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WORLD HEALTH ORGANIZATION ~ WHO/VBC/80.766 VBC/BCDS/80.09 ORGANISATION MONDIALE DE LA SANTE ENGLISH ONLY

DATA SHEET ON THE BIOLOGICAL CONTROL AGENT(l) INDEXED Romanomermis culicivorax (Ross and Smith 1976)

Romanomermis culicivorax is an obligatory endoparasitic nematode, the parasitic larvae of which develop inside larval . It has little genus and species specificity within the Culicidae faplily. A total of 87 species (including stephensi, An. albimanus, An. gambiae and many other vector species) have been exposed to it either in the laboratory or in the field and were infected.

R. culicivorax has been extensively studied for the past 10 years. It can be easily mass produced, is safe to mammals and other non-target organisms, and its environmental limitations are well documented. This parasite is effective when used in water habita~s with the following characteritstics: fresh and non polluted, semi-permanent or permanent, temperature rarely exceeding 40°C, and little water movement. Several natural predators among the aquatic organisms likely to dwell in pools have been shown to prey on R. culicivorax; but the size, amounts of open water, pH, vegetation densities and host densitie; are not significant factors in the successful use of this biological control agent. This nematode should now be operationally evaluated against vectors during large scale field trials in endemic areas.

1. Identification and Synonymy

Nematoda:

Romanomermis culicivorax (Ross and Smith, 1976), is a segregate of a complex known previously as Reesimermis nielseni (Tsai and Grundmann, 1969). R. nielseni is now restricted to a species known only from Wyoming. All published observation; of and data referring to Romanomermis sp.auct. (in relation to the North American mosquitos) and Reesimerrnis nielseni auct., are referable to Romanomermis culicivorax with the exception of the following papers: 1969-3, 1969-4. (1976-10, 1976-14, 1979-11).

2. Origin

R. culicivorax was originally found parasitizing the larvae of thirteen species of mosquitos in five freshwater ponds in southwestern Louisana, USA. The highest levels of parasitism occurred when the water level of a pool fluctuated but the pool did not dry up completely. The most heavily parasitized hosts were larvae of Psorophora columbiae and Uranotaenia sapphirina and then Aedes atlanticus and Anopheles crucians (1967-1, 1968-1, 1968-2, 1969-1). The original laboratory colony of~· culicivorax was established from larvae collected in the infested ponds (1968-1). R. culicivorax was also found parasitizing An. crucians and an Uranotaenia sp. in damp and-marshy areas near Gainsville, Florida (1971-2).

3. Natural geographical distribution

Southern United States of America: Louisiana and Florida (1971-2). The distribution of R. culicivorax is apparently somewhat restricted; the nematode does not appear to be abundant in any one locality (1979-11).

1 Information document produced by the WHO Division of Vector Biology and Control May 1980

The issue of this document does not constitute Ce document ne constitue pas une publication. formal publication. It should not be reviewed, ll ne doit faire l'objet d'aucun compte rendu ou abstracted or quoted without the agreement of resume ni d'aucune citation sans l'autorisation de the World Health Organization. Authors alone !'Organisation Mondiale de Ia Sante. Les opinions are responsible for views expressed in signed exprimees dans les articles signes n'engagent articles. que leurs auteurs. WHO/VBC/80.766 VBC/BCDS/80.09 page 2

4. Biological characteristics

4.1 Description (1972-4, 1974-2, 1974-9, 1974-10, 1976-14, 1979-11).

~· culicivorax is a whitish nematode with an elongated body. Females have an average body length of 17 mm; males are much shorter, with a length of 10 mm. The mouth opening is terminal. The trophosome (stored food reserves) is white and contains numerous small spheres. The body surface of the parasitic stages of ~· culicivorax is composed of three membranes overlying a layer of hypodermal cells. In all three membranes, there are pores ranging from 70-110~ in diameter. The pores are large enough for the passage of ferritin particles and thus can allow passage of all nutrients required for growth. (see para. 4.2, life-cycle) (1977-15).

Amphids are large. The tail of both sexes is bluntly rounded. The males have two long separated spicules and the females a characteristic barrel-shaped vagina and no vulval flap. The eggs are spherical in shape and usually about 80)Um in diameter. The preparasitic stage has an elongated form and a stylet connected to the pharyngeal tube. The characteristic elongated intestine and tail are probably modifications for rapid movement through water in search of mosquito larvae. The postparasitic stage has a long and pointed caudal appendage.

The external anatomy of parts of the male and infective stage of R. culicivorax has been described using the scanning electron microscope (1974-7).

4.2 Life-cycle (1972-4, 1973-6, 1974-2, 1974-10, 1975-5, 1976-10, 1978-8).

The eggs of ~· culicivorax are laid at the bottom of the mosquito pool. In the laboratory, most of the mature eggs hatch within 7 hours after cultures are flooded (see para. 7: production). A first moult occurs in the egg and the shed cuticle is frequently carried outside the egg by the emerging juvenile. The preparasitic juvenile (infective stage) swims to the surface of the pool and penetrates a mosquito larva. The preparasite of R. culicivorax is short lived and must find a suitable host within 36-48 hours after hatching in order to proceed with its development. The preparasite enters by cuticular penetration and often migrates to the thorax of the host mosquito larva. Experimental infection of larvae showed that the nematode increased in length by 18-fold and in width by 16-fold during its relatively short 6- to 8-day parasitic phase at 27°C. Most of the nematode growth is limited to the latter half of the infective period. The nematode obtains nourishment from the haemolymph, causing a depletion of metabolites within the host storage tissues.

Histochemical investigations have shown that R. culicivorax accumulates storage products primarily in the trophosome from the fourth day of-parasitic development onwards. Lipids constitute the predominant storage metabolite, glycogen the next abundant and proteins quantitatively only a minor storage product (1977-7).

The nematode moults 3-4 days after infection as indicated by the abtupt acceleration of its growth rate and concomitant morphological changes (loss of stylet and acquisition of caudal appendage) evident at this stage of development. As the nematode embarks upon its rapid growth phase, the trophosome (stored food reserves) becomes the most prominent structure, occupying a major portion of the pseudocoelom and obscuring other structures. The nematode does not possess a stylet to facilitate emergence from its host. The post parasite always kills the mosquito larva when it emerges. It is then free-living. It requires only a moist environment to moult to an adult and reproduce.

Maturation of R. culicivorax to the adult stage in sand cultures begins by the tenth day after emergence of the nematodes from their hosts at ambient temperatures (24-27°C) when the third and fourth moults occur simultaneously. A majority of the postparasitic males and females have reached the adult stage after 50 and 70 days, respectively. The first females exhibiting egg development and oviposition are observed 25-30 days after emergence but some oviposition is still taking place 150 days later. R. culicivorax lays an average 2480 eggs WHO/VBC/80.766 VBC/BCDS/80.09 page 3 per female over an 18-day oviposition period.

4.3 Host-parasite relationship

Laboratory studies conducted with larvae have shown that first­ and second-instar ~. guinguefasciatus are invaded most readily by R. culicivorax. Third­ instar larvae are slightly less susceptible and limited parasitism ~ccurs in fourth-instar larvae. All parasitized larvae failed to pupate. When pupal structures are partially developed or are beginning to develop, parasitism usually results in the early death of the host and the parasite. Almost 100% parasitism was obtained when parasites to host ratios in Cx. quinquefasciatus exceed 3:1 (1970-2).

Regression analysis on the relation between percentage parasitism and the ratio of preparasites to host (Cx. pipiens molestus) indicated that the ratio resulting in high incidence of parasitism varies with host density. The ratios attained 95% parasitism; they were decreased as host density rose. Extremely high ratios of preparasites to larvae proved unfavourable to the recycling of the nematodes in the habitat, due to early death of the host with consequent inhibition of parasite development (1979-2).

Water volume is not a major factor in determining the incidence of paras~t~sm by R. culicivorax. Preparasitic !· culicivorax are thigmotactic and negatively geotactic; they concentrate near the surface of the water and these responses greatly enhance the chances that the parasite will contact a suitable host since most mosquito species exhibit similar responses (1973-5).

The ratio of male to female R. culicivorax increases as the number of parasites per host increases. Hosts with a single nematode produced about 9% males compared with essentially 100% males in hosts with more than seven parasites. Males of R. culicivorax ~merge generally before females because of the earlier death of multiple-infect;d mosquitos. The species of the host mosquito influenced the sex ratio but the size of a specific host at the time of invasion did not. Singly infected hosts from starved populations produced 92% males compared with 13% in normally fed groups (1972-5).

Histological observations on Ae. aegypti larvae have shown that the most serious effects on the hosts occur between the 4th and 6th day after infection. During this time, the nematodes develop most rapidly, depleting host storage tissues while accumulating storage material in their trophosomes. The severity of the effects depends on the intensity of infection (1973~1, 1977-7).

High levels of DNA in fat body nuclei of Ae. aegypti parasitized by !· culicivorax for 6 days may indicate a high metabolic activity in parasitized mosquitos (1973-1).

