Biochemical effects of Steinernema feltiae, Steinernema riobrave and Heterorhabditis bacteriophora on Spodoptera littoralis larvae

the Egyptian Society for Biological Sciences ,Department of Entomology ,Faculty of Sciences Ain Shams University . logy & molecular biology journal is one of the series issued twice by the Egyptian Physio Academic Journal of Biological Sciences, and is devoted to publication of original papers that iological elucidate important biological, chemical, or physical mechanisms of broad phys significance.


INTRODUCTION
The Egyptian cotton leaf worm, S. littoralis (Boisd) is one of the most destructive phytophagous insect pests in Egypt, not only to cotton, but also to other field crops and vegetables (Kandil et al., 2003).These caterpillars are very polyphagous, causing important economic losses in both greenhouses and open field on a broad range of ornamental, industrial and vegetable crops.
Besides many populations have acquired resistance towards most insecticide groups (Alford, 2000).Therefore, there is always need for finding out new material shaving specific modes of actions to replace the conventional insecticides.
Among the most suitable biological control agents for controlling the cotton leafworm are the entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae, which are considered as good biocontrol agents because they cause rapid death of the insect host without side effects on mammals or plants (Poinar, 1986).Infective third-stage juveniles of these nematodes, which are capable of long-term survival without feeding, carry symbiotic bacteria, Xenorhabdus sp.In their intestine to be released into the host`s haemocoel leading to septicemia followed by death of the host insect species, the nematodes, then reproduce within the cadaver (Molyneux et al., 1983).
The present study aimed to evaluate the pathogenic action of the three nematode species, S. feltiae, S. riobrave and H. bacteriophora on S. littoralis larvae and to study the physiological and biochemical activities of some enzymes in fourth larval instars in laboratory.

MATERIALS AND METHODS Insect rearing technique:
The stock colony of S. littoralis was maintained in the laboratory at 25±2ºC and 65±5% RH.Adults were fed on 20% sucrose solution, while larvae were fed on castor oil leaves, Ricinus communis.

Pathogenicity of the nematodes to S. littoralis larvae Bioassay procedure:
The 4 th larval instar of S. littoralis was used for this purpose.The inoculum of IJs from S. riobrave, S. feltiae and H. bacteriophora was carried out by placing 4 th larval instar in 1.5 ml Eppendorf tube lined with filter paper.The latter was contaminated with 5, 10, 20 and 40 IJs.Each concentration level was replicated five times, ten larvae per each replicate.Control experiment of non-infected larvae was also carried out.Mortality records were made after 48 hr, and were corrected against natural mortality that was obtained from control using Abbott's formula (Abbott, 1925).The data were statistically analyzed according to Finney (1971) to obtain estimate of LC 50 value.The cadavers were dissected for nematode development and progeny production.

Biochemical Studies:
The biochemical studies of 4th larval instars were measured after 48 hours of treatment.Total protein, lipid and carbohydrate and protein contents were measured according to the methods described by Singh and Sinh (1977) and Bradford (1976), respectively.The total lipids were determined by the phosphovanilin method of Barnons and Blackstock (1973).
Determination of amylase, invertase and trehalase enzymes according to the method described by Ishaaya and Swiriski (1970).Acid and alkaline phosphtase activities were determined by the method described by Laufer and Schin (1971).

RESULTS AND DISUSSION Susceptibility of S. littoralis larvae to S. feltiae, S. riobrave and H. bacteriophora nematodes:
The Pathogenicity of S. feltiae, S. riobrave and H. bacteriophora nematodes against the fourth larval instar of S. littoralis.H.
bacteriophora.seemed to be comparatively more pathogenic than S. feltiae and S. riobrave to the tested instar larvae (Table 1).Estimated of LC 50 values were 5.01, 8.57 and 16 IJs/larva for H. bacteriophora, S. riobrave,and S.feltiae respectively.Thus, H. bacteriophora was about 3 times as pathogenic as S. feltiae to S. littoralis larvae at the LC 50 level.

