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Parasitism of the mosquito Culex pipiens by the nematode Heterorhabditis bacteriophora
JOURNAL
OF INVERTEBRATE
Parasitism
PATHOLOGY
39, 382-387 (1982)
of the Mosquito Culex pipiens by the Nematode Heterorhabditis bacteriophora GEORGE0. POINAR, JR., AND H. N. KAUL’
Division
of Entomology
and Parasitology,
University
of California,
Berkeley,
California
94720
ReceivedFebruary23, 1981;acceptedDecember 21, 1981 Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culexpipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts’ defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed. KEY WORDS: Heterorhahditis bacteriophoru; Culexpipiens; insect, melanization in; nematodes; Xenorhabdus
luminescens:
Pseudomonas
aeruginosu.
INTRODUCTION
The insect parasitic nematode Heterorhabditis bacteriophora was first described from a pupa of the noctuid moth Heliothis punctigera collected at Brecon in South Australia (Poinar, 1976). It was subsequently shown to be capable of infecting insects from 13 families, representing four major orders, including Diptera (Poinar, 1979). The infective third-stage juveniles enter through the natural openings of the host, then penetrate into the body cavity where they initiate development. Each infective stage, characteristically, develops into a hermaphroditic female which deposits a number of juveniles inside the host. These juveniles develop into males and females which mate. Their progeny become infective stages which leave the host and enter the environment. The infective stages of H. bacteriophora carry cells of the luminescent, symbiotic bacterium Xenorhabdus luminescens in their alimentary tract (Poinar et al., 1977; ’ Visiting WHO Fellow from the National Institute of Virology, Pune, India. 382 0022-201 l/82/030382-06$01.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
Thomas and Poinar, 1979). These cells are released soon after the nematode initiates development in the host. The bacteria multiply in the hemocoel, kill the insect within 48 hr, and provide conditions for nematode multiplication. The present paper describes the effect of these nematodes on larvae of the mosquito Culex pipiens. MATERIALS
AND METHODS
The nematodes were cultivated in wax moth larvae, Galleria mellonella, and the infective stages were stored at 20°C in a 0.25% saline solution. Larvae of Culex pipiens were reared on a mixture of yeast powder and dog biscuits in the insectary at the Entomology Department in Berkeley, California. Infection experiments involving second-, third-, and fourth-instar larvae were conducted at 25 -+ 3°C in 6-cm-diameter Petri dishes containing 20 ml of distilled water. The concentrations of infective nematodes used in these experiments ranged from 100 to 6000 per dish. At each nematode concentration, three replicates, com-
Heterorhahditis
prising 30 individuals of second, third, and fourth instars of C. pipiens larvae, respectively, were tested. A small amount of lactalbumin hydrolysate powder was added to each container as a feeding stimulant and observations were made 22-23 days following introduction of the nematodes. The LCSO and LCS9 values were obtained from the results of larval mortality at different concentrations. Controls contained mosquito larvae and feeding stimulant without nematodes.