When haemolymph composition of parasitized fourth-instar larvae of an autogenous strain of Culex ~i~ was examined, results indicate that parasitism causes major changes in the protein (1978-13). Immune responses of Ae. aegypti against!· culicivorax were atudied as both mosquitos and chironomid midges react to a number of metazoan parasites by forming a melanotic capsule around them. Of a total of 81 larvae of Ae. aegypti parasitized by both Neoaplectana carpocapsae and!· culicivorax, 75 of these were still capable of encapsulating !· carpocapsae. !· culicivorax waa never encapsulated even in larvae in which the reaction was initiated against N. carpocapsae. These results indicate that R. culicivorax does not actively suppress the encapsulation reaction, unless the suppression is-affected locally at the surface of the cuticle, and that specificity of the reaction resides not only in the activation step but also in the actual deposition of the capsular material (1975-3). A few mosquitos are able to partially or completely block normal development of R. culicivorax. Melanization was observed with Mansonia uniformis, an important vector of malaya~ filariasis in Thailand (1974-4) and Anopheles sinensis (1974-6). Similar reactions were observed with Aedes triseriatus and Culex territans larvae (1969-2, 1975-4).

Laboratory studies have shown the presence of two categories of insect immunity against WHO/VBC/80.766 VBC/BCDS/80.09 page 4 the juveniles of !· culicivorax: (1) humoral melanization as exhibited by larvae of Ae. triseriatus; this reaction involves the formation of melanin around the parasite by components in the non-cellular portion of the blood; (2) immune response as exhibited by Cx. territans: simple encapsulation accompanied by some pigment (probably melanin) formation. In both hosts, the response is lethal for the nematodes (1979-13).

The development of resistance by Cx. quinquefasciatus to !· culicivorax has been demonstrated. A laboratory culture of this nematode was maintained for 8 years through 300 generations without the introduction of additional field material by providing as a host a laboratory colony of Cx. quinquefasciatus. The practice of using pupae from exposed but uninfected mosquitos larvae to maintain adult populations exerted extensive pressure for selection for resistance to the parasite. A study of the laboratory colony made after some 50 mass rearing generations showed no resistance when compared with native populations of Cx. quinquefasciatus (1975-4). However after ca.ll5 generations, it became necessary to increase the parasite-host ratio from 12 to 15:1 to maintain a high level of parasitism in the mass-rearing system (1978-7). After some 300 generations, parasitism was 32-42% higher in a native strain than in the laboratory strain of the host under similar conditions. Differences were even greater when the number of successful penetrations (multiple parasitism) was measured. No evidence of humoral defense mechanisms were found and the mode of resistance has not been determined (1978-6).

4.4 Sensitivity to environmental factors

(a) temperature: R. culicivorax is active when water temperatures are above 18 0 C 0 - (1971-1). At 17 C, 11-12 weeks are required for 90% of the females to mature and 23 weeks are required for the first females to complete oviposition. At 23°C, the nematodes mature in 2-3 weeks and the females complete oviposition in 3-7 weeks (1976-10). The rate of parasitism of Cx. p. molestus by the mermithid decreased when temperatures were decreased from 30° to 15°C (1976-S). However, survival of preparasitic stages of!· culicivorax is prolonged by low temperatures (1974-5, 1976-5). The preparasites of R. culicivorax are infective at 12° to 33°C with the optimum infectivity at 21-33°C. Lower-temperatures decrease the percent infectivity but increase the time that the nematodes remain infective. The time required for host infection increased as the preparasitic larvae aged (1976-12, 1977-1). The optimum temperature range for development of the parasit~c stage in Cx. pipiens is 20 to 32°C. The threshold for development was calculated as 10.4 C (1976-12, 1977-6). A study was carried out in Manitoba, Canada, with Aedes dorsalis larvae which develop under parly spring snow-melt conditions and Culex tarsa~arvae which develop later in the season at higher temperatures. Successful invasion by R. culicivorax declined linearly from 93.6 to 1.5% in ~ tarsalis and from 73.1 to 1.6% in Ae~ dorsalis larvae exposed in the laboratory at 18, 16,-r4, 12 and 10°C. Larvae of Cx. tarsalis-were more susceptible than those of Ae. dorsalis at 18 and 16°C but this relationship was reversed at 12°C. Results of this experiment indicate difficulties in ~he application of R. culicivorax against certain mosquito species at low temperatures (1977-5). R. culicivorax established and overwintered in Maryland (USA) where temperatures went down to ~19°C (1976-7, 1979-6).

Laboratory tests conducted to determine the effects of high temperatures suggest that R. culicivorax has little value in habitats with temperature in excess at 40°C (1977-9). The nematode is killed with no significant change in appearance by a 3 sec. immersion in water at 65°C (1979-1).

(b) acidity: pH under normal conditions is not a limiting factor in obtaining infections in most mosquito habitats. R. culicivorax remains infective over a wide range of pH values (3.6-8.6) and infectivity is-enhanced by acid pH values (1979-7). Moreover, low pH inactivates the zoospores of Catenaria anguillulae, an hyperparasite which kills the postparasitic stages of R. culicivorax (see para. 4.3 predators of!· culicivorax and 7. production).

(c) salts: salinity is an important limiting factor for!· culicivorax. Even mild salinity (0.04 M NaCl) is inhibitive, and a sharp d~op in levels of parasitism is evident at concentrations between 0.015 and 0.030 M NaCl (1970-2). The effects of common inorganic ions WHO/VBC/80.766 VBC/BCDS/80.09 page 5 found in fresh water on the infectivity of the nematode and the survival of its host, Cx. pipiens were tested. In general the median lethal concentrations found for R. culicivorax were greater than the reported median concentrations of these ions in fresh water but less than the reported maximum natural concentrations. The ion toxicity for R. culicivorax (on a molar basis) increased in the following order: sodium

Although R. culicivorax will infect Aedes sierrensis under laboratory conditions (1968-2), it is unable to tolerate the water in tr~es, the normal habitat of Ae. sierrensis and consequently cannot be used as a biological control agent for the control of this species (1969-5, 1974-11).

(d) oxygen: Populations of R. culicivorax preparasites can survive low (1.8 and 3.1 3.1 mg/litre) levels of oxygen for-at least 36 hours and still remain infective. Low oxygen concentrations show losses of infectivity. of preparasitic R. culicivorax. Losses of infectivity are similar under aerobic (6 mg/1) and microaerobic (0.4 mg/1) conditions for the first 24 hours but thereafter are slower in the microaerobic group, suggesting a beneficial effect mediated by a quietent state of reduced metabolism. No interactions between temperature and oxygen were found over the ranges tested (15-27°C, 0.4-6.0 mg0 /l) (1978-2). Preliminary 2 biochemical evidence indicates that the post-parasitic larval stage of !· culicivorax (bottom of pools dweller) is able to function metabolically under anaerobic conditions (1976-12).

(e) photoperiod: Although Cx. pipiens larvae were found more susceptible to infection by!· culicivorax in total darkness (1974-1), no significant effect of photoperiod was detected in the study with Ae. dorsalis and Cx. tarsalis larvae and no interaction between temperature and photoperiod was evident (1977-5).

4.5 Susceptibility to pesticides, algacides and insect growth regulators

Insecticide susceptibility tests carried out in Taiwan have shown that temephos, and }r-HCH do not adversely affect the infective capacity of!· culicivorax at concentrations which could be lethal to mosquito larvae (1~74-6). Laboratory studies have shown that the (Altosid ), applied at doses ranging from 5-50 pg/kg did not affect the infectivity of the preparasitic, parasitic or postparasitic development of R. culicivorax. Host mortality was considerably increased when the mermithid and the IGR ;ere used concurrently against mosquitos (1976-3, 1977-4, 1979-6). The infectivity of R. culicivorax was not impaired by 1 mg/1 copper sulfate but higher concentrations were detrimental. Copper based organic algacides at concentrations of 0.5 mg/1 copper and Endothal 273 at-6.7 mg/1 did not affect the infectivity of the nematode. It appears that the effectiveness ofjk culicivorax would not be compromised by algacides since the above concentrations represent the usual levels of the compounds used for algae control (1976-12).

Susceptibility tests against preparasitic R. culicivorax indicate that 24 h exposure to temephos (Abate), 0.021 mg/kg; 0.100-mg/kg; 0.003 mg/kg; 0.001 mg/kg; Dimilin 0.005 mg/kg; and methoprene 0.005 mg/kg; produced no apparent detrimental effects to the viability and infectivity of R. culicivorax preparasities, or to the viability of the resulting postparasites. However, ~oncentrations of 0.0035 and 0.004 mg/kg fenthion, and 0.004 mg/kg chlorpyrifos, resulted in significantly lower preparasite infectivity when compared to controls even through acute mortality of preparasites was not evident at these concentrations (1977-10, 1979-6). WHO/VBC/80.766 VBC/BCDS/80.09 page 6

Dosage-mortality tests conducted with preparas~t~c stages of !· culicivorax showed that concentrations of 0.0021 mg/1 chlorpyrifos, 0.018 mg/1 , 0.36 mg/1 , 1.06 mg/1 temefos, 1.10 mg/1 fenthion, 2.95 mg/1 methoprene, 4.36 mg/1 and 6.78 mg/1 malathion caused a 50% loss of swimming activity and consequently impaired the ability of the nematode to locate and infest mosquito larvae. Chlorpyrifos was significantly more toxic and malathion caused significantly less mortality to the infective stages than the other tested. Treatment of parasitized larvae 24 h after infection with insecticides had no adverse effect on the viability of the postparasites that emerged. Juvenile nematodes less than 7 days after emerging, moulted to adults which mated and laid viable eggs after treatment with insecticides. The rate of egg hatching after exposure to malathion, fenthion and methoprene was significantly lower than that of eggs exposed to propoxur and naled. However, no significant differences in per cent of egg hatch existed between eggs treated with insecticides and untreated eggs. Insect growth regulators such as methoprene and diflubenzuron have the advantage of allowing more of the postparasitic stage nematodes to emerge from the mosquito larvae than conventional insecticides because the IGR's , tend to lengthen the larval life cycle allowing the development of immature parasites before the treated larvae die (1978-15).