Infective juvenile production
As shown in Fig.
(1), the total number of juveniles produced /a single S. littoralis larvae varied between the nematode species.
The highest progeny was produced from larvae infected by H. bacteriophora (at conc. 40 IJs/larva) which gave 7000 IJs/larva).In the present investigation, the mortality percentage increased with the increase of the parasite density.This is in accordance with the findings of Sikora et al. (1979) who stated that most developmental stages of S. littoralis were highly susceptible to N. carpocapsae infection, and the mortality was positively correlated with the parasite density.Similar findings were also reported by several authors (Ahmed, 1982;Abdel-Kaway, 1985;Kondo and Ishibashi, 1987;Choo et al., 1988 andGhally et al., 1988).Ghally et al. (1991 ) found that the rate of development of S. feltiae was faster and the rate of reproduction was higher in S. littoralis than in Musca domestica.Also, Hatsukade and Grey (1996) showed a higher infectivity of S. carpocapsae to larvae of S. littoralis.Khlibsuwan (1996) obtained a relationship between S. carpocapsae concentration and the number of nematodes invading S. litura larvae, percentage invasion increased with the exposure time.Likewise, Mogahed (1996) showed that the efficacy of H. heliothidis and H. bacteriophora increased with the increase of concentration and period after treatment of different stages of S. littoralis.
The efficacies of H. bacteriophora (HP88), H. bacteriophora (EASD98), S. riobrave and H. indicus (EAS59) against S. littoralis were tested by Shamseldean et al. (1996).All the tested nematodes attained almost 100% mortality at 4, 10 and 25ºC, but at 35ºC H. bacteriophora (HP88) achieved the least mortality (64%).Also, Reyad (2001) showed that the tested ionculum levels of S. carpocapsae and H. bacteriophora were effective against the larval instars of S. littoralis, and the level 40 infective juveniles/ml distilled water caused 100% mortality of the host.Elawad et al. (1997) isolated S. abbasi from soil in alfalfa fields and showed that this nematode species could be used as a biological control agent in high temperature against S. littoralis, with LD 50 value of 60.3 IJs/larvae.Also, Abbas and Saleh (1988) studied the efficiency of S. riobrave against 4 th instar larvae of the same insect species, with LD 50 value of 49.6 IJs/larva.The highest mortality (91.7%) was obtained in the 3 rd day post-treatment.
S. littoralis larvae infected by H. bacteriophora produced infective juvenile more higher than these infected by S. feltiae and S. riobrave.These may be due to that the nutrional requirements of H. bacteriophora nematodes were more higher than those of S .feltiaeand S. riobrave as evidenced in the present study.Obtained results agree with the work of Selvan et al. (1993) who reported that percentage of penetration of S. carpocapsae and H. bacteriophora to G. mellonella larvae declined in spite of an increase in the number of invading nematodes with the increase of the dose.Infective juvenile production was reduced at densities above and below100 nematodes/host.They thought that the effects of increased density of nematodes resulted from competition for limited nutrients within the host.Shannag et al. (1994) found that larval mortality and penetration of infective juveniles of S. carpocapsae, S. feltiae and H. bacteriophora into pickle worm Diaphania nitidalis were positively correlated to host exposure time.Smaller nematodes were more infective and induced mortality more quickly.Further, Shannag and Capinera (1995) determined that S. carpocapsae was the most pathogenic nematode species to the same insect species, followed by H. bacteriophora, S. felitae, S. anomaly and S. glaseri.

Biochemical influences of S. feltiae , S. riobrave and H. bacteriophora. nematodes on S. littoralis larvae: Total protein, lipid and carbohydrate contents:
The data obtained (Table 2) show that 48 hr post-infection of 4 th instar larvae of S. littoralis by the three nematode species significantly decreased the total content of protein, lipid and carbohydrate of larvae, as compared to control.The highest decrease was recorded in case of infection by S. riobrave and H. bacteriophora.Nematodes.