IN
Culrx
383
Because of their smaller oral aperture, second-instar larvae rarely ingested whole nematodes, more often crushing them with their mandibular teeth. Once the nematode cuticle was broken, the parasite perished. (3) Host reaction. C. pipiens larvae are able to melanize nematodes that have entered their hemocoels (Figs. 2-4). The melanization process is much more rapid and strong in third- and fourth-instar larvae than in second. In the latter, a newly entered nematode is often able to initiate developRESULTS ment and liberate the bacterium beThe infective stages of H. bacteriophora fore being melanized (Fig. 4). In late are heavier than water and slowly sink to third- and fourth-instar C. pipiens, the bottom of the container. However, the the first four to seven nematodes are mouth brushes of the mosquito larvae more often melanized so quickly that they or less keep the nematodes in suspension as have no time to liberate the bacterium. they are drawn forth to the oral cavity and Only after the maximum melanization number is reached can additional then either swallowed or rejected. For innematodes develop freely in the hefection to occur, the nematodes had to be uninjured after ingestion, at which time mocoel and release their bacteria. they could attempt to penetrate into the (4) Entrance offoreign bacteria. In situbody cavity. If the nematode was deposited ations where one or more nematodes in the midgut, its chances of penetration entered the host but were rapidly were very slight since there was no evimelanized and killed before releasing dence they could penetrate the peritrophic their symbiotic bacterium (Xenomembrane. Penetration through the alirhabdus), the host frequently develmentary tract was observed in the memoped septicemia and died. In the great branous neck region just behind the head. majority of cases, this condition was Penetration usually occurred after the due to the bacterium Pseudomonas nematode exsheathed in the lumen of the aeruginosa, which was present in the alimentary tract (Figs. 1, 2), although a few alimentary tract of the mosquito larnematodes exsheathed after entering the vae. Bacterial cells entered through body cavity. the entrance wound made by the Several factors governed the degree of nematode and multiplied in the hemoinfection obtained in these experiments. lymph, resulting in a lethal septicemia. (1) Nematode concentration. When the The results of the mortality experiments nematode concentration increases, a are shown in Figure 5. Mortality in the higher number are ingested and more controls was always below 5%. Host death reach the body cavity of the host could arise directly or indirectly from the larvae. nematode. Initial nematodes that entered (2) Size (stage) of host. Parasitism in fourth-instar C. pipiens larvae were rapidly general was highest in fourth-instar melanized and killed without having the larvae. This was because the larger opportunity of releasing their bacterium. hosts could more readily ingest Usually, the host would then continue to nematodes without damaging them. develop, carrying the melanized nematode
384
POINAR AND KAUL
bacteriophora in the anterior portion of the alimentary FIG. 1. An infective stage of Heterorhabditis tract of a Culex pipiens larva. Note exsheathed cuticle (C). x 161. FIG. 2. Infective stage oflfeterorhabditis bacteriophora penetrating through the anterior portion of the alimentary tract of a C&x pipiens larva. Note deposit of melanin on the head of the nematode. P, Penetration hole. x328.
through .the pupal and into the adult stage. However, about 30% of host larvae died from P. aevuginosu brought into the host through the actions of the penetrating
nematode. killed as nematode pattern of
Thus, the mosquito larvae were an indirect consequence of infection. The normal direct Heterorhabditis infection also
Heterorhahditis
IN Culex
385
FIG. 3. A completely melanized infective stage of Heterorbabditis bacteriophoru in the head capsule of a Culex pipiens larva. X75. FIG. 4. Cells of Xenorhabdus luminescens multiplying in the hemocoel of an infected Culex pipiens larva. Dark object represents a melanized Heterorhabditis bacteriophora juvenile. X 1200.
occurred when a nematode entered a mosquito that had already melanized four to seven previous parasites. The nematodes could initiate development and liberate cells of X. fuminescens into the hemolymph (Fig. 4). These cells could then cause a lethal septicemia. However, conditions were
never suitable for nematode development since contaminant bacteria (Pseudomonas) soon entered the cadavers, eventually destroying the nematodes. The LC,, for third- and fourth-instar C. &ens larvae was approximately 80 and 63 nematodes per host, respectively, while the
386
POINAR AND KAUL
1-1 mj B
2nd 3rd 4th
instar instar instar
NEMATODE CONCENTRATION FIG. 5. Percentage mortality of second-, third-, and fourth-&tar C&x pipierzs larvae at different bacteriophora. Mortality was measured concentrations (nematodes per container) of Heterorhabditis at 48 hr after infection. The controls for all groups were identically treated (no nematodes).