4.6 Predators and parasite of Romanomermis culicivorax

During an ultrastructural examination of preparasitic juveniles of!· culicivorax, particles morphologically resembling viruses were observed in the hypodermal cords, cells associated with the amphidial nerves, and spaces of the pseudocoelom. The particles found in the hypodermal cords of the nematode were isometric and developed in crystalline arrays in the cytoplasm. Damage to hypodermal cords was evident through cell disruption and lysis. On the basis of size, shape and location, the particles resembled viruses of the Togavirus family and Alphavirus group (1976-13, 1977-16).

Disease epizootics in laboratory cultures of !· culicivorax have been caused by the chytridiomycetous fungal parasite Catenaria anguillulae. Techniques used for mass-rearing the nematode provided ideal conditions for the dispersal of zoospores and multiplication of the fungus. f· anguillulae attacked the postparasitic stages of !· culicivorax within a short time after their emergence from the host and up to 90% of the postparasites of !· culicivorax were parasitized. No temperature suitable for mass rearing the nematodes but unsuitable for the fungus could be found. Adjustment of pH or a short period of chilling (0°C) were the most promising control measures. At acid pH (~5), zoospore motility and germination and infection of postparasites was suppressed (1976-12, 1978-10, 1978-12, 1978-14). Previous attempts to control~ anguillulae with six fungicides were unsuccessful:

Benomyl, cycloheximide and griseofulvin suppressed the mycelial growth but their activity against the fungus was very poor in tapwater. Benomyl appeared to be nematicidal since some fungus-free nematodes died. The fungal attack was not very severe in cycloheximide and griseofulvin treatments but inhibited the development of the nematode (1978-3).

Observations of high populations of aquatic in rice fields prompted studies on the potential of various aquatic invertebrates as predators of R. culicivorax. An ostracod, Cyprinotus dentatis, locally abundant in the rice fields of Taiwan was shown in the laboratory to prey upon preparasitic stages of R. culicivorax and reduce infection rates in mosquitos (1972-3, 1974-6).

Pre- and postparasitic studies have shown that copepods (Cyclops vernalis) are avid predators.

Single preparasites of R. culicivorax and arthropods were placed in small volumes of water and observed over 48 hours; the preparasites were attacked by C. vernalis and eaten within one hour. Ostracods were predatory but the time for prey location and ingestion was longer than that for copepods. Cladocerans (Simocephalus vetulus) consumed preparasites within 24 hours and the young of a gammarid (Hyallela azteca) attacked the preparasites within 48 hours. Green hydra did not attack the preparasites. WHO/VBC/80.766 VBC/BCDS/80.09 page 7

Single postparasites of R. culicivoraX were placed in 250 ml of water and observed over 72 hours. Diving beetle~ (Laccophilus terminalis) and garnmarids (~. azteca) attacked the postparasites and consumed the prey within one hour. Dragonfly and damselfly naiads and small crayfish (Procarnbarus clarki) ate the postparasites within 24 hours. Isopods (Asellus sp.) attacked the postparasites only after 48 hours. Planarians did not attack the postparasites.

These observations demonstrate the importance of biotic factors in the survival of R. culicivorax during application of this mermithid as a biological control agent (1978-10, l978-ll). Unpublished studies showed that starved copepods (C. vernalis) attack the infective larvae much more than satiated copepods and the attack rate is inversely proportional to the volume of water. Alternate food sources and vegetation in test containers did not reduce the ability of copepods to destroy infective larvae of R. culicivorax.

5. Effectiveness against target organisms

5.1 Under natural conditions

17 species of mosquitos are known to be natural hosts of R. culicivorax: Anopheles crucians, An. punctipennis, Culiseta inornata and Psorophora columbiae have a very high susceptibility to the parasite. Aedes atlanticus, Ae. mitchellae, Ae. sollicitans, Ae. tormentor, Culex erraticus, Cx. restuans, Uranotaenia lowii and ~· sapphirina are highly susceptible with no observed humoral resistance. Ae. vexans and An. quadrimaculatus are moderately susceptible and exhibit frequent physiological resistance. Cx. salinarius is moderately resistant, probably humoral in nature. Ps. discolor was also found infected by ~ culicivorax in nature (1979-9).

5.2 Under laboratory conditions

Seventy-eight species of mosquitos have been exposed to R. culicivorax in the laboratory (1979-9). Cx. quinquefasciatus is a good laboratory host for the nematode and is used to rear R. culicivorax in the laboratory.

Laboratory tests conducted in Taiwan showed that infection rates ranged from 90-100% when Cx. quinquefasciatus was exposed to the nematode at a ratio of 10:1 or greater (1974-6, 1976-8). Infection rates ranging from 67-73% were obtained in Cx. tritaeniorhynchus surnrnorosus at a ratio of 20:1 and from 5-47% when the exposure ~tio was 10:1. R. culicivorax also developed in Cx. fuscanus, Cx. fuscocephalus and Cx. rubithoracis. Observations on An. sinensis indicated that preparasites which invaded this species were melanized and failed to develop to maturity (1974-6).

Susceptibility of 18 species of mosquitos to the infective stage of !· culicivorax were compared to that of Cx. quinquefasciatus. An. albimanus, An. ~adrimaculatus and An. freeborni were all highly susceptible to!· culicivorax. These species were 6-9 times more susceptible to penetration and development than were Cx. quinquefasciatus when the incidence of parasitism was below 10% in the latter.

Ae. sollicitans was the most susceptible of the six Aedes spp. tested and was about three times-;ore susceptible than Cx. quinquefasciatus when parasitism was low. Ae. nigromaculis, Ae. taeniorhynchus and Ae. tormentor were about two times more susceptible than the control and A~~ aegypti was slightly less susceptible. Phystological resistance was high with Ae. triseriatus (parasitic development never exceeded 9%) and was attested by the presence of melanized parasites in older larvae. Cx. tarsalis and Cx. salinarius were three times more susceptible and Cx. £· pipiens was mor;-than twice as susceptible as Cx. quinquefasciatus to both penetration and development by~. culiciyorax.

No physiological resistance was noted in Cx. tarsalis, Cx·£· p~p~ens or native Cx. quinque­ fasciatus. However, penetration was consistently higher 24 h after treatment in Cx. salinarius and some resistance (about 10%) was evident at very late stages of host development. Cx. territans was the only species in which no parasite development was observed even though the preparasites penetrated up to 67% of the larvae. Cs. inornata, the most susceptible of WHO/VBC/80.766 VBC/BCDS/80.09 page 8 the culicine species, was about 9.5 times more susceptible than Cx. quinquefasciatus at low levels of parasitism and had no resistance to development of the parasite. Ps. columbiae and Ps. varipes were highly resistant to the development of~· culicivorax (1975-4).

~· culicivorax is currently being tested in the laboratory for the control of the vector species Ae. samoanus in Pandanus leaf axils. The results available so far indicate that application is effective up to a period of one month during which the mean average of mosquito larvae per leaf-axil was reduced from 9.8 to 6.5. There was also evidence of the parasite having become established in the habitat and maintaining a parasitism rate of 11% at the end of the period (1978-4).

The susceptibility of five common species of mosquitos in Japan to preparasites of R. culicivorax was studied. The incidence of parasitism was the highest in Cx. E· molestus, followed by Cx. E· pallens, Ae. albopictus, Ae. togoi and Armigeres subalbatus. 2400 pre­ parasitic nematodes were released in a 6.5 x 120 em stagnant water pool. Parasitism was higher when the nematodes were mixed in overall water and the mosquito larvae held in several locations. No parasites were recovered when the nematodes were introduced at one end and the larvae located at the other end of the pool (1979-3).

Field evaluation of R. culicivorax carried out in El Salvador (see para. 5.3) have shown that even a modest wave a~tion along the lake shore could considerably reduce the effectiveness of this mermithid against mosquito larvae (1978-9).

There was therefore very little chance that~. culicivorax could constitute a promising blackfly control agent. This matter received nevertheless some consideration because mosquito mermithids are much more easily mass-produced than blackfly mermithids.

Preliminary investigations made in still water at 27°C showed that after a four hours exposure preparasitic ~· culicivorax had infected first-, second- and third-instars of 3 species of North American blackflies: Simulium venustum, S. vittatum and S. decorum. The former species were more receptive than the latter and for each species, first- and second-instar larvae were more infected than third-instar larvae (1975-1).

Subsequent laboratory studies carried out in Western Africa indicated that preparasitic R. culicivorax in still water could pentrate a high proportion of first-, second- and third­ instar blackfly larvae belonging to the~ damnosum complex. The proportion of infected larvae decreased slightly when the experiment was done in water moving at a velocity of 2 em/sec and a much sharper decrease in infection rate was observed at higher water velocities (1976-4, 1978-5).