Carbohydrases activity (amylase, invertase and trehalase):
The effects of LC 50 of S. feltiae, S .riobraveand H. bacteriophora on the activity of the carbohydrate digestive enzymes, 48 hr post-infection of 4 th instars larvae of S. littoralis were shown in Table (3).The results revealed that amylase activity was significantly increased, as compared to control, due to infection of 4 th instar larvae of S. littoralis by S. feltiae, S. riobrave.Whereas, the activity of this enzyme was decreased insignificantly in case of infection by H. bacteriophora.
Infection by the two nematode species, S. riobrave and H. bacteriophora significantly increase the activity of invertase of S. littoralis larvae, as compared to control.
The highest increase was recorded in case of infection by H. bacteriophora.Infection by the three nematode species increased significantly trehalase activity as compared to control.It is well known that the pathology of the entomopathogenic nematodes beings immediately after reaching the insect's haemocoel.The symbiotic bacteria when released into the haemocoel, rapidly multiply causing a lethal septicemias to the insect host (Dutley, 1959;Nickle and Welch, 1984).So, biochemical changes in the haemolymph composition are expected, since the haemolymph is the main site of action.In the present study, the total protein content decreased due to parasitism of two nematodes to the fourth instar larvae of S. littoralis.This result agrees with that obtained by El-Bishry et al. (1997) who found that 30 hr post-infection of 6 th instar larvae of A. ipsion markedly produced the haemolymph protein in case of the three tested isolates; HP88 strain, Also isolates (H.bacteriophora) and AS2 isolate CH.Indicus).Also , Thong and Webster (1975) found that the total protein level decreased with the parasitism of the sphaerulariid nematode Contortylenchus reverses during the maturation of female scolytid beetle Dendroctonus pseudotsugae.Also, they found that the parasitism by this nematode did not affect haemolymph trehalose.Level.This finding is in contrast to somewhat with the results obtained in the present study where the nematode species tested decreased the total carbohydrate in S. littoralis larvae.Sahota (1970) obtained depletion of protein in the haemolymph of mature, but not in callow adult females of D. pseudotsugae, following infection by the nematode C. reverses.This suggests that during the maturation of the latter, the normal active incorporation of haemolymph protein into ovarian protein occurs concurrently with nematode withdrawal of host haemolymph protein, thus upsetting the haemostatic mechanism for the maintenance of the haemolymph protein levels.Nematode utilization of host protein probably also occurs in the callow female, but the homeostatic, control of haemolymph protein levels is able to cope with the rate of depletion, without the developing eggs drawing from the available protein.Alternatively, during the beetle diapause, the nematode may enter a state of inactivity and decrease its rate of metabolism and, hence, of protein utilization.Such effects have been observed in the nematode Heterotylenchus autumnalis parasitizing the face fly, Musca autmnalis.
It is also possible that the protein depletion measured may be the indirect consequence of changes in the insect fat body caused by the nematode Mermis nigrescens, for example, done not affect the taotal haemolymph protein level in the desert locust, Schistocerca gregaria, but depletes both fat body protein and amino acids (Gordon and Webster, 1971).Gordon et al. (1978) showed that the mermithid nematode, Neomesomermis flumenalis reduced the level of most amino compounds and depleted most protein fractions in haemolymph of both larval blackeflies, Prosimulium mixtum/fuscum and Simulium venustum, together with a significant decrease of haemolymph glucose levels.
However, blood trehalose concentration was not affected.This effect contrast with M. nigrescens which caused an overall reduction of blood carbohydrates in S. gregaria (Gordon and Webster, 1971), and which may be attributed to lowered trehalose levels .
Glucose, but not trehalose, is assimilated from the host's haemolymph in a transcuticutar manner by M. nigrescens (Rutherford and Webster, 1974;Rutherford, Webster and Barlow, 1977).Thus, depletion of blood glucose and exhaustion of fat body glucogen (Candon and Gordon, 1977) in mernithid-parasitized simuliids results from the nematode's nutritional demands for glucose and are symptomatic of accelerated glucogenolysis and/or impairedglycogensis by the host fat body.They added also that the utilization of haemolymph glucose by mermithid parasites could favor the production of more glucose via increased trehalose activity of the host.This activity, in turn, could increase fat body glycogenolysis and/or lower glycogensis to maintain adequate concentrations of trehalose in the haemolymph.Dahlman and Greene, (1981), Thompson (1982 a & b), Kawai et al. (1983), Cook et al. (1984), andKarnavar (1984) stated that haemolymph proteins and lipids exhibited quantitative variations by endoparasitism.Milstead (1979) while studying the path physiological influences of the nematode, Heterorhabditis bacteriophora complex on the seventh instar larvae of Galleria mellonella, reported that shortly after the nematode penetration into haemocoel of larvae began feeding upon the fat body.Thompson and Barlow (1983) reported that an extreme depression of de novo glyceride synthesis would allow the parasite to use host's fat after partial digestive hydrolysis and its own fatty acids for rapid triglyceride synthesis, thereby minimizing the energy cost of fat synthesis.Schmidt and Platzer (1979) found that the concentrations of total carbohydrates, protein, glucose and trehalose in the haemolymph of 4 th instar of Culex pipiens infected by the nermithid nematode Romanomermis culicivorax were reduced.These results agrees with those obtained in the present study regarding total carbohydrate and protein in nematodeparasitized S. littoralis larvae.
The flight ability of Locusta nigratoria was reduced by infection with M. nigrescens .Concomitantly, haemolymph level of carbohydrates was elevated and protein concentration was lowered during parasitism.Fat body carbohydrate, protein, and lipid were also reduced as was the amount of fat body tissue in L. nigratoria.
Based on the forgoing findings, it can be concluded that the interaction the nematodes tested in this study with S. littoralis larvae appears to be primarily nutritional.The parasite absorbs small molecular weight components from the host depriving the larvae of nutrients necessary for development.Growth of the nematode proceeds while the nutritional status of the host larvae deteriorates, i.e., the host become in a state of physiological starvation.
Many nematodes secrete chemicals that facilitate penetration and migration through host tissues, feeding, and avoidance of host immune responses.These chemicals include digestive enzymes and toxins (Lee and Atkinson, 1976).Proteases are digestive enzymes that catalyze the cleavage of peptide bonds in proteins.Some animal parasitic nematodes secrete proteases to assist in skin and tissue penetration (Von Brand, 1973).It has been proposed that these proteases are essential for the Pathogenicity of Steinernema kraussei.An inhibitor present in the haemolymph of Galleria mellonella inhibits both S. kraussei and its symbiotic bacteria proteases unevenly.The inhibitor is produced during the second period of infection when the larval defense system has already been overcome and infection is established (Kucera and Mracek, 1989).Morover, Abu Hatab et al. (1995) found that when the nematodes S. glaseri were treated with protease inhibitors and injected into G. mellonella gut, the percentage mortality of G. mellonella was reduced as compared to control, and nematode penetration of G. mellonella gut was reduced.

