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ORGANISATION MONDIALE DE LA SA WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 ~~1 ' ENGLISH ONLY DATA SHEET ON THE BIOLOGICAL CONTROL AGENT(l)~~Li~:i~} Lagenidium giganteum (Couch 1935) ~rvv~

L. giganteum is a facultative parasitic fungus which can grow vegetatively either as a saprophyte in the aquatic environment or as a parasite of mosquito larvae. Its life cycle is typical of the genus Lagenidium with both asexual (zoospore) and sexual (oospore) stages The zoospore of the fungus is the infectious agent. The mos­ quito larva is killed when the fungus invades theh~mocoel and forms an extensive mycelium throughout the body.

Several isolates of L. giganteum have been reported and differences have been noted in their pathogenicity and mosquito host-range although in general the species has been shown to infect a wide range of Aedes and Culex species with some activity against Anopheles, Psorophora and Culiseta. The specificity of the fungus appears to be limited to mosquitos and one gnatspecies (to a lesser extent); no evidence of pathogenicity was found in non­ target organisms: 1196 aquatic invertebrates (30 species,7 orders) tested in the laboratory a wide variety of aquatic crustaceans and insects found associated with Cx. tarsalis, as well as fish, birds and rats.

Basic studies on the biochemistry of ~· giganteum infection in mosquito larvae have been carried out, which include carbohydrates sources, nitrogen, lipids and, most recently, sterols requirements.

The practical use of L. giganteum for biological control of mosquito larvae requires a simple and rapid meansof mass production of infective zoospores. In vitro culture of the asexual stage of ~· giganteum has been developed and this is a great improvement over the in vivo culture that was used in the earliest studies. Significant progress has also been made on the development of a medium for in vitro production of o 3pores (sexual stage). This is important because it is generally thoughtthat the oospore, as opposed to the zoospore, is responsible for the preservation and subsequent recycling of the fungus during seasons of adverse conditions.

Although~· giganteum has very little tolerance for salinity, organic pollution and has somewhat narrow temperature limits, it has important attributes (facultatively parasitic nature, specificity for mosquito larvae, ability to recycle every three days under suitable conditions and a dormant stage which enables it to survive seasons of adverse conditions) which ma¥e this fungus a most promising biological control agent (1980-1, 1981-2, 1983-1, 1984-7).

1. Identification and synonyms

Class Oomycetes, order Lagenidiales

Lagenidium giganteum Couch (1935-1)

Keys to the identification of the fungus and photographs of hyphae and sporangia can be found in reference 1978-6.

(1) Information document produced by the WHO Division of Vector Biology and Control. This is the first revised edition of the data sheet which appeared as WHO/VBC/79.753 and VBC/BCDS/79. 02 . May 1985 The issue of this document does not constitute Ce document ne constitue pas uue publication formal publication. It should not be reviewed, II ne doit faire l'objet d'aucun compte rendu ou abstracted, quoted or translated without the resume ni d'aucune citation ou traduction sans agreement of the World Health Organization. l'autorisation de !'Organisation mondiale de Ia Authors alone are responsible for views expressed Sante. Les opinions exprimees dans les articles in signed articles. signes n'engagent que leurs auteurs.

------WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 2

2. Origin

L. giganteum was described originally from larvae of mosquitos and from copepods and daphnTds collected in North Carolina and Virginia, respectively, USA (1935-1, 1960-1).

Most of the observations recently reported were made with a strain obtained from an undetermined species of Culex from Oregan County, North Carolina, USA (infective only to Culicidae) (1970-1, 1972~

This aquatic fungus was provisionally named L. culicidum (1970-1) until taxonomic re­ vision indicated the organism to be a strain of~~ giganteum (1973-5).

3. Natural geographical distribution (1981-2, 1982-3)

USA: North Carolina, Virginia, Louisiana (1935-1, 1978-4); UK: Lake District (1969-1); Uganda: Lake George (1969-1); Antarctica: South Orkneys islands (1969-1) (these last three strains were assigned to ~· giganteum because of morphological similarities with~· giganteum Couch; however, there are morphological as well as significant biochemical differences between the two ~ (1982-3); - India: (1973-1).

A related Lagenidium sp. was found parasitizing eggs of Armigeres dentatus Barraud sent from Malaysia (1972-1, 1972-2, 1977-8).

A mosquito-pathogenic species of Lagenidium (with close resemblance to L. giganteum) has been isolated into pure culture from a dead larva of Ae. pseudoscutellarTs in Fiji, but neither sporangia· nor oospores were observed.

More recently, an oomycetous fungus closely related to L. giganteum, isolated from the nematode Romanomer.mis iyengari in India, was found to be pathogenic to larvae of both culi­ cine and anopheline mosquitos (1984-1).

Thus, the existence of these various strains and isolates and their differences imply that specific strains maybe more suitable than others in a given situation and that consi­ derable potential exists for the selection of infectivity, virulence and specificity (1982-3).

4. Biological characteristics

~· giganteum is a facultative parasitic fungus which can grow vegetatively either as a parasite of mosquito larvae or as a saprophyte in the aquatic environment (1935-1, 1972-3, 1973-1), where it apparently prefers a littoral habitat (1969-1).

4.1 Life cycle (1935-1, 1972-3, 1973-1, 1973-2, 1973-3, 1976-4, 1982-3, 1983-3)

The life ~ycle of the organism is relatively simple but involves two separate cycles (sexual and asexual reproduction).

In the aquatic environment, the asexual cycle maintains the fungus. The parasitic phase is initiated by a motile, laterally biflagellate zoospore which is the infective unit. Zoospores either encyst on the insect cuticle or are ingested and attach to the anterior portion of the digestive tract, usually the region of the pharynx. Less frequently the zoos­ pores penetrate at the base of the anal gills or randomly on the body. Once fixed, the zoospores form a germ tube which penetrates the cuticle and grows inward as a hypha. It has been suggested (1974-2) that a proteolytic or lipolytic enzyme in conjunction with mechani­ cal pushing through weaker layers might be operating as means of penetration. Characteristic WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 3 melanization occurs on the cuticle on these sites of attack. Later, once the initial hypha reaches the haemocoel, it branches into a nonseptate mycelium which ramifies throughout the head and body cavity. Then mycelium vegetative growth ceases and reproduction begins (Figure 1). Larval death occurs at this stage, ca.48-72 h after infection (should the larva die early during infection, the fungus will also die without reproducing). The mycelium then forms septa, each segment becoming either an asexual sporangium, an antheridium, or an oogonium.

When asexual reproduction occurs, which is the most common case, the sporangia swell and become spherical in shape; exit tubes are then developed which grow out through the cuticle and form terminal zoosporogenic vesicles. These vesicles begin vibrating with the movement of the enveloped zoospores until the membrane ruptures and the zoospores swim away, actively seeking a suitable host or subtrate to start the cycle again (Figure 1).

Figure 1. * h· giganteum life cycle. A. Zoospores. 8-E. Zoospores encyst and hyphae develop saprophytically. F. Hyphae segment into sporangia. G-I. Discharge_tube develops, cytoplasm discharged into vesicle, and_zoospores c~eaved. J. Ves1cle rup­ tures, re1easing zoospores. K. Zoospores encyst1ng on_mosq~1to la~va. L-M. Hyphae ramify throughout body and form sporangia. N. Sporang1 urn d1 scharg1 ng zoospores· The saprophytic cycle and parasitic cycle only differ by the nature of the mycelium subtrate: living host or organic matter.

When sexual reproduction takes place, (Figure 2) antheridia and oogonia from the same or different mycelium fuse and form thick walled oospores, the resistant stage.

* This illustration was originally published in reference 1976-4 and reproduced with kind permission of Dr. Thomas M. Me Innis. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 4

Figure 2.* Motile biflagellate zoospores (A) selectively encyst on mosquito larva and mycelium proliferates throughout the body of the host (B). Each nypaal segment can undergo reproductive development in one of two ways. Asexual zoos­ pores can be produced from a single cell after differentiation in a vesicle at the tip of an exist tube (C). Alternatively, two adjacent cells can fuse (D) during sexual development and produce a dormant oospore (E). After maturation, oospores can be activated by hydration to pro­ duce zoospores, morphologically identical to asexual zoospores, and a new infection cycle can begin. /

A detailed description of gametangia! copulation and oosporogenesis is given in reference 1985-1.

