110 Journal of Nematology, Volume 11, No. 1, January 1979

discovery necessitates a modification o[ our key to Leptonchus species, which is given below.

37 #m long .......................... g r a n u l o s u s Cobb, 1920 Prerectum 449--450 #m long; cuticle not loose; spicules 28 ,am long . . . . . b a c c a t u s Siddiqi, 1970

Key to Species of Leptonchus

1. Spear robust, lumen wide ...................................... _. p a t u l i h a s t u s Goseco, Ferris and Ferris, 1974

Spear slender, lumen narrow .................................... 2 2. Tail longer (c = 38.6-62.5); ventromedian sup-

plements closely spaced ........................................... ......................................... t r a n s v a a l e n s i s Heyns, 1963 Tail shorter (c = 72-106); ventromedian sup- plements widely spaced ....................................... 3

3. Prerectum 562-775 #m; cuticle loose; spicules

L I T E R A T U R E CITED

1. GOSECO, C. G., V. R. FERRIS, and J. M. FERRIS. 1974. Revisions in Leptonchoidea (Nematoda: Dorylaimida). Leptonchus, Pro- leptonchus, Funaria, and Meylis n. gen. in Leptonchidae, Leptonchinae. Purdue Univ. Research Bull. 911 : 1-32.

2. HEYNS, J. 1963. Five new species of Lepton- chidae (Nemata: Dorylaimoidea) from South Africa. Proc. Helminth . Soc. Wash. 30:7-15.

Immune Responses of Mosquitoes against Romanomermis culicivorax (Mermithida: Nematoda)

GEORGE O. POINAR, JR., 1 ROBERTA

T h e mosquito-parasitic nematode Ro- manomermis culicivorax has been used successfully in the field against a variety of mosquitoes and can develop in at least 50 spec]es of culicines (Poinar, 1979). However, there are a few mosquitoes which are able to partially or completely block normal de- velopment of this parasite. Kerdipibule et al. (1) failed to infect first-stage Mansonia uniformis with this nematode. In some cases, a dark pigment, presumably melanin, is deposited on the developing parasite, halting further development. Mitchell et al. (2) noted that R. culicivorax was melanized and killed soon after entering larvae of Anopheles sinensis in Taiwan. Similar re- actions were observed by Petersen et al. (4) and Petersen (3) against ,4edes triseriatus and CuIex territans larvae. T h e present study examines the host reactions elicited by R. culicivorax in the latter two mosquito species and describes, for the first time, a case of cellular encapsulation in a mosquito host.

First-stage and early second-stage larvae o[ C. territans and .4. triseriatus were placed in water with sand containing the mature eggs of R. culicivorax. After 5 hours, the

Received for publication 26 July 1978. a Division of Entomology and Parasitology, University of California, Berkeley, California 94720.

2Gulf Coast Mosquito Laboratory, U.S. Department of Agri- culture, Avenue J, Cbennault, Lake Charles, Louisiana 70601.

T. HESS) and JAMES J. PETERSEN"

mosquito larvae were removed and main- tained in nematode-fi'ee containers. Every 12 hours, one infected larva of each species was removed and dissected, and the devel- oping nematodes were examined by light microscope. In addition, for electron mi- croscopy, three to five infected larvae of C. territans were fixed in 4% glutaraldehyde in Millonig's phosphate buffer for 1 h and then transferred to a 1% buffered solution of osmium tetroxide for 1 h at 4 C. Follow- ing fixation, the capsules were rinsed in buffer, dehydrated in an alcohol series, and embedded in Araldite 6005. Sections made with glass knives mounted in a Porter-Blum MT-2 microtome were stained with satu- rated aqueous uranyl acetate followed by lead citrate and examined with an RCA-3F and a Philips EM-300 electron microscope.

T h e types of immune responses dis- played in the two species of mosquito larvae were different. With .4. triseriatus, nematode penetrat ion and development proceeded as normal until the fourth day, when the parasite usually exhibits a sudden increase in growth. Then , patches of melanin appeared on the developing nema- tode, and, by the sixth day, the parasites in the thoraxes and abdomens of the mosquito larvae were completely covered by a pig- mented layer (presumably melanin) (Fig. 1). T h e reaction killed the developing nematodes, and the mosquitoes continued

Mosquitoes Immune to Romanomermis: Poinar et al. 111

!

FIG. 1. Melanized parasitic juvenile of R. culicivorax (arrow) in the hemocoel of A. triseriatus (insert shows detail of nematode tail).

FIG. 2. Normal parasitic juvenile of R. culicivorax (arrow) inside the head capsule of A. triseriatus.

their growth and pupated. When the nema- todes initiated development in the mosquito head capsule, there was usually no host response (Fig. 2). However, sttch nematodes never completed their development, appar- ently for lack oI space rather than from any

host response, since they appeared normal in all other respects.

Ti le response to R. culicivorax by larvae of C. territans differed from the above and represented a more unusual reaction. In this host, nematode development seemed to

112 Journal o[ N e m a t o l o g y , V o l u m e 11, No. 1, January 1979

FIG. 3. Developing parasitic juvenile of R. culicivorax 2 (lays after entry into a C. territa~ts larva. Note atlached host blood cells.

lqG. ,t. Dead parasitic juvenile of R. culicivorax 3 days after entry into a C. territans larva [note wrinkled appearance of nematode head (arrow)].