Complementary investigations carried out with infected larvae of ~· damnosum and S. venustum from the above mentioned experiments showed that~ culicivorax initiated development in these hosts which died before the nematodes completed their development (1979-12). When complementary studies were carried out under less artifical conditions with first-instar larvae of S. damnosum exposed in troughs with a 5° slope and a water flow of 250 to 280 ml/sec, even very high rates of preparasitic R. culicivorax failed to induce significant levels of parasitism or cause the detachment and drifting of the Simulium larvae. A thorough analysis of the results further suggested that the low rate of infection observed was not due to any direct attack of the blackfly larvae by preparasites of the nematode while in running water but resulted from subsequent penetration of the blackfly larvae in still water containing pre­ parasitic nematodes during post-exposure observations. When drift samples were preserved in alcohol immediately after removal from the troughs, no infected blackfly larvae were found (1979-8).

In another experiment 100 young larvae of ~· vittatum were exposed to preparasites of R. culicivorax in moderately agitated water at 25 C. Dozens of the preparasitic nematodes were ;wallowed alive and packed in the gut. But noneentered the body cavity through the digestive tract or through the cuticle. Most of the blackflies pupated and became adults (1979-6). P'

WHO/VBC/80.766 VBC/BCDS/80.09 page 9

These experiments confirm-beyond any doubt that R. culicivorax neither offers any promise for the control of blackfly larvae nor constitutes an-acceptable model for studying mermithid­ blackfly relationships.

5.3 Under field conditions

33 species of mosquitos have been exposed to Romanomermis culicivorax in the field (1979-9). A mean of 65, 58 and 33% of the second, third and fourth-instar larvae of the Anopheles spp. respectively, were parasitized by the nematode R. culicivorax when 11 natural sites in Louisiana, USA, all semi-permanent to permanent and l~w in salinity, previously free of the nematode, were treated 20 times with preparasitic juveniles of R. culicivorax. Since tests showed that preparasites could be passed through a compressed air sprayer with little or no loss of infectivity, all preparasites were disseminated by using a compressed air sprayer with a standard fan nozzle. A dosage rate of 1000 preparasites per sq. metre of water surface resulted in 94% parasitism in second-instar Anopheles larvae and 64% parasitism in all Anopheles sampled. Three sites treated with postparasites produced mean parasitism of Anopheles larvae ranging from 20 to 24% with monthly highs of 100, 34 and 27 per cent. All sites produced natural infections at least once after treatment and R. culicivorax appeared to have become established in at least seven of the sites (1972-8).

~· culicivorax was introduced into paddies in two rice fields in central California in an attempt to parasitize larvae of Cx. tarsalis and An. freeborni. Cx. tarsalis was not found to be parasitized but its population was low. Fifty and 80-85% of first, second and third­ instaz larvae of ~· freeborni became parasitized when preparasites were applied at rates of 500/m and 1000/m , respectively (1972-6).

Three field treatments of drains and ditches in Bangkok, Thailand, by preparas~t~cs of 2 R. culicivorax at dosages up to 252,000/m for the control of ~~· ~pinquc;L:sciatus gave 0-21% infection rates. No evidence of reduction of the pupal populati0n or recycling of the parasite in nature was obtained (1972-2).

Attempts made in Taiwan to infect mosquito populations in their natural habitats met with mixed results and infection rates were generally low. There was no evidence that the parasite had become established in nature. Reduced water temperature prevented infection of Cx. quinquefasciatus in the field and an ostracod Cyprinotus dentatis was abundant in the rice fields: see 4.6 predators of R. culicivorax (1972-3, 1974-6).

When first-instar larvae of Ps. columbiae were exposed toJL. culicivorax in a series of 6 x 15 m rice field plots in Louisiana, mean parasitism ranged from 10 to 38 per cent as rates 2 of_R. culicivorax were incre~sed from 180 to 1450 perm of surface area; an estimated 3900 preparasitic nematodes per m would therefore be required to produce 95+ per cent parasitism of the species. Mean parasitism in first-instar larvae of ~· quadrimaculatus exposed in the s~e way ranged from 16 per cent for 181 preparasite2 per m to 61% for 725 preparasites per m ; an estimated 1300 preparasiti.csR. culicivorax/m would therefore be required to produce 95+ per cent parasitism in this species (19~3-7). ~· culicivorax was released 30 times (15 at rates of 1000 and 15 at rates of 2000 per m surface area) from a compressed air sprayer with a standard fan nozzle, in attempts to control Anopheles mosquito larvae, principally An. crucians in natural habitats in Louisiana. At the lower rate, 76% of the hosts were infected and parasitisru averaged 60, 80, 86, and 71% in lst-instar through 4th-instar hosts, respectively. At the higher rate, 85% of the An()pheles larvae were infected and 10 of the 15 treatments produced levels of parasitism in excess of 90%. In general vegetation and depth of water had little influence on ultimate levels of parasitism (1974-8).

A follow up of the 1971 and 1973 tests'carried out in Louisiana (1972-8, 1973-7, 1974-8) showed that R. culicivorax often becomes established in many permanent and semi-permanent water sites and pr~duced high levels of infection in Anopheles mosquitos for an indefinite period. Eight of the sites produced significant levels of parasitism for over 2 years and two of the sites showed continued activity for over 4 years after introduction (1975-6, 1976-9).

-- J WHO/VBC/80.766 VBC/BCDS/80.09 page 10 l Cultures containing R. culicivorax were introduced into habitats of floodwater mosquitos in Louisiana as a pre-hatch treatment. Significant levels of parasitism were achieved when cultures were placed in damp habitats that possessed adequate amounts of vegetation or organic debris and were subjected to minimum flushing action when flooded. 52% of Ae. atlanticus, 59% of Ae. tormentor, 38% of Ps. columbiae (= P. confinnis) and 51% of P. howardii were parasitized in 39 larval collections from 13 h;bitats. R. culicivorax ;as observed to penetrate but failed to develop in larvae of Ps. ferox. -Though the number of~. vexans were low, the levels of parasitism (5%) indicated that this species is somewhat resistant to the attack of R. culicivorax. Also levels of parasitism in Ae. fulvus pallens (13%) were low, probably b;cause the characteristic delayed hatch of this species allows it to miss the brunt of the infective stage nematodes (1976-11). 2 In Taiwan, two small scale field trials were conducted in 4.5 and 5 m ground pools (clear water but highly organic polluted bottom) with preparasites and postparasites. All mosquitos 4 sampled in the pool were identified as Cx. quinque~asciatus. A total of 1 x 10 preparasitics 2 (2000/m ) were introduced into one site and 1 x 10 postparasitic nematodes were released into the other site. The first recycling of R. culicivorax occurred on the tenth week after treatment. Recyclings of the nematode r;appeared and a few infected larvae of Cx. quinquefasciatus were found repeatedly on the 238th day and in the following samplings although the paras1t1sm was quite low, ranging from 0.1 to 0.9%. No infected larvae could be found in the treatment with postparasites (1976-8).

An attempt was made to use R. culicivorax for the control of spring Aedes mosquitos in Manitoba, Canada. Temperature was the primary factor limiting infection by the nematode in mosquito larvae which develop in snow-melt pools. When preparasitic nematodes were applied to pools containing mosquito larvae, onl6 about 20% of Aedes canadensis and Ae. pionips were infected at maximum temperatures of 5-10 C. No infection occurred at similar temperatures in larvae of Ae. dorsalis or Ae. spencerii. A few larvae of Ae. communis were infected at 0-5°C. Eight to 20% of the larvae~f Ae. vexans which appeared after an early spring rain were infected when preparasites wer;-introduced into the pool. Survival of R. culicivorax during parasitic development was dependent upon host species and degree of sup;rparasitism. Melanized nematodes were discovered in larvae of Ae. vexans and Ae. canadensis; the nematodes that had survived the winter and infected larvae of Ae-.-communis were all melanized (1976-2). 3 2 R. culicivorax was released at a dosage rate of ca. 3.6 x 10 preparasites/m of water surface with a compressed air sprayer in an attempt to control mosquitos breeding in a grassy 6 field+in S.W. Florida. Results from the release of ca. 9 x 10 preparasites indicated that 96.5 - 2.8% of the first-fourth instar larvae of Ps. columbiae, Ps. ciliata, Cx. nigripalpus and Ae. taeniorhynchus from 8 potholes and ditches in. the field were parasitized by R. culicivorax and marked reductions in host population were observed in samples 24 h post­ treatment. No larvae were found in two additional sampling sites 24 h post-treatment that contained high populations of 1st and 2nd-instar Ps. columbiae and Cx. nigripalpus prior to preparasite release (1977-12).

Tests were conducted in Sanibel Island, Florida, to determine if R. culicivorax would effectively parasitize and develop in Culex quinquefasciatus larvae br;eding in an abandoned sewage settling tank. Field releases of preparasites directly into two of the sewage settling compartments of the main system resulted in 37.3 and S3.7% parasitism of host larvae at estimated parasite-to-host ratios of 3.4:1 and 4.6:1 (1977-11).