Phosphatases activity (acid and alkaline).
The activity 48 hr post-infection of 4 th instar larvae of S. littoralis by S. feltiae, S.
riobrave and H. bacteriophora.was significantly increased as compared to control (Table 4).The highest increase was recorded in case of infection by S. riobrave The same pattern was also obtained for the activity of alkaline phosphatase in the larvae infected by S. riobrave and H bacteriophora.
Whereas, alkaline phosphatase activity in the larvae infected by S. feltiae was insignificantly decreased.: Not significant ** : Highly significant at P< 0.01 ≠ IU: International unit (the amount of enzyme which under defined assay conditions will catalyze the conversion of one micromole of substrate per minute).Xia et al. (2000) suggested that acid phosphates, as a lysosomal enzyme, may have a role in autophagy and cell turn over as well as defense.Therefore, it appears that the enhancement of acid phosphates activity in S. littoralis larvae infected with S. riobrave, Hetrorhabditis sp. and B. bassiana is an attempt by the insects to deferred or them self was against the invasion of the three pathogens.These authors also added that phogocyteosis is known to stimulate the production of lysosomal enzymes of which acid phosphates is a key component.Acid phosphate had been found in insect haemocytes and shown to be released into the plasma (Lai-Fook, 1973;Rowley and Ratclifte, 1979).Cheng (1983) reported hyper synthesis of acid phosphates by haemocytes of the mollusk, Biomphalaria globrata during phagocytoses.The enzyme was subsequently released into the plasma where its role is unknown although alteration of surface procedures of foreign particles recognition although a direct role of acid phosphates in cell killing can not be ruled out.
On the other hand, alkaline phosphates of secreting products across cell boundaries.
In the present study, acid phosphates activity was higher than Alkaline phosphates activity in non-infected nematode larvae.The predominance of acid phosphates activity could be correlated to an active range (Pant and Lacy, 1969).
In agreement with our results, Soliman (2002) found that acid and alkaline phosphates activity in last instar Ceratitis capitata infected with S. riobrave and Heterorhabditis bacteriophora.

