate, from 5 to 10

Figure 7.-Ellecl olla..al (Lv) population density(Culex resluans) on Ihe level 01 Inlecllon byLagenidium giganteum at various concentra­tions of inoculum. Note that 1.0 million zoosporesequals 6 unlls. From Umphlell and Huang, 1972.

Figure 6.-ENecl 01 larval (Lv) age In Culex res­luans an Ihe quanllfy 01 Lagenldlum glganleumInoculum required 10 kfll larvae. Nole Iha11.0 mil­lion zoospores equals 6 units. From Umphlell andHuang, 19n.

c:.!!U~ 50c:

Sampling Day ofplot treatment 3 4

Test 1 411 0 0Test 2 321 3' 0Tesl3 309 0 0Conlroll 367 75 24

percent, was obtained. McCray, Umph­lett, and Fay, (1973) extended theknown host range of L. giganteumto include Aedes aegypti, Ae.mediovittatus, A e. taeniorhynchus,A e. triseriatus, A e. so//icitans, Culexquinquefasciatus, Cu. tarsa/is, Cu.fatigans, and Cu. nigripalpus.

In a recent small field test with L.Riganteum (McCray, Womeldorf, etaI., 1973), two distinctly differenthabitats in California were utilized. Thesite near Hanford was intermittentlydry and flooded irrigated pasture land inwhich Aedes nigromaculis was theprincipal mosquito species present. Thefungus was applied in the test area byspraying sporangia into the water from aback-pack sprayer. To every squarefoot of water surface a number ofsporangia approximating the numberproduced by the fungus in one infectedfourth instar larva was applied, a poten­tial of about 250,000 zoospores persquare foot. Infection of the naturalpopulations of Ae. nigromaculis in thetest areas did occur, and all infectedspecimens died. Field populations weredramatically reduced within three daysafter treatment (Table I). At this sametest si te, larvae of C. tarsalis appearedin the treated areas subsequent to thetest. These larvae became infected bythe fungus, and all animals collectedwere found to be infected and subse­quently died.

The second study site, near Colusa,Calif., was in the vicinity of rice fieldsand associated drainage ditches. Thetest sites were not in the rice fieldsproper, but rather were isolated ditchesnearby. C. tarsalis was the target or­ganism in this area and three experimen­tal sites were chosen. The water in SiteNo. I contained a high level of dissolvedsolids and had a pH of 10.0, while SiteNo.2 had a pH of approximately 8.0,and water qualities here resembledthose of the rice fields and drainage

'All three larvae died and were infecled withLagenidium.

Post-treatment days

Table 1.-The number of living Aedes nlgromacullslarvae collecled and lound inlecled lollowlng Inlroduc­tlon 01 Lagenldlum g/ganteum allhe site near Hanford,CallI., 1972. From McCray, Womeldorf, el aI., 1973.

'0

9

___e

•

100 Lv~-_.

6 8Lv Age (Days 1

6Units of Inoculum

3

~• ~Olv

.-

4

~~'----~'~\9units •

.~~6units

3unifs"/ ~

:~:

20

10

'0

20

90

100

90

80

'00

c.go~50c

infect larvae of Aedes aegypti, but thisline of work was not pursued until 1969when an isolate of L. giganteum wasobtained from a culicine larva from oneof the original habitats.

An infection of a mosquito by L.giganteum results in the developmenlof ~

mycelium consisting of narrow, branch-ing hyphae (Fig. I) which soon increasein diameter and become septate. Thehyphal segments resulting from the sep­tations swell, thereby producing hyphaethat are constricted at the septa (Fig. 2).Within 72 h after infection has oc­curred, the coelomic cavity of the larvais about filled with mycelial growth, andin many instances hyphae can be seengrowing in the aorta of the insect. Deathof the larva occurs at this time. About 24h after an infected larva is dead, zoo­spore production is initiated by thefungus. The hyphal segments producethin discharge tubes that penetrate theexoskeleton of the dead insect (Fig. 3).Through these tubes the cytoplasm con­tained in the segments is discharged tothe outside where it is retained for a fewminutes in a membranous vesicle.Cleavage of the cytoplasm occurs in thevesicle, and the biflagellate zoosporesformed there escape when the vesiclebreaks down (Fig. 4). The zoospore isthe infectious agent (Fig. 5).

In the first report of experimentationwith L. giganteum against mosquitolarvae, Umphlett and Huang (1972)noted that this isolate behaved as a vir­ulent parasite of C. restuans in labora­tory tests. They found that the level ofinfection in larval populations variedwith the amount of inoculum which wassupplied as zoospores. Over 90 percentof 4-day-old larvae subjected to ca. 0.5million zoospores (3 units) per larvalculture were killed within 72 h afterinoculation, whereas 10-day-old larvaewith the same quantity of inoculumwere stricken only at a 5 percent level(Fig. 6). However, in tests using ca. 1.5million zoospores (9 units) per larvalculture over 90 percent of larvae at allages tested up to 10 days were killed(Fig. 6). It was noted also that when thehost population was doubled and held inthe same size container, larval mortalitywas three times that of the control when0.5 (3 units) or 1.0 million zoospores (6units) were utilized. When 1.5 millionzoospores (9 units) were applied, mor­tality above 90 percent prevailed in alltests regardless of host density or larval

63

Table 3.-Mean dally pre-and post-treatment collections of living Culex larsalls larvae and pupae from Colusa site#2 inoculated with Lagenldium glganfeum. From McCray, Womeldorf, et al.. 1973.