The sexual cycle maintains the fungus through periods of adverse seasons (winter and summer drought). If free water should no longer be available and dessication sets in, the oospores depOsited on the sub~rate will survive until reflooding, then germinate, produce a mycelium and, subsequently zoospores which will initiate the cycle for the aquatic environ­ ment.

4.2 Physiological and biochemical investigations on L. giganteum

Several aspects of the physiology and biochemistry of~· giganteum have been examined.

Nutritional studies have shown that trehalose is by far the best single carbon source for L. giganteum with glucose, mannose, fructose, maltose and glycerol also promoting good growth.

* This illustration was originally published in reference 1984-6 and reproduced by kind permission of Drs. Kerwin and Washino. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 5

Nitrate and ammonium tartrate are not used, but aspartate, glutamate and glutamine are the best nitrogen sources. The fungus is vitamin autotrophic although thiamine enhances growth (1971-1).

L. giganteum is unable to synthesize alkanes from glucose or palmitate nor do it meta­ bolizes some n-alkanes (1981-5).

L. giganteum possesses an intracellular (3-D-glucosidase and a trehalase (1971-1, 1973-4, 1974-3).

The~-D-glucosidase ftom Lagenidium shows preferential act1v1ty for p-nitro-phenyl-{>­ glucopyranoside (PNPG), indicating the best in vivo subtrate would be an aromatic glycoside, releasing glucose from subsequent uptake and catabolism.

Trehalase which catalyses the hydrolysis of the disaccharide~ .~'-trehalose to glucose has been found in microorganisms, fungi and higher plants; its occurence is most widespread in insects however (the sugar~ ,ol.l-trehatose is the major carbohydrate of insects).

Suggested physiological roles for the trehalase include the breakdown of stored treha­ lose in the mycelium and zoospores, and the digestion of hemolymph trehalose in the infected mosquito larvae. Comparison of the properties of the trehalases from Lagenidium and Culex mosquitos strongly suggests that during infection, trehalose in the mosquito larvae is pre­ ferentially utilized by the fungal parasite. Parasitism by Lagenidium involves a general "physiological starvation" of the host (1974-2) in which trehalose represents an important part of the nutrients which are depleted. As trehalose levels in the larvae drop, the energy reserves of the larvae are decreased and this, along with the depletion of other nutrients, result in larval death (1976-3, 1978-3).

L. giganteum produces extra cellular proteases when grown in peptone-yeast extract-glu­ cose medium. The production of the enzymes requires a suitable inducer such as protein and is repressed by glucose. When the growth medium is supplemented with sterol ~holesterol,sitosrerol campesterol or ergosterol), the cultures will double to triple the protease output and the growth time will be halved (1978-1). These proteases may conceivably function to facilitate penetration of the cuticle by the encysting zoospore. They may additionally permit ready emergence of the zoosporangial discharge tube through the partially digested cuticle of the cadaver. The proteases of ~· giganteurri are capable of digesting mosquito cuticle in vitro, yet no evidence has been produ·ced to indicate that these enzymes are acting in either an invasive or destructive manner in the larvae (1983-2).

Two decades ago, it was found that addition of sterols to basal growth media changed the mode of growth of fungi belonging to the Pythiaceae (Oomycetes: Peronosporales) from vegetative proliferation to reproductive development (1964-1).

Similar requirements have been foundfor asexual (zoosporogenesis) (1976-2, 1977-1, 1977-4. 1977-5. 1977-6, 1981-1) and sexual (oosporogenesis) reproduction for L. giganteum (1983-8).

It was first observed in 1971 that hemp seed (Cannabis sativa) oil had some effect on zoospore growth (1971-1, 1974-1). Oil-rich materials were found to be very effective inducers for zoospores and this oil requirement'was associated with sterols. The best individual exo­ genous sterols for in vitro zoosporangial growth are sitosterol and campesterol and less effectively ergosterol and cholesterol (1977-6). L. giganteum (and L. callinectes, a Crustacean parasite) possesses a cycloartenol-based sterol synthetic pathway; -when the organism is given mevalonic acid (or other suitable sterol precursors) in its growth medium, zoospores are produced and cholesterol ii identified as the sterol found. The apparent metabolic route leading to cholesterol is cycloartenol-~ fucosterol ~cholesterol, which is similar to WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 6 the overall metabolic sequence observed in many algae (1981-4, 1983-11). It should be noted that the four major sterols extracted from six species of 4th instar mosquito larvae (Ae. aegypti, Ae. albopictus, Ae. epactius, ~· taeniorhynchus, ~· quinquefasciatus, An. -­ stephensi) after growth in a non-sterile environment with a complex undefined diet were those which maximally induced zoosporogenesis in L. giganteum: the 24- alkyl sterols and cholesterol (1984-5).

Under appropriate conditions, Lagenidium will produce viable oospores in vitro also when appropriate sterols are incorporated into a basal growth medium. Following prolonged mainte­ nance on sterol-free medium, the fungus retains its ability for zoosporogenesis, but oospore­ genesis does not occur. Oospore yield is affected by changes in the sterol side chain, espe­ cially at C-24, and by the degree and position of ins~turation in the sterol ring. Oospore yield and viability can be enhanced by incorporating fatty acids in the growth medium, espe­ cially in the form of triglycerides. Oosporogenesiswhen induced by fatty acid sterol esters is much reduced relative to that obtained using free sterols (1983-8).

Research on the role of cyclic nucleotides in regulating oosporogenesis has been com­ pleted. Incorporation of phosphodiesterase inhibitors, which cause the accumulation of cyclic nucleotides, into basal growth media limits the induction of oosporogenesis by L. giganteum (and related sterol auxotrophic pythiaceous fungi). Cyclic AMP (adenosine monophosphate) en­ hanced while cyclic GMP (guanosine monophosphate) antagonized the effects of these compounds. Adenosine, guanosine, and their 5'- monophosphate derivatives acted primarily by reducing oospore maturation rather than induction. Asexual reproduction was generally promoted by cyclic AMP and phosphodiesterase inhibitors. It is suggested that cyclic nucleotides and sterols play a pivotal role in the induction of meiosis and that sterols and other factors affecting reproductive development by these fungi may act in part by regulating cellular cyclic nucleotide levels (1984-4, 1984-6).

4.3 Sensitivity to environmental factors

a) Temperature

It was first reported (1971-1) that vegetative growth of the fungus in simple liquid media had a narrow optimum around 30°C.

The temperature optimum for zoosporogenesis in solicl media was found to be 2l-270C (1983-7). In liquid culture, zoospore production occurred at a temperature range of 17-280C with the optimum production at 25-28°C (1982-2).

From field studies (1977-9, 1982-1, 1982-3) data indicate that transmission of the fungus is limited to a range of 20-34°C. Overwintering studies in North Carolina revealed that when water temperature dropped to below 18°C, infection of mosquito larvae by the fungus ceased, and that Lagenidium did not survive a winter during which water temperatures fell to 4°C and surface ice formed on the experimental lagoons (1983-6). Also, if the temperature is much above 30°C for appreciable periods of time, the fungus will not be effective against mosquito larvae (1982-1). Laboratory studies with the North Carolina (NC) and Louisiana (LA) isolates of L. giganteum have shown that the highest larval infection rates occur at 21-29°C for both isolates. There was a marked decrease in the infection rate above 29°C and below 21°C. This temperature range corresponds to the range for zoospoiQgenesis (1983-7).