Field tests were conducted in California with R. culicivorax against four species of mosquito larvae in i natural and 2 arti~ical sites. Infective nematodes were disseminated at 706 to 25 000/m surface area. An average 87.5% of An. franciscanus were infected in the artifical ponds. Lower levels of infection were found in Cx. tarsalis and Cs. inornata which were found to be about equally susceptible. The infection level in all species increased as dosage was increased. Since anopheline larvae tend to spend more time at the surface than larvae of culicines, it is reasonable that the preparasites infect \ anophelines in naturel habitats more readily because the preparasites are located mainly in F

WHO/VBC/80.766 VBC/BCDS/80.09 page 11

surface water (1977-2). 2 R. culicivorax was mass-produced for the treatment of 144 000 m of Anopheles albimanus breeding area in El Salvador (see para. 7, production) (1978-8). The mosquito breeding area of Lake Apastepeque, El Salvador was treated 11 times over a 7 weeks' period with R. culicivorax to control An. albimanus and An. pseudopunctipennis. 6 The study called for the release of 51.4 x 10 preparasitic nematodes over all of the Anopheles breeding area (1.2 ha) around the margin of Lake Apastepeque twice weekly for six weeks. To accomplish this, 40 cultures were to be flooded 14-20 h prior to each application for the first 6 planned treatments; and for the last treatments 20 cultures were to be flooded and the 40 cultures from the previous treatment were to be reflooded.

Cultures were flooded with ca. 700 ml of chlorine-free water during the evening of the day prior to the planned application. A 19-litre backsprayer was used to apply the nematodes. Parasitism averaged 58% but varied greatly from treatment to treatment and from site to site. It was found that wave action on the lake produced an average 86% parasitism. No significant differences in susceptirility toR. culicivorax were found between instars or between species. Also no correlation was found bet;een nematode dosage rates and levels of parasitism. Some Cx. erraticus and Ur. lowii present in the habitat were also infected but there was no evidence of any effect~non-target organisms. Though the parasitism averaged about 60% of the desired level, Anopheles populations dropped from more than 10 per dip at the beginning of the release programme to 0.6 per dip at the end of the release period (a 94% reduction). There was no significant recycling of!· culicivorax in Lake Apastepeque (1978-9).

A follow-up of this study to determine the efficacy of R. culicivorax against larvae of An. albimanus was made in the more typical breeding sites in-the coastal area of El Salvador. Treated areas were sites such as flooded pastures, roadside ditches, ponds and swamps that contained larval populations of An. albimanus. Dosage used ranged from 2400 to 4800 preparasites/ sq.m of surface breeding area. All treatments gave excellent parasitism of An. albimanus regardless of dosage or instar. Multiple parasitism of larvae was common. Overall parasitism following the treatment averaged 96% and mean parasitism for 1st to 4th instars was 95, 95, 98 and 95% respectively. Two treatments made just prior to a heavy rain produced infection levels of only 74 and 89%. Occasionally larvae of Ur. lowii and Cx. erraticus were presented and infected (1980-1).

Tests were conducted to determine if the preparasitic stage of R. culicivorax could be applied from a helicopter via a low profile aerial spray system. Preparasites suffered no apparent adverse effects from impact within the spray system and nozzle assemblies (1976-6, 1977-8).

Thus an experimental release of preparasites of !· culicivorax was made in 3 ponds via 2 helicopter at the rate of ca. 4629/m of water surface to cOntrol natural populations of An. quadrimaculatus, An. crucians and Cx. erraticus larvae in S.W. Florida. Mean levels of parasitism of 52.4, 66.7 and 43.8% of the 1st-4th instar Anopheles spp. and 39.2, 40.0 and 53.3% of the 1st-4th instar larvae of Cx. erraticus from the three ponds, respectively, were obtained. Encapsulation and melanization of the nematode was observed in several 3rd and 4th instar Anopheles spp. larvae. The majority of the Anopheles spp. larvae examined 24 and 48 h after treatment were infected with more than one nematode. The aerial spray tests show that little or no inhibitory effect on infectivi~y and development of R. culicivorax could be expected as a result of the helicopter spray system (1979-4).

R. culicivorax was released in breeding areas in Maryland (USA) where it established and overwintered, killing mosquitos at the level of 50-100% even two years after treatment (1979-6).

Preliminary field trials were carried out with~. culicivorax against An. gambiae ln natural breeding sites around Kaduna, Nigera. Testing sites were chosen for two types of mosquito breeding habitats. The first dried up and the second held water throughout the wet season and became dry only during the dry season. l WHO/VBC/80.766 VBC/BCDS/80.09 page 12

Results from these field trials showed that the nematodes are probably killed by dry condition and thus will nor survive in breeding sites which dry up intermittently. The life cycle of the nematode takes 7 to 8 weeks while the larval life of An. gambiae is between 6 and 11 days. Besides, An. gambiae may oviposit daily. Thus, repeated-;nd frequent nematode applications are necessary so that the preparasitic forms are continuously available to infect the successive generations of An. gambiae larvae.

A fungus found in breeding sites apparently caused a fatal infection of the nematode, thereby preventing recycling under natural conditions (1979-14). Unpublished information sent to WHO gives the following details of further findings: R. culicivorax recycles readily in small field ponds. In the absence of suitable hosts, a single inoculation of postparasites continues to produce infective larvae for at least a year. Reinvasion of areas in which hosts were eliminated by massive inoculations of infective larvae may be suppressed as long as the postparasites have developed and were not destroyed by predators.

Studies in large field environments have shown that R. culicivorax can recycle readily. An early application of postparasites of the nematode dem~nstrated that they could mature to adults, mate and lay eggs in the rice field environment. Preparasites hatching from those eggs provided continuous partial control of larval An. freeborni and Cx. tarsalis.throughout the rice growing season. Control of An. freeborni was comparable to that obtained in the past by direct application of preparasites-;nd exceeded that previously achieved for Cx. tarsalis. Infection was observed up to 6 metres from the original point of application (1979-10, 1979-15).

6. Effectiveness against non-target organisms

6.1 Invertebrates

Tests were made on three aquatic invertebrates: the daphnid Daphnia magna, the amphipod Gammarus pseudolimnaeus and an oligochaete, Lumbricus sp. All were exposed to heavy populations of preparasitic larvae of R. culicivorax and were still living after 14 days of exposure. No evidence of penetration ~r damage was detected despite a thorough examination of the body wall, coxal gills and digestive tract (1973-3). A series of tests was conducted with larvae of Chironomus sp. and Psychoda sp. Preparasitic !· culicivorax failed to damage or penetrate Psychoda sp. larvae. Apparently preparasites can penetrate larvae of Chironomus sp. when the population of R. culicivorax is large but are unable to develop and survive (1973-3, 1979-12).

Preparasitic R. culicivorax were also tested against other non-target organisms that commonly occur in aquatic habitats: immature and/or adult stages of Diptera (Chaoboridae), Coleoptera (Hydrophilidae, Dystiscidae, Haliplidae), Hemiptera (Notonectidae, Corixidae and Belostomatidae), Odonata (Zygoptera, family not determined), Ephemeroptera (Baetidae) and crayfish (Cambarus sp.). Only larvae of Corethrella appendiculata, a treehole breeding chaoborid were susceptible to R. culicivorax and only when they were exposed to high numbers of preparasites (1300/host). No penetration occurred at a density of 60 preparasites/insect host. However, large numbers of preparasites (1300/host) either killed larvae of Corethrella brakeleyi because of multiple penetrations or failed to develop once they had successfully penetrated (1973-3).

When R. culicivorax was applied for the control of spring Aedes mosquitos in Manitoba, larvae of Mochlonyx velutinus and Dixa sp and nymphs of Ephemeroptera were not infected (1976-2).

Several non-target organisms found in Simulium damnosum habitats were exposed to R. culicivorax. One or more juveniles of six species of Tricoptera, 3 species of Chirono;idae, 1 species of Ceratopogonidae, 2 species of Ephemeroptera, 2 species of Simuliidae, 3 species of Odonata and one species of watermite were exposed to the nematode at a parasite to host ratio of 1000:1. Only one simuliid larva and two chironomid larvae were infected. First-instar Culex larvae exposed at a 10:1 ratio at the same time were heavily infected ( ;> 90%)(1979-8). WHO/VBC/80.766 VBC/BCDS/80.09 page 13

The diving bettles, Ranatra and Nepa species (Hemiptera) living in the same mosquito breeding habitats as An. gambiae were exposed to preparasitic !· culicivorax (about 200 nemas each for 5 ); no adverse effect was found on the exposed species (1979-14).

6.2 Poikilotherm vertebrates

Eggs, yolk-sac fry and swim-up fry of four species of freshwater fish: rainbow trout (Salmo gairdneri), large mouth bass (Micropterus salmoides), channel catfish (Ictalurus punctatus) and fathead minnows (Pimephales promelas) were exposed to preparasitic larvae of R. culicivorax. Exposure levels were 6.25 preparasitic larvae per ml of water which was a ratio of 1250:1 for nematode larvae to trout swim-up fry and 94:1 for all other species. Exposure times were 96 hours for bass and minnows and 10 days for catfish. Rainbow trout were exposed continuously from the eyed egg into the yolk-sac fry stage. Whole body examination of bass and minnows, and examination of the buccal cavity, gills, gut, skin surface and eyes of trout and catfish revealed no evidence of nematode invasion. In addition, nematodes could not be found in any body tissues (1973-3). Tadpole and Epiplatys fish found in An. gambiae breeding habitats were not infected by !· culicivorax at concentrations of approximately 800 nemas for each tadpole and 200 nemas for each 5 fish (1979-14).