FIG. 5. Parasitic juvenile of R. culicivorax nearly disintegrated by the host reaction in a larva of C. territaTts (note absence of host capsule),

Mosquitoes Immune to Romanomermis: Poinar et at. 118

FIG. 6. Parasitic juvenile of R. cl~licivorax within 12 h of entry into a C. te~ritans larva. Note absence of homogeneous deposit on nematode and entry hole (H) in host body wall.

FIG. 7. Parasitic juvenile of R. eulicivorax 30 h after entry into a C. te~'ritans larva. Note homogeneous deposit surrounding nematode (arrow) arid the similarity of this deposit to those surrounding the host tissues.

114 J o u r n a l o I N e m a t o l o g y , V o l u m e 1I , N o . I , ,]anuar~ 1979

FIG. I~. Cellular debris and dead blood cells adhering to the homogeneous deposit on the cuticle of an 14. culicivorax juvenile 48 h after entering a C. territans larva.

FIG. 9. Capsular wall surrounding R. culicivorax 5 days after entry into a C. territans larva, showing areas of pigment formation in the cellular debris (arrows).

Mosquitoes Immune to Romanomermis : Poinar et al. 115

proceed normally for 1-2 days after entry into the host. After about 60 h, however, host blood ceils accumulated on the surface of the nematode, which otherwise appeared normal (Fig. 3). During the following 24 h, more blood cells became attached to the initial layer, and the nematode became en- closed in a thick clear capsule (Fig. 4). At this stage, the nematodes became moribund and a series of folds or crinkles appeared in their cuticles. All the nematodes were dead and enclosed in large capsules on the fourth day. The parasites began to disinte- grate within their capsules on the fourth and fifth days, and it appeared that they were being "digested" by the surrounding cells. Eventually, the nematodes became shriveled and lacked internal definition, becoming difficult to locate inside the living host. At this time, the capsule seemed to diminish in thickness, with the disappear- ance of surrounding blood cells (Fig. 5). Further details in capsule formation were seen in the electron micrographs. When the preparasitic juveniles of R. culicivorax first entered C. territans larvae, they were free of any host coating (Fig. 6). Between 12 and 24 h after entry, a thin homogeneous deposit surrounded the cuticle of the para- site (Fig. 7). Shortly after this, host plasmatocytes became numerous and ad- hered to the homogeneous deposit on the cuticle of the juvenile (Fig. 8). After 4 days, most of the surrounding blood cells were necrotic and showed signs of losing their integrity. After 5 days, when the parasites had begun to disintegrate, the capsular wall consisted mainly of cellular debris, with small areas of pigment formation (presum- ably melanin) (Fig. 9). The capsules never became completely pigmented as did those formed around the nematodes in A. triseriatus.

The results demonstrate the presence of two categories of insect immunity against the juveniles of R. culicivorax. One of these is humoral melanization, as exhibited by larvae of A. triseriatus. Such a reaction in- volves the formation of melanin around the parasite by components in the noncellular portion of the blood. This system has been shown to operate in both mosquito and midge hosts (5). In the present situation, the reaction is delayed, and it is curious that the melanin does not form on the para-

sites immediately after entry, since this type of reaction is typically very rapid. The second type of immune response that was exhibited by C. territans is simple encap- sulation, accompanied by some pigment (probably melanin) formation. The amount of pigment produced, however, is not suf- ficient to be considered a case of melanotic encapsulation (5). This type of response has never been observed in mosquitoes previ- ously but may be common in C. territans because of its ability to produce large num- bers of plasmatocytes, again a rather unusual characteristic for mosquito larvae. Another unusual feature is the disintegra- tion and almost complete disappearance of tile parasite after it had been surrounded and killed by the host blood cells. The host cells appeared to digest the nematode. Since the final stage in the reaction was the partially disintegrated parasite without a surrounding cellular capsule, the capsular cells were themselves either removed or "digested" by host enzymes. In both hosts, the response was lethal for the nematodes. In A. triseriatus, the response occurred against older parasites in the body cavity and only rarely against nematodes that entered the head capsule. With C. territans, the response was noted throughout the mos- quito host and occurred 2-3 days after entry. From the practical standpoint, the present study shows that before R. culici- vorax is applied in nature, laboratory infections against the target insect should be conducted to determine whether host immunity is present.

LITERATURE CITED

1. KERDIPIBULE, V., T. DEES1N, S. SUCHARIT, and C. HARINASUTA. 1974. A preliminary study on the control of Mansonia uniformis by nematode parasitism (Reesimermis nielseni). Southeast Asian J. Trop. Med. Pub. Hlth. 5:150-151.

2. MITCHELL, C. J., P. S. CHEN, and H. C. CHAPMAN. 1974. Exploratory trials utilizing a mermithid nematode as a control agent for Culex mosquitoes in Taiwan. J. Formosan Med. Assoc. 73:241-254.

3. PETERSEN, J. J. 1975. Penetration and development of the mermithid nematode Reesimermis nielseni in eighteen species of mosquitoes. J. Nematol. 7:207-210.

4. PETERSEN, J. J., H. C. CHAPMAN, and O. R. WILLIS. 1969. Fifteen species of mosquitoes as potential hosts of a mermithid nematode

116 Journal of Nematology, Volume 11, No. 1, January 1979

Romanomermis sp. Mosquito News 29:198- in Immunol)iolt)gy. Vol. 4. E. L. Cooper, ed. 201. l'lenunl Press, New York. 167-178.

5. I'OINAR. G. O., JR. 1976. Insect immunity to 6. POINAR, (;. O., JR. 1979. Entomogeneous parasitic nematodes. In: Contemporary Topics nematodes for biological control. CR.C Press.