Sporulation by the NC isolate in larvae was optimal from 19 to 32°C, inhibited below 19°C and completely absent at 10°C. The LA isolate sporulated in larval cadavers less success­ fully; the optimum temperature was 18°C. At temperature above 21°C, the infected larvae were killed too rapidly for the fungus to attain maturation, and the Lagenidium perished. When cadavers with fungus of either isolate were subjected to 10°C for 4 days, and then returned to 25°C, the fungus successfully sporulated, but after 10 days at 10°C, no sporulation occurr­ o ed upon return to 25 C. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 7

The difference between the NC and LA isolates suggests that both high and low temperature tolerant isolates may exist in nature. If these different isolates can be found and cultured, then possibly a strain tailored for specific environmental tolerances could be selected for appropriate habitats (1983-5).

b) pH, light and salinity

Hydrogen ion concentration has little importance in zoospore production. L. giganteum readily infects mosquito larvae within the pH range of 4.5-8.4 in both laboratory and field experiments. This pH range includes most larval breeding habitats likely to be encountered in the field. (1982-1, 1982-4, 1985-3). Buffers, particularly phosphate buffer have are­ pressive effect on zoospore produdction (1982-2). Light has no effect on zoospore output, but the zoospores accumulate in the surface layer. This behaviour is significant as mosquito larvae remain largely in this region during their lives, and thus contact between zoospores and larvae is enhanced (1982-2).

Early studies have shown that L. giganteum had little tolerance to salinity (1973-]. NaCl concentration of 5;;/1 al:::ost completely suppressed zoospore production and concentrations above 15g/1 prevented saprophytic growth or produced a distored mycelium (1978-5, 1979-4),

Later laboratory studies showed that the highest concentration of salt water containing Lagenidium that produced an infection in Cx. tarsalis larvae was 5% artificial sea water (i.e. 1. 7g/l) which produced 14 infections out of 40 treated larvae (1980-2).

Since some important pestiferous and disease vectoring Aedes mosquito species are pro­ duced in brackish water habitats such as salt marshes and diked~ dredged~aterial disposal sites, the degree to which L. giganteum can be considered part of an integrated pest manage­ ment program (1979-1) for mosquitoes in coastal areas depends, in part, upon the tolerance of the fungus to salinity.

Laboratory experiments were thus conducted to determine the salinity range over which North Carolina (NC) and Louisiana (LA) isolates of the fungus grow vegetatively and infect Aedes larvae. The mycelial growth rates of the two isolates on nutrient agar were increased with 2.5 and 5.0 g/1 of NaCl added, and reduced with 7.5g/l or more NaCl. The LA and NC iso­ lates did not grow on agar containing 20 and 30 g/1 NaCl respectively. The concentrations of NaCl for 50% inhibition (Ic50 ) of mycelial growth of the LA and NC isolates (10.9 and 12.0g/l, respectively) were not significantly different. The ability of the two isolates of L. giganteum to infect larvae of Ae. taeniorhynchus decreased as salinity of the water in­ ~reased. The IC50 values for the inhibition of infection in mosquito larvae by the LA and NC isolates (0.52 and O.SSg/1 NaCl respectively) were not significantly different. Microscopic examination of the fungus in saline and distilled water showed that NaCl inhibited the pro­ duction of zoospores. In water containing l.Sg/1 there was two complete inhibition of zoospo­ rogenesis and mosquito infection in each isolate of L. giganteum. Zoosporogenesis was ca. 22 times more sensitive to salinity than was mycelial g;owth (1982-1, 1982-6). NaCl at a concen­ tration of 0,6g/l virtually eliminated zoosporogenesis. At 0.2g/l NaCl there was a significant reduction in four of the five fungal isolates tested in this particular study. Transmission of the fungus from Cx. pipiens to Ae. taeniorhynchus larvae was achieved in concentrations of up to 0.8g/l NaCl, whereas zoospores were not produced in vitro at this concentration, showing that zoosporogenesis in vivo is less subject to inhibition by NaCl than is zoosporogenesis in vitro (1985-3).

L. giganteum did not infect Ae. taeniorhynchus larvae in laboratory tests conducted in different sized containers filled-with dredged-spoil (water salinity ranged from 1.6 to 1.9g/D and in a field test in enclosures in a salt marsh pool (lOg/1 water salinity) (1982-7).

Dilute aqueous solutions (0.0005 M) of MgS04, MgC1 2, CaC1 2 • 2H2o and MnC12 • 4H20 were WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev, 1 page 8 also tested. Zoospore production was greatly or completely repressed when mycelia were immersed in these solutions rather than in water. KCl at the same concentration caused only moderate inhibition, while NaCl had little effect on zoosporogenesis (1982~2).

c) Organic water pollution Low levels of organic pollution prevented the establishment of Lagenidium in rice field water containing organic wastes (1977-9) and in pilot-scale animal waste lagoons (1982~4), The presence of organic pollution in the water prevented the formation of sporogenic vesicles by the fungus and drastically reduced the viability of any zoospores that were produced. Ammomium nitrogen (NH3N) and total phosphorus P were statistically significant predictors of pollution effects on infection of larvae by Lagenidium. Less than lOmg/1 of either drasti­ cally depressed infection rates. It should be noted that habitats in which Cx, quinquefascia­ tus oviposits may have much higher levels of organic pollution than that of the pilot scale lagoons or samples of polluted water used in this study (1982-1, 1982-3, 1982-4).

d) Oxygen, organic solutes and presence of other microorganisms

Total removal of oxygen from the water in ~hich L. giganteum mycelium was suspended ter­ minated zoosporogenesis. Reintroduction of atmospheric 02 within 7 days allowed the process to take place. Recovery from 02 deprivation was incomplete after deprivation for just one day and was diminished with time of anoxic treatment until, at 9 days, all of the fungus had died (1985-3).

Three sugars (glucose, arabinose, sucrose), a sugar alcohol (mannitol), three amino acids (methionine, glycine and glutamine) and peptone all reduced zoosporogenesis at levels that generally correlated positvely with their nutritional values for the fungus. Peptone, which can support lush vegetative growth of L. giganteum in the absence of other nutrients was far more effective in inhibiting zoosporogenesis than the other solutes (1985-3).

Five species of bacteria (Pseudomonas sp, Pseudomonas syringae, Escherichia coli, Bacillus thuringiensis var. israelensis, Corynebacterium nebraskense) inhibited zoosporogene­ sis at concentrations of 10l-1o8 cells/ml. The yeast Saccharomyces cerevisiae was inhibitory at 106 cells/ml.

These results are of great importance in the eutrophic habitats of many mosquito species, where L. giganteum must compete with saprophytes and scavengers to complete its life cycle be­ fore its host is decayed or eaten (1985-3).

e) Pesticide toxicity (1982-1, 1983-10)

Due to the use of certain insecticides as larvicides for mosquito control and herbicides for weed control, and the contamination of aquatic habitats by pesticide transport from agricul­ tural land&, the compatibility of L. giganteum with various pesticides in the aquatic environment may be a critical factor in its ability to survive and function as an effective microbial control agent for mosquitos. So laboratory experiments were conducted to determine the relative toxicity of certain insecticides, herbicides, and fungicides to the vegetative growth and zoospore pro­ duction in North Carolina (NC) and Louisiana (LA) isolates of L. giganteum. Zoospore production in both isolates was less tolerant than mycelial growth to most of the pesticides tested, s1nce zoospore production was often completely inhibited in lower pesticide concentrations than those which partially inhibited mycelial growth on agar, or resulted in mycelial plasmolysis in water. The most toxic pesticides to one or both isolates of L. giganteum were captan, gamma-BHC, DDT, toxaphene, chlorpyrifos, and fenthion. These pesticides produced at least 10% inhibition of mycelial growth rate or completely inhibited zoospore production at concentration below 5mg/l. The least toxic pesticides caused a 10% inhibition in mycelial growth rate and failed to inhibit zoospore production at concentrations greater tahn 50mg/l. These were diflubenzuron, WHO/VBC/79. 753, Rev. 1 VBC/BCDS/79.02. Rev. 1 page 9 permethrin, temephos and propoxur (LA isolate only), Pesticides of int~rmediate tox1c1ty were: malathion, carbaryl, methoprene, alachlor, andatmzine, The two isolates differed substantially in their tolerance of the following pesticides: malathion, chlorpyrifos, toxaphene, carbaryl, propoxur, permethrin, and methoprene. At their recommended rates of application for control of mosquito larvae, methoprene, fenthion, malathion and temephos would probably be compatible with both isolates of L. giganteum.