LC,, was between 170 and 220 nematodes per host for both instars. DISCUSSION
The present investigation showed that the infective stages of H. bacteriophora are able to enter the body cavity of larvae of C. pipiens. Mortality of mosquito larvae may occur from the direct action of the nematode through liberation of its associated bacterium, Xenorhabdus, or it may occur from a septicemia caused by bacteria, Pseudomonas, in the immediate environment that entered the hemocoel through the wound made by the penetrating nematode. It has been demonstrated that foreign bacteria can adhere to the cuticle of insectpenetrating nematodes (Poinar, 1979). The nematodes initiated development in the initial stages of Xenorhabdus infections, but foreign bacteria (mostly Pseudomonas sp.) soon entered and made conditions uninhabitable. X. luminescens produces defective phage particles that have been shown to be ex-
tremely
effective
in destroying
species of (Poinar et al., 1980). However, these particles are not as effective against Pseudomonas spp. Due to the contaminating bacteria and the rapid disintegration of the host tissue in water, the nematodes were not able to complete their development. This may be mostly due to the high incidence of foreign bacteria in the gut of the mosquito since Welch and Bronskill (1962) found that the related Neoaplectana carpocapsae, nematode, could complete its cycle in larvae of Aedes Bacillus in the same environment
aegypti.
The melanization process in the present investigation appears to be similar to that produced in mosquito hosts against infective stage juveniles of Neoapfectana cur1971); pocapsae (Poinar and Leutenegger, Welch and Bronskill, 1962). It is a humoral reaction involving the formation of pigment granules around the nematode. These results suggest that this means of humoral defense may be exhausted by extensive nematode parasitization. Fourth-instar lar-
Heterorhabditis
vae of C. pipiens are able to quickly melanize the first four to seven nematodes, but then the host reaction rapidly diminishes and additional nematodes are either slowly or only partially melanized. Therefore, in order to kill the mosquito larvae, this initial melanization reaction should be considered when determining the concentration of nematodes to be used. One advantage that H. bacteriophora has over the mosquito mermithid nematode, Romanomermis culicivorax, is its ability to be grown in alternate hosts (wax moth larvae) or on artificial media such as dog food agar (Poinar, 1979). A second advantage is the longevity of the inactive stages (between 3 and 6 months in water at room temperature). Thus, H. bacteriophora is a potential biological control agent against bottom feeding culicines in specialized habitats. ACKNOWLEDGMENTS The authors would like to thank R. Dadd and J. Kleinjan for supplying the mosquito larvae used in this study and G. Thomas for identification of the associated bacteria. The junior author gratefully acknowledges the World Health Organization for pro-
IN
387
Cu1e.x
viding him with a fellowship made this project possible.
training
award which
REFERENCES G. O., JR. 1976. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora ngen., n.sp. (Rhabditida: Heterorhabditae n. fam.). Nemarologica, 21, 463-468. POINAR, G. O., JR. 1979. “Nematodes for Biological Control of Insects.” CRC Press, Fla. POINAR, G. O., JR., AND LEUTENEGGER, R. 1971. Ultrastructural investigation of the melanization process in C&x pipiens (Culicidae) in response to a nematode. J. Ultrastruct. Res., 30, 149-158. POINAR, G. O., JR., HESS, R., AND THOMAS, G. 1980. Isolation of defective bacteriophages from Xenorhabdus spp. (Enterobacteriaceae). IRCS Med. Sci., 8, 141.
POINAR,
POINAR,
G. O., JR., G. M.,
THOMAS,
AND
HESS, R.
1977. Characteristics of the specific bacterium associated with Heterorhabditis bacteriophora (Heterorhabditae: Rhabditida) Nematologica, 23, 97- 102. THOMAS, G. M., AND POINAR G. O., JR. 1979. Xenorhabdus gen. nov., a genus of Entomopathogenie, nematophilic bacteria of the family Enterobacteriaceae. Int. J. Septemat. Bacferiol. 29, 352-360. WELCH, H. E., AND BRONSKILL, J. F. 1962.
Parasitism of mosquito larvae by the nematode, DD-136 (Nematodea: Neoaplectanidae). Canad. J. Zoo/.,
40, 1263-1268.