6.3 Homeotherm vertebrates

Suckling and adult mice and adult rats were subjected to either per~· intranasal, intraperitoneal or dermal exposure to R. culicivorax. Body weight gain and histological examination of tissues from re~eiving nematodes were essentially identical to those of untreated animals. Immune-depressed rats also were not susceptible. A few dead nematodes were detected in faeces from mice fed nematodes. R. culicivorax was not detected in urine samples (1974-3).

7. Production

Procedures have been developed for the exposure of larvae of Cx. quinquefasciatus (which are easily maintained in colony and highly susceptible to attacks by the nematode) to preparasitic R. culicivorax to obtain high yields of nematodes. The eggs of R. culicivorax are stored in-moist sand where they remain viable until needed. When the nematodes are needed for tests, the egg-sand cultures are flooded with chlorine-free water and hatched nematodes are exposed to mosquito larvae. !· culicivorax is produced most rapidly and economically 1n 51 x 137 x 5 em trays containing 20 000 hosts per tray, when hosts are exposed at a ratio of 14:1 parasites/hosts and hosts fed 0.30, 0.45 and 0.60 g of food per 1000 hosts on the 1st, 2nd and 3rd days respectively and 0.90 g each day thereafter. After 7-8 days the nematodes are removed from the rearing. trays and placed into nematode collecting trays. The screen bottom in these trays permits a quick and easy separation of the nematodes as they escape from their hosts. The nematodes are then washed, weighed and placed in aluminium cakepans containing clean sterile sand (1.5 em deep) covered with 1 em of chlorine-free water. The cultures are covered and stored. After about three weeks the water is carefully decanted and the culture stored for an additional 4-15 weeks before use (1972-1, 1972-7, 1973-4, 1978-7). This standard rearing procedure was slightly modified when necessary to control the chytridiomycetous hyperparasite Catenaria anguillulae (1978-12) (see 4.6, predators and parasites of!· culicivorax). 2 R. culicivorax was mass-produced for the treatment of 144 000 m of An. albimanus breeding area in ~1 Salvador. The production of the necessary inoculum required the exposure of 6 1.6 x 10 first-instar Cx. quinquefasciatus larvae to 137 x 10 preparasites (1:14 ratio) each week for six weeks. The method employed produced an average 13.7 g (ca. 2200/g) of post­ parasitic nematodes per rearing tray (20 000 mosquitos), a total of 6392 g fgr the six-week period and 425 cultures. Two persons easily handled the rearing of 1.6 x 10 mosquitos and the resulting nematodes each week (1978-8).

Experiments were made in the laboratory to grow R. culicivorax in in vitro cultures. Pre­ parasitic juveniles, hatched from sodium hypochlorite-treated (sterilized) eggs can develop over a 6 week period to a length of 6-7 mm when cultured in association with tissue culture cells (1973-8, 1973-9, 1976-1). When cultures were initiated using insect culture media together with l WHO/VBC/80.766 VBC/BCDS/80.09 page 14 inactivated calf serum, R. culicivorax grew to the stage of a nematode having all the basic developmental features of a six-day parasite in the mosquito host. The worms had a well­ developed cuticular tube and a vulval aperture but it took them six weeks to obtain a lengt~ of 12 mm. However they lacked a developed trophosome indicating a nutrient problem. The surv~val rate was estimated at 30%. None of the nematodes cultured in vitro differentiated into males (1977-3).

Proton-induced X-ray emission, an extremely sens~t~ analytical technique has been used to study the concentration of several major and trace elements of R. culicivorax and its hosts; this technique may be useful in elucidating important physiological and nutritional relations between the nematode and its host, e.g. in determing elemental co-factors necessary in the formation of an insect tissue-culture medium for successful in vitro culturing of R. culicivorax (1979-5).

8. Stability

The tests with R. culicivorax against Simulium damnosum were conducted at the OCCGE laboratory at Bouake, Ivory Coast. The mermithid cultures were produced in Louisiana 10 weeks prior to the proposed testing period. The cultures were carefully packaged in a manner that prevented shifting of the sand and reduced vibration. They were subjected to two long drives and air travel and arrived in apparent good condition. About a 50% loss in preparasite yield was experienced as a result of shipping and handling. These losses were moderately low considering the handling, shipping distance and age of cultures at the time of shipment (1979-8).

9. Formulation and specifications

None.

10. References

1967

1967-1 Chapman, H. C., Woodard, D. B. and Petersen, J. J. (1967). Nematode parasites of Culicidae and Chaoboridae in Louisiana. Mosq. News, 27: 490-492.

1968

1968-1 Chapman, H. C., Petersen, J. J., Woodard, D. B. and Clark, T. B. (1968). New records of parasites of Ceratopogonidae. Mosq. News, 28: 122-123.

1968-2 Petersen, J. J., Chapman, H. C. and Woodard, D. B. (1968). The bionomics of a mermithid nematode of larval mosquitoes in southwestern Louisiana. Mosq. News, 28: 346-352.

1969

1969-1 Chapman, H. C., Clark, T. B., Petersen, J. J. and Woodard, D. B. (1969). A two-year survey of pathogens and parasites of Culicidae, Chaoboridae, and Ceratopogonidae in Louisiana. Proc. N.J. Mosq. Exterm. Assoc., 56: 203-212.

1969-2 Petersen, J. J., Chapman, H. C. and Willis, 0. R. (1969). Fifteen species of mosquitoes as potential hosts of a mermithid nematode Romanomermis sp. Mosq. News, 29: 198-201.

1969-3 Tsai, Y.-H. and Grundmann, A. W. (1969). Reesimermis nielseni gen. et. sp. n. (Nematoda: Mermithidae) parasitizing mosquitoes in Wyoming. Proc. Helminthol. Soc. Wash., 36: 61-67. WHO/VBC/80.766 VBC/BCDS/80.09 page 15

1969-4 Tsai, Y.-H., Grundmann, A. W. and Rees, D. M. (1969). Parasites of mosquitoes in southwestern Wyoming and northern Utah. Mosq. News., 29: 102-110.

1969-5 Petersen, J. J. and Chapman, H. C. (1969). Chemical factors of water in tree holes and related breeding of mosquitoes. Mosq. News, 29: 29-36.

1970

1970-1 Chapman, H. C., Clark, T. B. and Petersen, J. J. (1970). Protozoans, nematodes and viruses of anophelines. Misc. Publ. Entomol. Soc. Amer., 7: 134-139.

1970-2 Petersen, J. J. and Willis, 0. R. (1970). Some factors affecting parasitism by mermithid nematodes in southern house mosquito larvae. J. Econ. Entomol., 63: 175- 178.

1971

1971-1 Petersen, J. J. & Willis, 0. R. (1971). A two-year survey to determine the incidence of a mermithid nematode in mosquitoes in Louisiana. Mosq. News, ~: 558-566.

1971-2 Savage, K. E. and Petersen, J. J. (1971). Observations of mermithid nematodes in Florida mosquitoes. Mosq. News, 31: 218-219.

1972

1972-1 Anonymous. (1972). Mass-produced nematodes down mosquitoes. Agri. Res., U.S.D.A., 20: 3-4.

1972-2 Chapman, H. C., Pant, C. P., Mathis, H. L., Nelson, M. J. and Phantumachinda, B. (1972). Field releases of the nematode Reesimermis nielseni for the control of Culex. ~· fatigans in Bangkok, Thailand. Mimeographed document WHO/VBC/72.412, Geneva, 7 pages.

1972-3 Mitchell, C. J., Chen, P. S. & Chapman, H. C. (1972). Exploratory trials utilizing a mermithid nematode as a control agent for Culex mosquitos in Taiwan (China). Mimeographed document WHO/VBC/72.410, Geneva:-rz-pages.

1972-4 Nickle, W. R. (1972). A contribution to our knowledge of the Mermithidae (Nematoda). J. Nematol., 4: 113-146.

1972-5 Petersen, J. J. (1972). Factors affecting sex ratios of a mermithid parasite of mosquitoes. J. Nematol., ~: 83-87.

1972-6 Petersen, J., J., Hoy, J. B. and O'Berg, A. G. (1972). Preliminary field tests with Reesimermis nielseni (Mermithidae: Nematoda) against mosquito larvae in California rice fields. Calif. Vector Views, 19: 47-50 (Mimeographed document WHO/VBC/72.357, Geneva, 4 pages.

1972-7 Petersen, J. J. and Willis, 0. R. (1972). Procedures for the mass rearing of a mermithid parasite of mosquitoes. Mosq. Ne~s, ~: 226-230.

1972-8 Petersen, J. J. and Willis, 0. R. (1972). Results of preliminary field applications of Reesimermis nielseni (Mermithidae: Nematoda) to control mosquito larvae. Mosq. News, ~: 312-316. WHO/VBC/80.766 VBC/BCDS/80.09 l i page 16

1973

1973-1 Bailey, C. H. and Gordon, R. (1973). Histopathology of Aedes aegypti (Diptera: Culicidae) larvae parasitized by Reesimermis nielseni (Nematoda: Mermithidae). J. Invertebr. Pathol., 22: 435-441.

1973-2 Hoy, J. B. and- P.etersen, J. J. (1973). · Fish and nematodes - current status of mosquito control techniques. Proc. Calif. Mosq. Control. Assoc., 41: 49-50.

1973-3 Ignoffo, C. M., Biever, K. D., Johnson, W. W., Sanders, H. 0., Chapman, H. C., Petersen, J. J. and Woodard, D. B. (1973). Susceptibility of aquatic vertebrates and invertebrates to the infective stage of the mosquito nematode Reesimermis nielseni. Mosq. News, 33: 599-602.