It should be noted that the physical nature of the culture medium may affect the fungi­ toxicity of certain pesticides to the fungus i,e, : mycelial plasmolysis in Lagenidium was observed in water containing either chlorpyrifos or DDT at concentrations far below their respective mycelial LC50 values in agar. Other factors which may affect the toxicity of cer­ tain pesticides to L. giganteum are the nutritional composition in which the fungus is ex­ posed and the possible interaction between the fungus, the pesticide and the organic solvent used to dissolve the pesticide, Final tests should thus be conducted under field conditions as data in this study were gathered from laboratory experiments on artificial media.

5. Effectiveness against target organisms

5.1 Under laboratory conditions

L. giganteum was originally described as a saprophyte with weak paras1t1c properties (1935-1) but the strains isolated since in the USA (1972-3, 1978-4) have shown greater patho­ genicity particularly for mosquitos. Host specificity seems to vary from strain to strain, one strain having less effectiveness against Anopheles larvae (1972-3) than against Culicine larvae.

Altogether the following species were infected (1935-1, 1972-3, 1973-1, 1973-2, 1975-1, 1976-6' 1978-4): - An quadrimaculatus, An. freeborni, An. punctipennis, An. stephensi; - Ae. aegypti, Ae, epactius, Ae. mediovittatus, Ae. nigromaculis, Ae. polynesiensis, Ae. sollicitans, Ae. taeniorhynchus, Ae. triseriatus; - Cx. quinquefasciatus, Cx. nigripalpus, Cx. tarsalis, Cx. territans, Cx. restuans; - Cs. inornata, Cs. incidens; - Psorophora sp.

Laboratory infection rates were usually very high, resulting in most instances in a complete kill of exposed larvae when infection took place during the first half of the IVth instar or earlier. Older larvae, pupae and adults were not susceptible (1973-2).

No accurate quantification of the fungal inoculates were made but orders of magnitude were determined on the basis of one dead infected mosquito larva producing about 178,640 zoospores (1972-3) to 250,000 zoospores (1973-3). 100% mortality level was obtained with inoculates comprised between 715,000 for 100 ml of water and 1,600,000 for 1 litre of water (1972-3, 1973-2).

Results of early laboratory studies on the susceptibility of An. freeborni to L. giganteum infection have been incosistent. 1974 data indicated a 72.6% infection rate, a series of trials conducted in 1977 resulted in infection rates of 10% to 60% (1977-9, 1978-2). An incon­ sistent pattern in the gena~1 infection rate (33 to 80%) was again observed in 1980 with laboratory and field strains of An. freeborni (1980-4),

Oospores of the fungus were evaluated in the laboratory for their pathogenicity to Cx. tarsalis larvae. First infections were observed seven days after rehydration of the oos­ pores. At the lowest dosage investigated (30,000 osspores/1), larval infection peaked at day seven of the test and persisted at rates of 50% for 20 days, after which activity quickly drop­ ped. For the two higher osspore dosages, infection rates remained at 80% or higher for 30 days, WHO/VBC/79.753. Rev. 1 VBC/BCDS/79,02. Rev. 1 page 10

with some activity persisting until the tes,t was terminated 51 days after rehydration (1983-12).

During evaluation of Lagenidium against larval mosquito populations in North Carolina, larvae of a gnat species in the family Chaoboridae: Chaoborus flavicans were also tested against the NC isolate of the fungus. In these larboratory tests, 82.7% of larvae of Culex quinquefasciatus became infected in four days at 20°C as well as 86.6% of the Ae, aeg~ larvae. None of the chaoborid larvae became infected. No zoospore encystment of the chaoborid's cuticles was evident by microscopic examination nor where there any signs of aborted penetra~ tion by the fungus in the chaoborids. However, it has been reported that L. giganteum gave partial control of the Clear Lake gnat C. astictopus (1975-1. 1977-2, 1979-2) which is consi­ dered surprising (1984-2) as both C. flavicans and C. astictopus are benthic/plantetonic pre­ dators of copepods, cmronomids,mosquitos, oligochaete and rotifus in ponds and lakes and these two species are ecologically and biologically similar. In this bioassay C. flavicans was not susceptible to the fungus.

5.2 Under field conditions

When about 21 million zoospores of L. giganteum were introduced in a 1.5 sq. metre pond in North Carolina, Cx. restuans was eliminated within a week, the few Psorophora sp. larvae were all infected and 8% of the anopheline larvae were infected (1972-3).

The introduction of L. giganteum as a spray into seepage ditches adjacent to rice fields in California at a dosage of about 3,000.000 zoospores/sq.m. resulted in the elimination of Cx. tarsalis from the ditch where environmental conditions (pH and salinity) were favourable to this fungus and low rates of infection in the other ditches. L. giganteum survived re­ peated drying in the ditches over the subsequent three years (1973-3).

In a Louisiana black gum swamp naturally infected by L. giganteum, Cx. territans was de­ cimated by epizootics year after year during the whole period of observation (1975-1978). During that time, the fungus was present in the mosquito population for 9 of 21 months. In­ fection levels among the weekly collections of larvae ranged from 38 to 100% with the average mortality exceeding 60%. The fungus recrudesced after at least two winters during which the mean daily temperatures were 6 to 14°C and after one four~onthdrought. It should be noted that the other mosquito species present in this swamp (An. crucians, Ae. atlanticus, Ae. tormentor, Cx. peccator, Ps. howardii, Uranotaenia sapphirina) were not infected (1978-4).

In California, numerous field trials have been conducted during the last 10 years (1975-3, 1976-7, 1977-3, 1977-7, 1978-7, 1979-3, 1981-7, 1983-9, 1983-12, 1984-6). These trials have yielded variable but generally effective control of mosquito populations in rice fields, e seepage ditches and flooded pastures. At the inoculum rates used, (0.5 and 1.5 1 of medium/10m2) the asexual stage of L. giganteum successfully infected An. freeborni and Cx. tarsalis in rice field habitats. As previously shown in the laboratory, An. freeborni larvae were more resistant to L. giganteum infections than Cx. tarsalis. Most plots exhibited 100% mortality of the later species and 60 tO 80% mortality of the former (1983-12).

Laboratory and field trials using the sexual and asexual stage of L. giganteum were carried out in Arkansas, USA, using Ps. columbiae and An. quadrimaculatus. With four to five times the inoculum dose used against Cx. tarsalis and An. freeborni in California, 40 to 90% mortality of sentinel larvae of the two southern mosquito species was documented over a six-day period; these higher doses may have been necessary because of strain susceptibility. Mortality due to oospores was 100% in the laboratory and 13 to 23% in the field (1984-6).

L. giganteum was evaluated for the control of the Clear Lake gnat C. astictopus in a 960 m2 agricultural pond in California. A week after the pond was inoculated, a 38% suppression of the larval gnats was observed in the treated pond as compared to a 50% increase in the WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 11 control pond. The suppression in the gnat population occurred at a dosage range 52% and 17% of that calculated for minimal and maximal suppression of the gnats from a dosage-response curve from previous studies. The results suggest that the probability of the fungus infecting gnat larvae increases with increasing gnat densities (1979-2).

6. Effect on non-target organisms

L. giganteum was described from strains obtained from mosquito larvae, copepods and daphnids (1935-1) which could indicate a broad spectrum of activity. Subsequent studies with more recently isolated strains failed however, to show any activity against Cyclops sp., Daphnia sp., Scapholeberis sp., other unidentified copepods and cladocerans, polychaete worms, dytiscid beetles, and chironomid midges (1973-3). The only non-mosquito arthropod which could be infected was the Clear Lake gnat Chaoborus astictopus (1975-1, 1977-2, 1979-2). In compari­ son to the control mosquitos tested concurrently, the length of time required for a patent infection to become established in C. astictopus larvae was significantly larger (i.e.: 4-12 days) than for the mosquito larvae (2-4 days) (1980-2).