Day -4 -3 -2 -1 0' +1 +2 +3 +4 +5 +17

Control' 110 122 102 125 88 72 93 80 123 112 111Test3 96 96 78 93 89 88 51 36 5 0 0

'Day of inoculation.2AII instars from two plots combined.3AII instars from three plots combined.

ditches. At Site No.3 the chloride ionconcentration was about 25 times thatofthe normal habitat in which C. tarsalisbreeds. Table 2 shows the number ofliving larvae of C. tarsalis collected andfound infected following the introduc­tion ofL. gigantellm in Sites l, 2, and 3.It can be seen that in Site 2, which mostnearly resembled the normal breedinghabitat of the mosquito, a single intro­duction of the fungus infected andeliminated the natural population of C.tarsalis. The effect of the fungus onmosquito larvae was reduced, though,in Sites 1 and 3 in which water analyseshad revealed conditions known to bedetrimental to the fungus. Table 3shows the mean daily pre- and post­treatment collections of living C. tar­salis larvae and pupae from Site 2 inocu­lated with L. gigantellm. It should benoted that on the fifth post-treatmentday no living larvae or pupae were col­lected, and none appeared as late as theseventeenth post-treatment day whenthe test was terminated.

During these studies more than 1,400aquatic non-target organisms (smallcrustaceans and insects) from thetreated sites were examined. No infec­tion was observed in any of these spec­imens. Results of recent pathogenicitytests using L. giganteum at the Center

for Disease Control, Atlanta, Ga., I

indicate that the fungus is not patho­genic to small mammals.

Umphlett and Huang (1972) offeredthe opinion that there is sufficient prom­ise to dictate that further studies aimedat realization of the full potential of L.giganteum as an agent for the biologi­cal control of mosquitoes are feasible

I AjeUo, L. Chief. Medical Mycology Section,Center for Disease Control, Atlanta, Ga. Pers.commun.

Table 2.-The number of living Culex tarsalis larvaecollected and found infected following introduction ofLagenldium glganfeum at sites near Colusa, Calif.,1972. From McCray, Womeldorl, et aI., 1973.

Dayaftertreatment 2 3 5 Total

Site #1Larvaecollected 388 399 206 198 1,191Larvaeinfected 0 100 105Percentinfected 0 25.5 1.4 1.0 8.8

Site #2Larvaecollected 146 101 255Larvaeinfected 146 101 255Percentinfected 100 100 100 100

Sile #3Larvaecollected 114 81 45 46 286Larvaeinfected 0 15 3 22Percentinfected 0 18.5 6.7 8.7 7.6

and desirable. McCray, Womeldorf, etat. (1973) stated that their studies re­vealed that the Umphlett strain of L.giganteum is an excellent candidate forfurther evaluation as a biological con­trol agent, and that more definitive testsare in order.

LITERATURE CITED

Bland, C. E., and H. V. Amerson. 1973. Ob­servations on Lagenidium caJlinecles: Isola­lion and sporangial development. Mycologia65:310-320.

Couch, J. N. 1935. A new saprophytic species ofLagenidium, with notes on other forms.Mycologia 27:376-387.

---,---,---,__. 1942. A new fungus on crab eggs.J. Elisha MitcheU Sci. Soc. 58: 158-162.

Giebel, P. E., and A. J. Domnas. In press. Infec­tion of Anopheles quadrimaculalus withLageflidillm sp. J. Invenebr. Pathol.

Johnson. T. W.,Jr. 1958. A fungus parasite in ovaof the barnacle Chthamalus fragilis denticulata.Bioi. Buli. (Woods Hole) 114:205-214.

Johnson, T. W., Jr., and R. R. Bonner, Jr.1960. Lagenidillm callinecles Couch in barnacleova. J. Elisha MitcheU Sci. Soc. 76: 147-149.

Lightner, D. V., and C. T. Fontaine. 1973. A newfungus disease of the white shrimp Penaeusseliferus. J. Invertebr. Pathol. 22:94-99.

McCray, E. M., Jr., C. J. Umphlett, and R. W.Fay. 1973. Laboratory studies on a new fungalpathogen of mosquitoes. Mosquito News33:54-60.

McCray, E. M., Jr., D. J. Womeldorf, R. C. Hus­bands, and D. A. Eliason. 1973. Laboratoryob­servations and field tests with Lageflidiumagainsl California mosquitoes. Proc. Calif.Mosq. Control Assoc. 41: 123-128.

Rogers-Talben, R. 1948. The fungus Lagenidiumcallinecles Couch (1942) on eggs of the blue crabin Chesapeake Bay. BioI. BuU. (Woods Hole)95:214-228.

Scott, W. w. 1962. The aquatic phycomycetousflora of marine and brackish waters in the vicinityof Gloucester Poinl, Virginia. Va. Inst. Mar.Sci., Spec. Sci. Rep. 36, 16 p.

Sparrow, F. K., Jr. 1960. Aquatic phycomycetes,2nd ed. Univ. Michigan Press, Ann Arbor, 1187p.

Umphlett, C. J., and C. S. Huang. 1972. Ex­perimental infection of mosquito larvae bya species of the aquatic fungus Lagenidium. J.Invertebr. Palhol. 20:326-331.

Willoughby, L. G. 1969. Pure culture studies onthe aquatic phycomycete, Lagetlidium gigan­lcum. Trans. Br. Mycol. Soc. 52:393-410.

MFR Paper 1148. From Marine Fisheries Review, Vol. 37, Nos. 5-6,May-June 1975. Copies ot this paper, in limited numbers, are availablefrom 083, Technical Information Division, Environmental Science Infor­mation Center, NOAA, WaShington, DC 20235, Individual copies of MarineFisheries Review are available from the Superintendent of Documents,U.S. Government Printing Office, WaShington, DC 20402 for $1.10 each.

64