1973-4 Petersen, J. J. (1973). Factors affecting mass production of Reesimermis nielseni a nematode parasite of mosquitoes. J. Med. Entomol., 10: 75-79.

1973-5 Petersen, J. J. (1973). Relationship of density, location of hosts, and water volume to parasitism of larvae of the southern house mosquito by a mermithid nematode. Mosq. News, 12: 516-520.

1973-6 Petersen, J. J. (1973). Role of mermithid nematodes in biological control of mosquitoes. Exp. Parasitol., 33: 239-247.

1973-7 Petersen, J. J., Steelman, C. D. and Willis, 0. R. (1973). Field parasitism of two species of Louisiana rice field mosquitoes by a mermithid nematode. Mosq. News, 33: 573-575.

1973-8 Roberts, D. W. and Van Leuken, W. (1973). Limited growth of a mermithid nematode in association with tissue culture cells. Abstr. Int. Coll. Ins. Path. Micro. Control., Oxford: p. 91.

1973-9 Sanders, R. D., Stokstad, E. L. R. and Malatesta, C. (1973). Aexenic growth of Reesimermis nielseni (Nematoda: Mermithidae) in insect tissue culture media. Nematologica, 19: 567-568.

1974

1974-1 Brown, B. J. and Platzer, E. G. (1974). The effect of temperature, light, larval age and exposure time on the infectivity of preparasitic larvae of Reesimermis nielseni. J. Nematol., ~: 137 (Abst.).

1974-2 Gordon, R., Bailey, C. H. and Barber, J. M. (1974). Parasitic development of the mermithid nematode Reesimermis nielseni in the larval mosquito Aedes aegypti. Can. J. Zool., 52: 1293-1302.

1974-3 Ignoffo, C. M., Chapman, H. C., Petersen, J. J., and Novotny, J. F. (1974). Lack of susceptibility of mice and rats to mosquito nematode Reesimermis nielseni Tsai and Grundmann. Mosq. News, 34: 425-428.

1974-4 Kerdpibule, V., Deesin, T., Sucharit, S. and Harinasuta, C. (1974). A preliminary study on the control of Mansonia uniformis by nematode parasitism (Reesimermis nielseni). South East Asian J. Trop. Med. Public Health, i= 150-151.

1974-5 Kurihara, T., Araki, J. and Ikemoto, T. (1974). The effects of temperature and other factors upon the establishment and survival of a mermithid nematode Reesimermis nielseni in mosquito larvae. Joint Conf. Parasit. Dis. Jpn. US Coop. Med. Sci. Program: pp. 20-21.

I,, -·· WHO/VBC/80.766 ·VBC/BCDS/80.09 page 17

1974-6 Mitchell, C. J., Chen, P. S. and Chapman, H. C. (1974). Exploratory trials utilizing a mermithid nematode as a control agent for Culex mosquitos in Taiwan. J. Formosan Med. Assoc., 73: 241-254. -----

1974-7 Nickle, w. R. and Hogger, c. H. (1974). Scanning, electron microscopy of the mosquito parasite Reesimermis nielseni (Nematoda: Mermithidae). Proc. Helminthol. Soc. Wash., 41 : 17 3-1 77 •

1974-8 Petersen, J. J. and Willis, 0. R. (1974). Experimental release of a mermithid nematode to control Anopheles mosquitoes in Louisiana. Mosq. News, 34: 316-319.

1974-9 Poinar, G. 0. Jr. and Hess, R. (1974). Structure of the pre-parasitic juveniles of Filipjevimermis leipsandra and some other Mermithidae (Nematodae). Nematologica, 20: 163-173.

1974-10 Poinar, G. 0. Jr. and Otieno, W. A. (1974). Evidence of four molts in the Mermithidae. Nematologica, 20: 370.

1974-11 Poinar, G. 0. Jr. and Sanders, R. D. (1974). Description and bionomics of Octomyomermis troglodytis sp.n. (Nematoda: Mermithidae) parasitizing the western tree hole mosquito Aedes sierrensis (Ludlow) (Diptera: Culicidae). Proc. Helminth. Soc. Wash., 41: 37-41.

1975

1975-1 Finney, J. R. (1975). The penetration of three simuliid species by the nematode Reesimermis nielseni. Bull. Wld Hlth Org., 52: 235.

1975-2 Galloway, T. (1975). Application of a mermithid nematode (Reesimermis nielseni, Tsai and Grundmann) from Louisiana for mosquito control in Manitoba. Proc. Alberta Mosq. Abat. Symp.: pp. 191-197.

1975-3 Hall, D. W., Andreadis, T. G., Flanagan, T. R. and Kaczor, W. J. (1975). Melanotic encapsulation of the nematode Neoaplactana carpocapsae by Aedes aegypti larvae concurrently parasitized by the nematode Reesimermis nielseni. J. Invertebr. Pathol. 26: 269-270.

1975-4 Petersen, J. J. (1975). Penetration and development of the mermithid nematode Reesimermis nielseni in eighteen species of mosquitos. J. Nematol., l= 207-210.

1975-5 Petersen, J. J. (1975). Development and fecundity of Reesimermis nielseni, a nematode parasite of mosquitoes., J. Nematol., l= 211-214.

1975-6 Petersen, J. J. and Willis, 0. R. (1975). Establishment and recycling of a mermithid nematode for the control of larval mosquitoes. Mosq. News, 35: 526-532.

1976

1976-1 Finney, J. R. (1976). The in vitro culture of the mermithid parasites of mosquitos and blackflies. Proc. Int. Colloq. Invertr. Path., Kingston, Canada: pp. 225-226.

1976-2 Galloway, T. D. and Brust, R. A. (1976). Field application of the mermithid nematode Romanomermis culicivorax Ross and Smith, for the control of mosquitos, Aedes spp., in spring in Manitoba. The Manitoba Entomologist, 10: 18-24.

1976-3 Gordon, R., Condon, W. J. and Finney, J. R. (1976). Endocrine relations between certain larval diptera and their mermithid parasites. Proc. Int. Colloq. Invert. Path., Kingston, Canada: pp. 268-271. WHO/VBC/80.766 VBC/BCDS/80.09 page 18

1976-4 Hansen, E. L. and Hansen, J. R. (1976). Parasitism of Simulium damnosum by Romanomermis culicivorax. IRCS Med. Sci., 4: 508.

1976-5 Kurihara, T. (1976). Population behaviour of Reesimermis nielseni, a nematode parasite of mosquitos with notes on the attraction of infective stage nematodes by mosquito larvae, Culex pipiens molestus. Jap. J. Parasitol., ~: 8-16.

1976-6 Levy, R., Murphy, L. J. Jr., and Miller, T. W. Jr. (1976). Effects of a simulated aerial spray system on a mermithid parasite of mosquitoes. Mosq. News, 36: 498-501·

1976-7 Nickle, W. R. (1976). Toward the commercialization of a mosquito mermithid. Proc. Int. Colloq. Invert. Path., Kingston, Canada: pp. 241-244.

I 1976-8 Paoshu, C. (1976). Study on Reesimermis nielseni for control of Culex pipiens I' fatigans in Taiwan. Bull. Inst. Zool. Acad. Sinica, J2: 21-28. I" 1976-9 Petersen, J. J. (1976). Recycling of the nematode Reesimermis nielseni during 1975 in Anopheles crucians in Louisiana. Proc. Utah Mosq. Abat. Assoc., 28: 17-18.

1976-10 Petersen, J. J. (1976). Comparative biology of Wyoming and Louisiana populations of Reesimermis nielseni, parasitic nematode of mosquitos. J. Nematol., ~: 273-275.

1976-11 Petersen, J. J. and Willis, 0. R. (1976). Experimental release of a mermithid nematode to control flood water mosquitos in Louisiana. Mosq. News, 36: 339-342.

1976-12 Platzer, E. G. and Brown, B. J. (1976). Physiological ecology of Reesimermis nielseni. Proc. Int. Colloq. Invert. Path., Kingston, Canada: pp. 263-267.

1976-13 Poinar, G. 0. Jr. and Hess, R. (1976). A virus infection of a nematode. IRCS Med. Sci., 5: 31.

1976-14 Ross, J. R. and Smith, S. M. (1976). Review of mermithid parasites (Nematoda: Mermithidae) described from North American mosquitos (Diptera: Culicidae) with description. Can. J. Zool., 54: 1084-1102.

1977

1977-1 Brown, B. J. and Platzer, E. G. (1977). The effects of temperature on the infectivity of Romanomermis culicivorax •. J. Nematol., 9: 166-172.

1977-2 Brown, B. J., Platzer, E. G. and Hughes, D. S. (1977). Field trials with the mermithid nematode Romanomermis culicivorax in California. Mosq. News, 12= 603-608.

1977-3 Finney, J. R. (1977). The development of Romanomermis culicivorax in in vitro culture. Nematologica, 23: 479-480.

1977-4 Finney, J. R., Gordon, R., Condon, W. J. and Rusted, T. N. (1977). Laboratory studies on the feasibility of integrated mosquito control using an insect growth regulator and a mermithid nematode. Mosq. News, 12= 6-11.

1977-5 Galloway, T. D. and Brust, R. A. (1977). Effects of temperature and photoperiod on the infection of two mosquito species by the mermithid Romanomermis culicivorax. J. Nematol., ~: 218-221.