OF INVERTEBRATE
Parasitism
PATHOLOGY
39, 382-387 (1982)
of the Mosquito Culex pipiens by the Nematode Heterorhabditis bacteriophora GEORGE0. POINAR, JR., AND H. N. KAUL’
Division
of Entomology
and Parasitology,
University
of California,
Berkeley,
California
94720
ReceivedFebruary23, 1981;acceptedDecember 21, 1981 Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culexpipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts’ defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed. KEY WORDS: Heterorhahditis bacteriophoru; Culexpipiens; insect, melanization in; nematodes; Xenorhabdus
luminescens:
Pseudomonas
aeruginosu.
INTRODUCTION
The insect parasitic nematode Heterorhabditis bacteriophora was first described from a pupa of the noctuid moth Heliothis punctigera collected at Brecon in South Australia (Poinar, 1976). It was subsequently shown to be capable of infecting insects from 13 families, representing four major orders, including Diptera (Poinar, 1979). The infective third-stage juveniles enter through the natural openings of the host, then penetrate into the body cavity where they initiate development. Each infective stage, characteristically, develops into a hermaphroditic female which deposits a number of juveniles inside the host. These juveniles develop into males and females which mate. Their progeny become infective stages which leave the host and enter the environment. The infective stages of H. bacteriophora carry cells of the luminescent, symbiotic bacterium Xenorhabdus luminescens in their alimentary tract (Poinar et al., 1977; ’ Visiting WHO Fellow from the National Institute of Virology, Pune, India. 382 0022-201 l/82/030382-06$01.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
Thomas and Poinar, 1979). These cells are released soon after the nematode initiates development in the host. The bacteria multiply in the hemocoel, kill the insect within 48 hr, and provide conditions for nematode multiplication. The present paper describes the effect of these nematodes on larvae of the mosquito Culex pipiens. MATERIALS
AND METHODS
The nematodes were cultivated in wax moth larvae, Galleria mellonella, and the infective stages were stored at 20°C in a 0.25% saline solution. Larvae of Culex pipiens were reared on a mixture of yeast powder and dog biscuits in the insectary at the Entomology Department in Berkeley, California. Infection experiments involving second-, third-, and fourth-instar larvae were conducted at 25 -+ 3°C in 6-cm-diameter Petri dishes containing 20 ml of distilled water. The concentrations of infective nematodes used in these experiments ranged from 100 to 6000 per dish. At each nematode concentration, three replicates, com-
Heterorhahditis
prising 30 individuals of second, third, and fourth instars of C. pipiens larvae, respectively, were tested. A small amount of lactalbumin hydrolysate powder was added to each container as a feeding stimulant and observations were made 22-23 days following introduction of the nematodes. The LCSO and LCS9 values were obtained from the results of larval mortality at different concentrations. Controls contained mosquito larvae and feeding stimulant without nematodes.