Preliminary investigations carried out with fish (Poecilia and Gambusia), birds (chicken and quail) and rats did not show any sign of pathogenicity of L. giganteum for these animals (1976-6, 1981-2).

Attempts were made to develop patent infections in the following organisms: Crayfish, mayfly, two species of dragonflies, damselfly, two water bugs, three beetles and a soldier fly. All results were negative (1977-9).

Over one thousand aquatic (carnivore, herbivore and omnivore) arthropods, representing over thirty ~xa, associated with mosquito larvae were collected from test sites in the Sacra­ mento Valley (Northern California) and tested with negative results, except for C. astictopus (see above) (1980-2, 1980-3). These are (1983-3):

Class insects:

Callibaetis montanus (Ephemeroptera: Bae~idae) Corisella sp. (Hemiptera: Corixidae) Belostoma flumineum (Hemiptera: Belostomatidae) Hygrotus medialis (Coleoptera: Dytiscidae) Laccophilus decipiens (Coleoptera: Dytiscidae) Laccophilus tristernalis (Coleoptera: Dytiscidae) Thermonectus basilaris (Coleoptera: Dytiscidae) Enochrus cuspidatus (Coleoptera: Hydrophilidae) Chironomidae (Diptera) Ephydridae (Diptera) Syrphidae (Diptera) Class Crustacea: Daphnia pulex (order Cladocera) Cyclops sp. (order Cyclapo ida) Order Podocopa (subclass Ostracoda) Procambarus clarki (order Decapoda)*

* It should be noted that none of the sixty crayfish (P. clarki) tested became infected, as a major concern to private industries dealing with commercial marine and fresh water crustaceans is that the introduction of Lagenidium species for biological control purpose might cause great harm to the shrimp, crab, lobster and crayfish industry. This concern is based on the knowledge that there is at least one marine species of Lagenidium (i.e. L. callinectes) that parasitises the eggs and larvae of blue crabs (Callinectes sapidus) and larval lobster (Homarus americanus) (1975-2, 1976-1, 1976-5).

------~ WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02 page 12

7. Production

The practical use of L. giganteum for biological control requires a simple and rapid means of mass producing infective zoospores.

In the earliest studies (1972-3, 1973-2, 1973-3, 1976-7), the fungus was produced in vivo on larvae of Cx. restuans, Cx. tarsalis or Cx. quinquefasciastus. Such methods were time consuming, labor intensive and posed problems in the quantification of doses.

In vitro zoospore production was reported in liquid culture (1982-2). In this process, the fungus was grown for 4 to 6 days on a peptone, yeast extract and glucose medium, trans­ ferred to a medium of hempseed extract, wheat germ, yeast extract and glucose for 6 days, then washed and transferred to water. Zoospore production at 25 ± 1°C began at 18h, peaked between 25-28h and ceased at 36h; 13700-21000 zoospores were produced per ml. An LDso of 1400 zoospores per ml was obtained with II instar larvae of Cx. quinquefasciatus.

On solid media the fungus was grown for 7 days in a liquid medium of wheat germ, hemp­ seed, yeast extract and glucose, then placed onto hemp-seed agar. Zoosporogenesis was induced on agar by immersing the fungal cultures into water. Zoospore production began lOh postimmer­ sion, peaked at 18h and ceased by 36h. A single 10 em Petri dish of fungus on hemp-seed agar produced 1.7-3.8 x 107 zoospores during the 26h of zoosporogenesis. Optimal zoospore produc­ tion occurred with 4-7 days old cultures; cultures older than 10 days produced few zoospores. The temperature range for zoosporogenesis was 15-35°C. The extent of zoosporogenesis was directly related to the volume of water used to induce zoospore formation and inversely pro­ portional to agar thickness. Bioassay of zoospores against 2nd instar Cx. quinquefasciatus larvae yielded an LDso of 400 zoospores/ml (1983-7).

A protocol for· mass production of Lagenidium consisting of a two phase system (Figure 3) has been presented. (1) large amounts of vegetative mycelium are produced in an aqueous ex­ tract of edible sunflower seed (Helianthus annus), although it may be possible to substitute

Figure 3. '7 .R . lOOm I --+ 6C, d"hos

E3l~ ~8~~SFE LIQUid E!_oducJ.!_QQ ~t.ill!d.C! ~ (subculture weekly) SFE Agar Sol1d Culture

~. ./ (Petlro d•sh•;::~~g~wk 25-27'C ~ Up tol2wks 15-16'C ,, nece~sory. HomoQen1ze and apply . to hab1tot ~ ~

. ~ ' PYG LIQUid Res~~ ~!:!ILL!!.~ (Subculture weekly}

Fig. 3.*Diagr;un of silllplilicd jHildtHtion ...,)...,l<'lll fot l.u,::,nzirli11m p,t,::,tutfl'lll/1. *This diagram was originally published in reference 1984-3 & reproduced by kind permission of Dr. Axtell. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 13 other substances such as certain types of beans (1985-2) and (2) the mycelium is transferred to solid agar base medium for storage until used in the field. With this biphasic system, the fungus can be stored for several weeks on agar medium, which allows flexibility in synchroni­ zing production of the fungus and its introduction into mosquito breeding habitats (the fungus readily produces zoospores when grown on sunflower seed extract (SFE) which apparently provides the necessary sterols). Based on the authors field trials, an initial dose of two petri dishes of SFE agar culture of Lagenidium per m2 of water surface should be adequate in areas where mosquito larvae are observed. Temporary breeding habitats which dry within 3 days of introduction of L. gi~anteum should not be treated since there will be insufficient time for the fungus to cause larval mortality and become established in the habitat (1984-3).

It was recently found that L. giganteum will produce oospore in vitro when supplied with exogenous sterols (1982-5, 1983-8). The sexual (oospore) stage of the fungus is of special interest since it is amenable to long term storage and can be dissiminated easily using stan­ dard insecticide application technique. L, giganteum oospores were grown on a variety of media containing chlesterol, fatty acids, calcium and yeast extract. Oospore yield is affected by changes in the sterol side chain, especially at C-24, and by the degree and position of unsa­ turation in the sterol ring. Oospore yield is enhanced by incorporating fatty acids (1983-12). Fermentation production of oospore stage of L. giganteum has been achieved by using 12-litre batches. Current yields are one-third to one-half the optimum yields obtained in smaller scale (100 to 1000 ml) shake cultures. Oosporogenesis by the fungus in fermentation culture is very sensitive to aeration, medium pH, agitation rate, and concentrations of the basal medium com­ ponents. As found for shake cultures, both calcium and magnesium, along with sterols and esterified unsaturated fatty acids are necessary for oospore formation (1984-6).

8. Stability and persistence of L. giganteum

Zoospores are rather fragile and can only be preserved for a few days or weeks under simple environmental conditions (1981-8). The dormancy and resistance to dessication of oos­ pores make it possible to store them dry at room temperature for indefinite periods. In an evaluation of the effect of storage time on viability, several collection of oospores made in 1977 from in vivo material were rehydrated in enamel pans. After three weeks of quiescence, these oospores, which had been stored in soil at room temperature for seven years, began to germinate and kill sentinel larvae added to the pans (1984-6).

In long term studies in California, it was observed that L. giganteum persisted at least 8 years in a drainage ditch where it was introduced in 1973 (1973-3). The fungus failed to survive in two other sites (1980-5, 1981-6).