1977-6 Hughes, D. S. and Platzer, E. G. (1977). Temperature effects on the parasitic phase of Romanomermis culicivorax in Culex pipiens. J. Nematol., ~: 173.

1977-7 Ittycheriah, P. I., Gordon, R. and Condon, W. J. (1977). Storage material of the nematode Romanomermis culicivorax, a mermithid parasite of larval mosquitos. Nematologica, 23: 165-171.

i I, p

WHO/VBC/80.766 VBC/BCDS/80.09 page 19

1977-8 Levy, R.,Cornell, J. A. and Miller, T. W. Jr. (1977). Application of a mermithid parasite of mosquitos with an aerial spray system. Mosq. News, lZ: 512-516.

1977-9 Levy, R. and Miller, T. W. Jr. (1977). Thermal tqlerance of Romanomermis culicivorax, a nematode parasite of mosquitos. J. Nematol., ~: 259-260.

1977-10 Levy, R. and Miller, T. W., Jr. (1977). Susceptibility of the mosquito nematode Romanomermis culicivorax (Mermithidae) to pesticides and growth regulators. Environ. Entomol.,~: 447-448.

1977-11 Levy, R. and Miller, T. W. Jr. (1977). Experimental release of a mermithid nematode to control mosquitos breeding in sewage settling tanks. Mosq. News, 12: 410-414.

1977-12 Levy, R. and Miller, T. W. Jr. (1977). Experimental release of Romanomermis culicivorax (Mermithidae: Nematoda) to control mosquitos breeding in southwest Florida. Mosq. News, 37: 483-486.

1977-13 Malloy, D. and Jamnback, H. (1977). A larval blackfly control field trial using mermithid parasites and its cost implications. Mosq. News, 12: 104-108.

1977-14 Platzer, E. G. (1977). Biological control of mosquitoes with the mermithid nematode Romanomermis culicivorax. Univ. Calif. Mosq. Cont. Res., Annual Report 1977: pp. 33-36.

1977-15 Poinar, G. 0. Jr. and Hess, R. (1977). Romanomermis culicivorax: morphological evidence of transcuticular uptake. Exp. Parasitol , 42: 27-33.

1977-16 Poinar, G. 0. Jr. and Hess, R. (1977). Virus-like particles in the nematode Romanomermis culicivorax (Mermithidae). Nature, 266: 256-257.

1978

1978-1 Brown, B. J. and Platzer, E. G. (1978). Salts and the infectivity of Romanomermis culicivorax. J. Nematol., 10: 53-61.

1978-2 Brown, B. J. and Platzer, E. G. (1978). Oxygen and the infectivity of Romanomermis culicivorax. J. Nematol., 10: 110-113.

1978-3 Dhillon, M. S. and Platzer, E. G. (1978). Laboratory evaluation of fungicides for the control of Catenaria anguillulae, a fungus parasite of Romanomermis culicivorax (Nematode: Mermithidae). Proc. Annual Conf. Calif. Mosq. Vector Cont. Assoc., 46: 92-93.

1978-4 Medical Research Council of New Zealand (1978). Biological control of filariasis transmitting mosquitoes. Research review, 1978: 182.

1978-5 Mondet, B., Berl, D. and Prud'hom, J. M. (1978). Infestation de larves de Simulium damnosum s.l. (Simuliidae) par Romanomermis culicivorax (Mermithidae) parasite de moustique. Mission entomologique de l'ORST~M aupres de l'OCCGE, Institut de recherches sur l'onchocercose. Mimeographed document No. 16/0NCHO/RAP.78.

1978-6 Petersen, J. J. (1978). Development of resistance by southern house mosquito to the parasitic nematode Romanomermis culicivorax. Environ. Entomol., L: 518-520.

1978-7 Petersen, J. J. (1978). Observations on mass-production of Romanomermis culicivorax, a nematode parasite of mosquitos. Mosq. News, 38: 83-86.

1978-8 Petersen, J. J., Willis, 0. R., and Chapman, H. C. (1978). Release of Romanomermis I culicivorax for control of Anopheles albimanus in El Salvador.!. Mass production of nematode. Amer. J. Trop. Med. Hyg., 27: 1265-1267. L l ' WHO/VBC/80.766 VBC/BCDS/80.09 page 20

1978-9 Petersen, J. J., Willis, 0. R., Chapman, H. C. and Fukuda, T. (1978). Release of Romanomermis culicivorax for control of Anopheles albimanus in El Salvador.2. Application of nematode. Amer. J. Trop. Med. Hyg., ~: 1268-1273.

1978-10 Platzer, E. G. (1978). Biological control of mosquitoes with the mermithid nematode Romanomermis culicivorax. Univ. Calif. Mosq. Control Res. Annual Report 1978: pp. 47-51.

1978-11 Platzer, E. G. and McKenzie-Graham, L. L. (1978). Predators of Romanomermis culicivorax. Proc. Annual Conf. Calif. Mosq. Vector Cont. Assoc., 46: 93 (abstract).

1978-12 Platzer~ E. G. and Stirling, A. M. (1978). Improved rearing procedures for Romanomermis culicivorax. Proc. Annual Conf. Calif. Mosq. Vector Cont. Assoc., 46: 87-88.

1978-13 Schmidt, S. P. and Platzer, E. G. (1978). Hemolymph compos1t1on of mosquito larvae infected with a mermithid nematode. J. Nematol., 10: 299.

1978-14 Stirling, A. M. and Platzer, E. G. (1978). Catenaria anguillulae in the mermithid nematode Romanomermis culicivorax. J. Invertebr. Pathol., 32: 348-354.

1978-15 Winner, R. A., Schillin, P. E. and Steelman, C. D. (1978). Effects of selected insecticides on Romanomermis culicivorax, a mermithid nematode parasite of mosquito larvae. Mosq. News, 38: 546-553.

1979

1979-1 Curran, J. and Hominick, W. M. (1979). The effects of different heat-killing procedures on the morphology and morphometries of Gastromermis sp. and Romanomermis culicivorax (Nematoda: Mermithidae). Parasitology,~: 29-30.

1979-2 Kurihara, T. (1979). Numerical relationships between a nematode parasite Romanomermis culicivorax, and its host population Culex pipiens molestus larvae. Jap. J. Parasit., 28: 99-105.

1979-3 Kurihara, T. and Hata, K. (1979). Two factors affecting parasitism by Romanomermis culicivorax in mosquito larvae. Jap. J. Sanit. Zool., 30: 200-202.

1979-4 Levy, R., Hertlein, B. C., Petersen, J. J., Doggett, D. W. and Miller, T. W. Jr. (1979). Aerial application of Romanomermis culicivorax (Mermithidae: Nematoda) to control Anopheles and Culex mosquitoes in southwest Florida. Mosq. News, ~: 20-25.

1979-5 Levy, R., Van Rinsvelt, H. A. and Miller, T. W. Jr. (1979). Major and trace-elements analyses of a mermithid parasite and its mosquito host by proton-induced X-ray emission. J. Nematol. 11: 100-103.

1979-6 Nickle, W. R. (1979). Probable establishment and overwintering of a mermithid nematode parasite of mosquitos in Maryland. Proc. Helminthol. Soc. Wash., 46: 21-27.

1979-7 Petersen, J. J. (1979). pH as a factor in parasitism of mosquito larvae by the mermithid Romanomermis culicivorax. J. Nematol. 11: 105-106.

1979-8 Petersen, J. J. (1979). Romanomermis culicivorax as a potential biological control agent of Simulium damnosum. Report to WHO, 8 pages.

Petersen, J. J. and Chapman, H. C. (1979). Checklist of mosquito species tested against the nematode parasite Romanomermis culicivora~ ~· Med. Entomol., 15: 468-471.

1979-10 Platzer, E. G. (1979). Biological control of mosquitos with a mermithid nematode. Mosquito Research Highlights. University of California Cooperative Extension. October, 1979; 7 pages. ....

WHO/VBC/80. 766 VBC/BCDS/80.09 page 21

1979-11 Poinar, G. 0. Jr. (1979). "Nematodes for Biological Control of Insects". CRC Press, Boca Raton, Florida, USA. 277 pages.

1979-12 Poinar, G. 0. Jr., Hess, R., Hansen, E. and Hansen, J. W. (1979). Laboratory infection of blackflies (Simuliidae) and midges (Chironomidae) by the mosquito mermithid, Romanomermis culicivorax. J. Parasitol., 65: 613-615.

1979-13 Poinar, G. 0., Jr.; Hess, R. T. and Petersen, J. J. (1979). Immune responses of mosquitos against Romanomermis culicivorax (Mermithidae, Nematoda). J. Nematol., 11: 110.

1979-14 Prasertphon, S. (1979). A brief summary of biological control activities at WHO/ VBCRU I, Kaduna, Nigeria. Report to WHO, 8 pages.

1979-15 Westerdahl, B. B., Washino, R. K. and Platzer, E. G. (1979). Early season application of Romanomermis culicivorax provides continuous partial control of rice field mosquitoes. Proc. Annual Conf. Calif. Mosq. Vector Cont. Assoc., 46: 55.

1980

1980-1 Willis, 0. R., Chapman, H. C. and Petersen, J. J. (1980). Additional field testing of the mermithid parasite Romanomermis culicivorax against Anopheles albimanus in El Salvador. Mosq. News, 40: 71-73.