IN
Culrx
383
Because of their smaller oral aperture, second-instar larvae rarely ingested whole nematodes, more often crushing them with their mandibular teeth. Once the nematode cuticle was broken, the parasite perished. (3) Host reaction. C. pipiens larvae are able to melanize nematodes that have entered their hemocoels (Figs. 2-4). The melanization process is much more rapid and strong in third- and fourth-instar larvae than in second. In the latter, a newly entered nematode is often able to initiate developRESULTS ment and liberate the bacterium beThe infective stages of H. bacteriophora fore being melanized (Fig. 4). In late are heavier than water and slowly sink to third- and fourth-instar C. pipiens, the bottom of the container. However, the the first four to seven nematodes are mouth brushes of the mosquito larvae more often melanized so quickly that they or less keep the nematodes in suspension as have no time to liberate the bacterium. they are drawn forth to the oral cavity and Only after the maximum melanization number is reached can additional then either swallowed or rejected. For innematodes develop freely in the hefection to occur, the nematodes had to be uninjured after ingestion, at which time mocoel and release their bacteria. they could attempt to penetrate into the (4) Entrance offoreign bacteria. In situbody cavity. If the nematode was deposited ations where one or more nematodes in the midgut, its chances of penetration entered the host but were rapidly were very slight since there was no evimelanized and killed before releasing dence they could penetrate the peritrophic their symbiotic bacterium (Xenomembrane. Penetration through the alirhabdus), the host frequently develmentary tract was observed in the memoped septicemia and died. In the great branous neck region just behind the head. majority of cases, this condition was Penetration usually occurred after the due to the bacterium Pseudomonas nematode exsheathed in the lumen of the aeruginosa, which was present in the alimentary tract (Figs. 1, 2), although a few alimentary tract of the mosquito larnematodes exsheathed after entering the vae. Bacterial cells entered through body cavity. the entrance wound made by the Several factors governed the degree of nematode and multiplied in the hemoinfection obtained in these experiments. lymph, resulting in a lethal septicemia. (1) Nematode concentration. When the The results of the mortality experiments nematode concentration increases, a are shown in Figure 5. Mortality in the higher number are ingested and more controls was always below 5%. Host death reach the body cavity of the host could arise directly or indirectly from the larvae. nematode. Initial nematodes that entered (2) Size (stage) of host. Parasitism in fourth-instar C. pipiens larvae were rapidly general was highest in fourth-instar melanized and killed without having the larvae. This was because the larger opportunity of releasing their bacterium. hosts could more readily ingest Usually, the host would then continue to nematodes without damaging them. develop, carrying the melanized nematode
384
POINAR AND KAUL
bacteriophora in the anterior portion of the alimentary FIG. 1. An infective stage of Heterorhabditis tract of a Culex pipiens larva. Note exsheathed cuticle (C). x 161. FIG. 2. Infective stage oflfeterorhabditis bacteriophora penetrating through the anterior portion of the alimentary tract of a C&x pipiens larva. Note deposit of melanin on the head of the nematode. P, Penetration hole. x328.
through .the pupal and into the adult stage. However, about 30% of host larvae died from P. aevuginosu brought into the host through the actions of the penetrating
nematode. killed as nematode pattern of
Thus, the mosquito larvae were an indirect consequence of infection. The normal direct Heterorhabditis infection also
Heterorhahditis
IN Culex
385
FIG. 3. A completely melanized infective stage of Heterorbabditis bacteriophoru in the head capsule of a Culex pipiens larva. X75. FIG. 4. Cells of Xenorhabdus luminescens multiplying in the hemocoel of an infected Culex pipiens larva. Dark object represents a melanized Heterorhabditis bacteriophora juvenile. X 1200.
occurred when a nematode entered a mosquito that had already melanized four to seven previous parasites. The nematodes could initiate development and liberate cells of X. fuminescens into the hemolymph (Fig. 4). These cells could then cause a lethal septicemia. However, conditions were
never suitable for nematode development since contaminant bacteria (Pseudomonas) soon entered the cadavers, eventually destroying the nematodes. The LC,, for third- and fourth-instar C. &ens larvae was approximately 80 and 63 nematodes per host, respectively, while the
386
POINAR AND KAUL
1-1 mj B
2nd 3rd 4th
instar instar instar
NEMATODE CONCENTRATION FIG. 5. Percentage mortality of second-, third-, and fourth-&tar C&x pipierzs larvae at different bacteriophora. Mortality was measured concentrations (nematodes per container) of Heterorhabditis at 48 hr after infection. The controls for all groups were identically treated (no nematodes).