From 1974-1980, overwintering survival of L. giganteum was repeatedly demonstrated in aquatic situations associated with flooded rice fields in the Sacramento Valley in northern California. In two documented instances, the fungus persisted through two winters during which the field was drained, rice harvested, crop residue burned, the ground ploughed and re­ planted and the intervening summer without any irrigation water. In one instance (19747 the fungugal infectivity pattern in mosquito larvae was erratic and of low order in nature. In 1978, L. giganteum was recovered from laboratory-reared sentinel Cx. tarsalis larvae at the rate of 95% in one of the test sites ten days after flooding of the rice fields, Alternate drying and flooding within a season did not adversely affect fungal establishment in the habitats. This study did not confirm high temperature and soluble salts of the soil as limit­ ing factors for this organism. Within a continuous pond system, a minimum dispersal of 28-33m from the point of introduction was documented. In aquatic habitats situated nearby but separa­ ted by a physical barrier (e.g., dirt levee) no airborne dispersal was noted. L. giganteum was a highly virulent microbial agent of Cx. tarsalis larvae, but in comparative in situ studies appeared to be inconsistent against An freeborni larvae. No patent infections were observed in associated aquatic invertebrates collected from test sites (see para. 6) (1980-3, 1980-5, 1982-8, 1983-3). WHO/VBC/79. 753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 14

The North Carolina and Louisiana isolates. of L, e;i~anteum_were introduced into natural freshwater mosquito breeding sites in North Carolin~ in June 1982 and their establishment and persistence monitored through the remaining mosquito breeding season. The North Carolina iso­ late on agar media was introduced into a flooded woodland that had populations of An, punctipennis, Cx. restuans, Cx. territans, A~. vexans, Ps, ferox and Ps. columbiae:-A liquid culutre of the Louisiana isolate was added to a flooded depression that was a source of An. punctipennis, Cx. restuans, Cx. territans, and Ae. vexans. The fungus became established-at both sites, remaining at enzootic levels when larvae were scarce and recrudescing when larval populations increased after flooding. Infections during the observation period ranged from 0 to 100% among larvae collected from the sites and 20 to 100% among laboratory-reared larvae (Cx. quinquefasciatus) exposed in the sites for 24-48hr. Mosquito larvae added to water samples drawn from the sites and held in the laboratory became infected with the fungus even when natural larval populations were very low or absent. Results indicate that L. giganteum recycled for the entire season dispite periodic scarcity of hosts and short-term drought. Oospores of the fungus were observed in a large proportion of infected larvae. These oospores may have been responsible for the survival of Lagenidium when the water temperature dropped to below 18°C, infection of mosquito larvae by the fungus ceased. (see para. 4.3 a).

The two sites were samples for the presence of L. giganteum in June 1983, one year after the initial introduction of the fungus. Mosquito larvae (Culex spp.) collected from both sites were infected (resp. 15 out of 91 and 10 out of 35). Larvae (2d instar) of laboratory-reared Cx. quinquefasciatus which were added to water collected from one of the sites became infected (19 out of 95). These data show that the fungus survived overwinter in these sites (1983-6).

9. Formulations and Specifications

None to date. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 15

10. References

1935

1935-1 Couch, J.N. (1935). A new saprophytic species of Lagenidium with notes on other species. Mycologia, 27: 376-387.

1960

1960-1 Couch, J. (1960). Some fungal parasites of mosquitoes. In: (Proceedings of a Conference on the) Biological Control of Insects of medical Importance, Washington D.C., American Institute of Biological Sciences, pp. 35-48.

1964

1964-1 Elliot, C., Hendrie, M., Knights, B. and Parker, W. (1964). A steroid growth factor requirement in a fungus. Nature (London), 203: 427-428.

1969

1969-1 Willoughby, L.G. (1969). Pure culture studies on the aquatic phycomycete, Lagenidium giganteum. Trans. Brit. Mycol. Soc.,~: 393-410.

1970

1970-1 Umphlett, C.J. and Huang, C.S. (1970). Lagenidium culicidum as an agent of biologi­ cal control of mosquitoes. Bull. Assoc. Southeast. Biol., 17: 68.

1971

1971-1 Me Innis T. (1971). A physiological and biochemical investigation on the aquatic phycomycete Lagenidium sp., a facultative parasite of certain mosquito larvae. Ph.D. Dissertation, University of North Carolina, Chapel Hill, N.C. USA, 108 pages.

1972

1972-1 Mattingly, P. (1972). Mosquito eggs. XVII. Further notes on egg parasitization in genus Armigeres. Mosq. Syst., ~: 1-8.

1972-2 Mattingly, P. (1972). Mosquito eggs. XX. Egg parasitism in Anopheles with further notes on Armigeres. Mosq. Syst., 4: 84-86,

1972-3 Umphlett, C.J. and Huang, C.S. (1972). Experimental infection of mosquito larvae by a species of the aquatic fungus Lagenidium. J. Invertebr. Path., 20: 326-331.

1973

1973-1 Couch, J.N. and Romney, S.V. (1973). Sexual reproduction in Lagenidium giganteum. Mycologia, ~: 250-252.

1973-2 McCray, E.M., Umphlett, C.J. and Fay, R.W. (1973). Laboratory studies on a new fungal pathogen of mosquitoes. Mosquito News, 21= 54-60.

1973-3 McCray, E.M., Womeldorf, D.J., Husbands, R.C, and Eliason, D.A. (1973). Laboratory observations and field tests with Lagenidium against California mosquitoes. Proc. Pap. Ann. Conf. Calif. Mosq. Control Ass., 41: 123-128. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 16

1973

1973-4 Mcinnis, T. and Domnas, A. (1973). The properties of trehalase from the mos­ ~uito-parasitizing water mold, Lagenidium sp •• J. Invertebr. Path., 22: 313-320.

1973-5 Umphlett, C.J. (1973). A note to identify a certain isolate of Lagenidium which kills mosquito larvae. Mycologia, 65: 970-972.

1974

1974-1 B~swell, J., Umphlett, C. and Mcinnis, T. (1974). Zoospore production from axenic cultures of the mosquito parasite Lagenidium giganteum (Oomycetes). iull. Assoc. Southeast Biol., ~: 41.

1974-2 Domnas, A., Giebel, P.E. and Mcinnis, T. (1974). Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinque­ fasciatus following infection with Lagenidium giganteum. J. Invertebr. Path., 24: 293-304.

1974-3 Mcinnis T. and Domnas, A.J. (1974). An aryl beta-D-glucosidase of the aquatic fungus Lagenidium giganteum, a parasite of mosquito larvae. Arch. Microbiol., 101: 343-350.

1975

1975-1 University of California (1975). Research on mosquitos, 1974. UC Division of Agricultural Sciences, Special Publication 3014, p.9.

1975-2 Umphlett, C.J. and McCray, E.M. (1975). A brief review of the involvements of Lagenidium an aquatic fungus parasite, with arthropods. Marine Fisheries Review, 37: 61-64.

1975-3 Washino, R.K., McCray, E.M. and Lasko, J.F. (1975). Recovery of Lagenidium giganteum an aquatic fungal parasite of mosquitos, two years after field introduc­ tion. Pro~. N.J. Mosq. Control Ass., 62: 125.

1976

1976-1 Armstrong, D., Buchanan, D. and Caldwell, R. (1976). A mycosis caused by Lagenidium sp. in laboratory-reared larvae of the Dungeness crab, Cancer magister, and possible chemical treatment. J. Invert. Pathol., 28: 329-336.

1976-2 Domnas, A., Shipley, R. and Hicks, B. (1976). Sterol requirement for zoospore production by the mosquito parasite Lagenidium giganteum. In: Proc. 1st Int. Colloq. Invertebr. Pathol. & IX Annu. Meet. Soc. Invertebr. Pathol. (Kingston, Canada), p. 427.

1976-3 Giebel, P. and Domnas, A. (1976). Soluble trehalases from larvae of the mosquito, Culex pipiens and the fungal parasite Lagenidium giganteum. Insect Biochem. 6: 303-311.

1976-4 Mcinnis, T. (1976). Biochemical basis of pathogenesis of fungal diseases of vectors. In: Biological Regulation of Vectors: the saprophytic and aerobic Bacteria and Fungi-.-U.S. DHEW Publ. No. (NIH) 77-1180, pp. 95-110.

1976-5 Nilson, E., Fisher, W. and Shleser, R. (1976). A new mycosis of larval lobster (Homarus americanus). J. Inv. Pathol., 27: 177-183. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 17

1976

1976-6 Umphlett, C.J. (1976). Pathogenicity as a consideration in the systematics of fungus pathogens of invertebrates. In: Biological Regulation of Vectors: the saprophytic and aerobic Bacteria an~Fungi. US. DREW Publ. No. (NIH) 77-1180, pp. 125-134.

1976-7 Washino, R.K., Fetter, J., Fukushima, C.K. and Gonot, K. (1976). The establish­ ment of Lagenidium giganteum, an aquatic fungal parasite of msoquitos, three years after field introduction. Proc. and Papers Calif. Mosqu. Control Ass., 44: 52.

1977

1977-1 Boswell, J. (1977). Zoosporogenesis in Lagenidium giganteum, a fungal parasite of mosquito larvae, in response to nutritional supplements. Diss. abstr. Int. B. Sci. Eng. J 38 (4): 1528B -1529B.

1977-2 Brown, J.K. and Washino, R.K. (1977). Developments in research with the Clear Lake gnat Chaoborus astictopus in relation with the fungus Lagenidium giganteum. Proc. and Papers 45th Ann. Conf. Calif. Mosq. and Vector Control Ass.: 106.

1977-3 Christensen, J.B., Fetter-Lasko, J.L., Washino. R.K., Husbands, R.C. and Kauffman, E.E. (1977). A preliminary field study employing Lagenidium giganteum, a fungus, as a possible biological control agent against the pasture mosquito Aedes nigro­ maculis. Proc. and Papers 45th Ann. Calif. Conf. Mosq. and Vector Control Ass.:, 105.

1977-4 Domnas, A. (1977). Biochemical aspects of mosquito infection by Lagenidium giganteum. Abstr. Pap. Xth Ann. Meet. Soc. Invertebr. Pathol. (Michigan), p.l.

1977-5 Domnas, A. and Hicks, B. (1977). Properties of mosquito infection with Lagenidium giganteum. Abst. Pap. Ann. Meet. Soc. Invertebr. Pathol. (Michigan). p. 4.

1977-6 Domnas, A.J., Srebro, J.P. and Hicks, B.F. (1977). Sterol requirement for zoospore formation in the mosquito-parasitizing fungus Lagenidium giganteum. Mycologia, 69: 875-886.

1977-7 Fetter-Lasko, J.L. and Washino, R.K. (1977). A three year study of the ecology of Lagenidium infections of Culex tarsalis in California. Proc. and Papers 45th Ann. Conf. Calif. Mosq. and Vector Control Ass.: 106.

1977-8 Mattingly, P. (1977). Lagenidium sp.: a fungal parasite ofmosquito eggs. Trans. Roy. Soc. Trop. Med. Hyg., !l= 383.

1977-9 Washino, R.K. (1977). The ecology of mosquitos and mosquito control agents in California, July 1976-June 1977. University of California Mosquito Control Research, Annual Report 1977: 39-46.

1978

1978-1 Dean, D. and Domnas, A. (1978). Influence of sterols on protease production by the mosquito pathogen Lagenidium giganteum. In: Int. Colloq. Invertebr. Pathol. and XI Annual Meet. Soc. Invertebr. Pathol.,-prague, Czechoslovakia, p. 27.

1978-2 Fetter-Lasko, J. and Washino, R. (1978). Patterns of experimental infection of mosquito larvae by Lagenidium giganteum. Proc. and Papers, 46th Ann. Conf. Calif. Mosq. and Vector Control Assoc., p. 90, WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 18

1978

1978-3 Giebel, P. and Domnas, A. (1978). Carbohydrate and amino acid metabolism in Culex pipiens quinquefasciatus infected with Lagenidium giganteum. In: Int. Colloq-.---­ Invertebr. Pathol. and XI Annual Meet. Soc. Invertebr. Patho~ Prague, Czechoslovakia, p. 43.

1978-4 Glenn, F.E. and Chapman, H.C. (1978). A natural epizootic of the aquatic fungus Lagenidium giganteum in the mosquito Culex territans. Mosquito Ne~ 38: 522-524.

1978-5 Insect affecting Man and Animals Research Laboratory, Gainesville, Florida, USA.

1978-6 Poinar, G. and Thomas, G. (1978). Diagnostic Manual for the identification of Insect Pathogens. Plenum Press, New York and London, 218 pages.

1978-7 Washino, R. and Fukushima, C. (1978). Assessment of biological control agents against mosquito larvae in Northern California: a progress report. Proc. Pap. Annu. Con£. Calif. Mosq. Vector Control Assoc., 46: 89.

1979

1979-1 Axtell, R. (1979). Principles of integrated pest management in relation to mosquito control. Mosquito News, ~: 709-718.

1979-2 Brown, J. and Washino, R. (1979). Evaluating the fungus Lagenidium giganteum for the biological control of the Clear Lake gnat, Chaoborus astictopus in an agricul­ tural pond in lake country, California. Proc. and Papers 47th Ann. Conf. Calif. Mosq. and Vector Control Assoc.: 37.

1979-3 Fetter-Lasko, J. and Washino, R. (1979). Lagenidium giganteum and mosquitoes as­ sociated with irrigated pastures. Proc. and Papers 47th Ann. Calif. Con£. Mosq. and Vector Control Assoc.: 55.

1979-4 Insect affecting Man and Animals Research Laboratory, Gainesville , Florida, USA.

1980

1980-1 Federici, B., Fetter-Lasko, J., Soares, G. and Tsao, P. (1980). Fungi show promise in biological control. Calif. Agric., 34: 25-27.

1980-2 Fetter-Lasko, J. and Washino, R. (1980). Impact of Lagenidium gigan~ on non­ target organisms asaociated with mosquito larvae. Salt and brackish water tolerance of Lagenidium giganteum Couch. Annual progress report, October 1, 1979 through September 30, 1980. Ecology of mosquitoes and mosquito control, U. of California.

1980-3 Washino, R. (1980). Recycling of Lagenidiumgi~nteum Couch and other microbial agents in relation to mosquito control in rice fields and other cultivated systems in California, USA. Abstract llS-1,6, XVI International Congress of Entomology, Kyoto, Japan, 3-9 &gust 1980.

1980-4 Washino, R. (1980). Experimental host range of Lagenidium giganteum Couch: Anopheles freeborni Aitken. Annual progress report, October!, 1979 through September 30, 1980. Ecology of mosquitoes and mosquito control, University of California.

1980-5 Washino, R. (1980). Lo~ term persistence of Lagenidium giganteum Couch in certain mosquito breeding habitats of California. Annual progress report, October 1,1979 through September 30, 1980. Ecology of Mosquitoes and mosquito control. U. of California. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 19

1981

1981-1 Domnas, A. (1981). Biochemistry of Lagenidium giganteum infection in mosquito larvae. In: Pathogenesis of invertebrate microbial diseases. E. Davidson (Ed.), Allanheld, Osmun, Totowa, N.J., USA, pp. 425-449.

1981-2 Federici, B. (1981). Fungi: mosquito control by Culicinomyces, Lagenidium and Coelomomyces. In: Burges, H. (Ed.). Microbial Control of Pests and Plant Diseases 1970-1980., Academic Press, N.Y. pp. 555-572.

1981-3 Toohey, M., Goettel, M. and Pillai, J. (1981). A review of the prospect of using biological control against mosquito vectors of subperiodic filariasis and arbovi­ ruses in Polynesia. S. Pac. J. Nat. Sci.,~: 4-43.

1981-4 Warner, S. and Domnas, A. (1981). Evidence for a cycloartenol-based sterol synthe­ tic pathway in Lagenidium spp .. Exp. Mycol., ~: 184-188.

1981-5 Warner, S., Graham, M., Sovocool, G. and Domnas, A. (1981). Alkane contamination of lipids extracted from Lagenidium giganteum and Lagenidium callinecte~ Lipids, 16: 628-630.