LC,, was between 170 and 220 nematodes per host for both instars. DISCUSSION
The present investigation showed that the infective stages of H. bacteriophora are able to enter the body cavity of larvae of C. pipiens. Mortality of mosquito larvae may occur from the direct action of the nematode through liberation of its associated bacterium, Xenorhabdus, or it may occur from a septicemia caused by bacteria, Pseudomonas, in the immediate environment that entered the hemocoel through the wound made by the penetrating nematode. It has been demonstrated that foreign bacteria can adhere to the cuticle of insectpenetrating nematodes (Poinar, 1979). The nematodes initiated development in the initial stages of Xenorhabdus infections, but foreign bacteria (mostly Pseudomonas sp.) soon entered and made conditions uninhabitable. X. luminescens produces defective phage particles that have been shown to be ex-
tremely
effective
in destroying
species of (Poinar et al., 1980). However, these particles are not as effective against Pseudomonas spp. Due to the contaminating bacteria and the rapid disintegration of the host tissue in water, the nematodes were not able to complete their development. This may be mostly due to the high incidence of foreign bacteria in the gut of the mosquito since Welch and Bronskill (1962) found that the related Neoaplectana carpocapsae, nematode, could complete its cycle in larvae of Aedes Bacillus in the same environment
aegypti.
The melanization process in the present investigation appears to be similar to that produced in mosquito hosts against infective stage juveniles of Neoapfectana cur1971); pocapsae (Poinar and Leutenegger, Welch and Bronskill, 1962). It is a humoral reaction involving the formation of pigment granules around the nematode. These results suggest that this means of humoral defense may be exhausted by extensive nematode parasitization. Fourth-instar lar-
Heterorhabditis
vae of C. pipiens are able to quickly melanize the first four to seven nematodes, but then the host reaction rapidly diminishes and additional nematodes are either slowly or only partially melanized. Therefore, in order to kill the mosquito larvae, this initial melanization reaction should be considered when determining the concentration of nematodes to be used. One advantage that H. bacteriophora has over the mosquito mermithid nematode, Romanomermis culicivorax, is its ability to be grown in alternate hosts (wax moth larvae) or on artificial media such as dog food agar (Poinar, 1979). A second advantage is the longevity of the inactive stages (between 3 and 6 months in water at room temperature). Thus, H. bacteriophora is a potential biological control agent against bottom feeding culicines in specialized habitats. ACKNOWLEDGMENTS The authors would like to thank R. Dadd and J. Kleinjan for supplying the mosquito larvae used in this study and G. Thomas for identification of the associated bacteria. The junior author gratefully acknowledges the World Health Organization for pro-
IN
387
Cu1e.x
viding him with a fellowship made this project possible.
training
award which
REFERENCES G. O., JR. 1976. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora ngen., n.sp. (Rhabditida: Heterorhabditae n. fam.). Nemarologica, 21, 463-468. POINAR, G. O., JR. 1979. “Nematodes for Biological Control of Insects.” CRC Press, Fla. POINAR, G. O., JR., AND LEUTENEGGER, R. 1971. Ultrastructural investigation of the melanization process in C&x pipiens (Culicidae) in response to a nematode. J. Ultrastruct. Res., 30, 149-158. POINAR, G. O., JR., HESS, R., AND THOMAS, G. 1980. Isolation of defective bacteriophages from Xenorhabdus spp. (Enterobacteriaceae). IRCS Med. Sci., 8, 141.
POINAR,
POINAR,
G. O., JR., G. M.,
THOMAS,
AND
HESS, R.
1977. Characteristics of the specific bacterium associated with Heterorhabditis bacteriophora (Heterorhabditae: Rhabditida) Nematologica, 23, 97- 102. THOMAS, G. M., AND POINAR G. O., JR. 1979. Xenorhabdus gen. nov., a genus of Entomopathogenie, nematophilic bacteria of the family Enterobacteriaceae. Int. J. Septemat. Bacferiol. 29, 352-360. WELCH, H. E., AND BRONSKILL, J. F. 1962.
Parasitism of mosquito larvae by the nematode, DD-136 (Nematodea: Neoaplectanidae). Canad. J. Zoo/.,
40, 1263-1268.