1981-6 Washino, R. (1981). Biocontrol of mosquitoes associated with California rice fields with several reference to the recycling of Lagenidium giganteum Couch and other microbial agents. In: M. Laird (Ed.), Biocontrol of medical and veterinary Pests. Praeger Publishers-,-New York, USA, pp. 122-139.

1981-7 Washino, R. and Brown-Westerdahl, B. (1981). Effectiveness of Bacillus thuringien­ sis var.. israelensis (H-14), Lagenidium giganteum, and Romanomeimis culicivorax in controlling rice field mosquitoes. U. of California, Mosquito c~ntrol Research, annual Report 1981, pp. 75-77.

1981-8 Washino, R., Kerwin, J. and Dritz, D. (1981). In vitro culture and subsequent pathogenicity of the infective stage of Lagenidium giganteum. U. of California, Mosquito Control Research, Annual report 1981, pp. 87-88.

1982

1982-1 Axtell, R., Jaronski, S. and Merriam, T. (1982). Efficacy of the mosquito fungal pathogen Lagenidium giganteum (Oomycetes: Lagenidiales). Proc. and Papers. Calif. Mosq. and Vector Control Assoc .., 50: 41-42.

1982-2 Domnas, A., Fagan, S. and Jaronski, S. (1982). Factors influencing zoospore pro­ duction in liquid cultures of Lagenidium giganteum (Oomycetes:Lagenidiales). Mycologia, ~: 820-825.

1982-3 Jaronski, S. (1982). Oomycetes in mosquito control. Proceedings, IIIrd International Colloquium on Invertebrate Pathology, Brighton, United Kingdom, 6-10 September 1982, pp. 420-424. '"

1982-4 Jaronski, S. and Axtell, R. (1982). Effects of organic water pollution on the in­ fectivity of the fungus Lagenidium giganteum (Oomycetes: Lagenidiales) for larvae of Culex quinquefasciatus (Diptera: Culicidae): field and laboratory evaluation. J. Med. Entomol., ~: 255-262.

1982-5 Kerwin, J. and Washino, R. (1982). Artificial culture of the sexual and asexual stages of Lagenidium giganteum. Proc. Calif. Mosq. Vector Control Assoc., 50: 43. WHO/VBC/79.753. Rev. 1 VBC/BCDS/79.02. Rev. 1 page 20

1982

1982-6 Merriam, T. and Axtell, R. (1982). Salinity tolerance of two isolates of Lagenidium giganteum (Oomycetes: Lagenidiales), a fungal pathogen of mosquito larvae. J. Med. Entomol., ~: 388-393.

1982-7 Merriam, T. and Axtell, R. (1982). Evaluation of the entomogenous fungi Culici­ nomyces clavosporus and Lagenidium giganteum for control of salt marsh mosquito Aedes taeniorhyncus. Mosq. News, ~: 594-602.

1982-8 Washino, R. (1982). Biocontrol of mosquitoes associated with California rice fields with special reference to the recycling of Lagenidium giganteum Couch and other microbial agents. In: M. Laird (Ed.). Biocontrol of medical and veterinary Pests. Praeger Publ., Ne;-York, USA, pp. 122-139.

1983

1983-1 Davidson, E. and Sweeney, A. (1983). Microbial control of vectors: a decade of progress. J. Med. Entomol., 20: 235-247.

1983-2 Dean, D. and Domnas, A. (1983). The extracellular proteolytic enzymes of the mos­ quito-parasitizing fungus Lagenidium giganteum. Exp. Mycol., 2= 31-39.

1983-3 Fetter-Lasko, J. and Washino, R. (1983). In situ studies on seasonality andre­ cycling pattern in California of Lagenidium giganteum Couch, an aquatic fungal pathogen of mosquitos. Environ. Entomol., ~: 635-640.

1983-4 Goettel, M., Toohey, M. and Pillai, J. (1983). Preliminary laboratory infection trials with a Fiji isolate of the mosquito pathogenic fungus Lagenidium. J. Invert. Pathol., il= 1-7.

Jaronski, S. and Axtell, R. (1983). Effects of temperature on infection, growth and zoosporogenesis of Lagenidium giganteum, a fungal pathogen of msoquito larvae. Mosq. News., 43: 42-45.

1983-6 Jaronski, S. and Axtell, R. (1983). Persistence of the mosquito fungal pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) after introduction into natural habitats. Mosq. News., 43: 332-337,

1983-7 Jaronski, S., Axtell, R., Fagan, S. and Domnas, A. (1983). In vitro production of zoospores by the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) on solid media. J. Invert. Pathol., il= 305-309.

1983-8 Kerwin, J. and Washino, R. (1983). Sterol induction of sexual reproduction in Lagenidium giganteum. Experim. Mycol., 2= 109-115.

1983-9 Kerwin, J. and Washino, R. (1983). Field evaluation of Bacillus thuringiensis var. israelensis, Lagenidium giganteum and Romanomermis ~ulicivorax in California rice fields. Proc. and Papers Calif. Mosq. and Vector Control Assoc.,~: 19-25.

1983-10 Merriam, T. and Axtell, R. (1983). Relative toxicity of certain pesticides to Lagenidium giganteum (Oomycetes: Lagenidiales), a fungal pathogen of mosquito larvae. Environ. Entomol., ~: 515-521.

1983-11 Warner, S., Sovocool, G. and Domnas, A. (1983). Comparative utilization of sterols by the oomycetes Lagenidium giganteum and Lagenidium callinectes. Exp. Mycology, 7: 227-232. WHO/VBC/79.753. Rev, 1 VBC/BCDS/79.02. Rev. 1 page 21

1983

1983-12 Washino, R. and Kerwin, J. (1983). Evaluation of the sexual and asexual stage of Lagenidium giganteum for the control of rice field mosquitoes. University of California, Mosquito C~ntrol Research annual Report 1983, pp. 68-69.

1984

1984-1 Galagali, J., Balasubramaniane, R. and Balaraman, K. (1984). Indigenous isolation of F-17- an oomycetous fungus pathogenic to mosquito larvae. Indian Journal of Medical Research, 80: 95-102.

1984-2 Jaronski, S. and Axtell, R. (1984). Non-susceptibility of Chaoborus flavicans (Chaoboridae) to the mosquito pathogen Lagenidium giganteum (Oomycetes). Mosq. News., 44: 81-82.

1984-3 Jaronski, S. and Axtell, R. (1984). Simplified production system for the fungus Lagenidium giganteum for operational mosquito control. Mosq. News., 44: 377-381.

1984-4 Kerwin, J. and Washino R. (1984). Cyclic nucleotide regulation of oosporogenesis by Lagenidium giganteum and related fungi. Exp. Mycol., ~: 215-224.

1984-5 Warner, S., Sovocool, G•• Domnas, A. and Jaronski, S. (1984). The composition of sterols in mosquito larvae is optimal for zoosporogenesis in Lagenidium giganteum. J. Invert. Path., 43: 293-296.

1984-6 Washino, R. and Kerwin, J. (1984). Factors affecting zoospore and oospore produc­ tion and germination by Lagenidium giganteum. University of California, Mosquito Control Research annual Report 1984, pp. 72-74.

1984-7 WHO (1984). UNDP/World Bank/WHO Special Programme for Research and training in tropical diseases. Report of the seventh meeting of the scientific working group on biological control of vectors, Geneva, 5-9 March 1984. TDR/BCV/SWG-7/84.3, p. 18.

1985

1985-1 Brey, P. (1985). Observations of in vitro gametangia! copulation and oospore- genesis in Lagenidium giganteum. J. Inv. Pathol., 45: 276-281.

1985-2 Jaronski, S., Fagan, S. and Domnas, A. (1985). Simple media for zoospore production by the msoquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) - submitted to publication.

1985-3 Lord, J. and Roberts, D. (1985). Effects of slinity, pH, organic solutes, anaerobic conditions and the presence of other microbes on production and survival of Lagenidium giganteum (Oomycetes: Lagenidiales) zoospores. J. Inv. Pathol., 45: 331- 338.