Transaminases activity (GOT & GPT):
Data in table (5) show the effect of the nematode species, S. feltiae, S. riobrave, H.bacteriophora on the activity of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), 48hr post-infection of 4 th instar larvae of S. littoralis.The results indicated that infection by the nematode species S. riobrave, H.bacteriophora ( decreased significantly the activity of GOT and GPT as compared to control.Whereas, insignificantly decreased.in the larvae infected by S. feltiae.(Chen, 1966;Gilbert, 1967;Plant and Morris, 1972) and with protein catabolism in certain others (Asmore et al., 1964;Wergedal et al., 1964;Knox and Greengard, 1965).In the present work, the significant decline of GOT in S. littoralis larvae after 48 hr post-infection by H.bacteriophora and S. riobrave, as compared to control treatment, may be attributed to the significant decline in free amino acids content, as has been pointed out by Kaur et al. (1985).They added that the quantum of free amino acids directly influenced the activity of transaminase at the time of protein synthesis.Thus, both GOT and GPT may play a direct role in protein synthesis of S. littoralis larvae and may explain the coincidence in the activity of this transaminase with the total protein content in non-infected S. littoralis larvae in the present study.The increased ratio in GPT: GOT in S. littoralis larvae showed that GPT was comparatively more active than GOT in noninfected by nematode infected larvae reflecting that there was a better rate of interplay between alanine and glutamate, as has been suggested by Kaur et al. (1985).They added that the fact that higher activities of GOT and GPT were simultaneous to the increased deposition of glycogen content is suggestive of the possible role of these two enzymes in incorporating free amino acids into carbohydrates via transamination reactions to bring about the metabolism of waste nitrogen products and in gluconeogensis.Soliman (2002)

Table 1 :
Pathogenicity of S .feltiae, S. riobrave and H. bacteriophora against 4 th larval instar of S. littoralis

Table 2 :
Effect of LC 50 of S. feltiae, S. riobrave and H. bacteriophora on the total content of protein, lipid and carbohydrate 48h post-infection of 4 th instar larvae of S. littoralis.

Table 3 :
Effect of LC 50 of S. feltiae, S. riobrave and H. bacteriophora on the carbohydrate digestive enzymes 48h post-infection of 4 th instar larvae of S. littoralis.: Significant at P< 0.05 ≠ IU: International unit (the amount of enzyme which under defined assay conditions will catalyze the conversion of micromole of substrate per minute). *

Table 4 :
Effect of LC 50 of S. feltiae, S. riobrave and H. bacteriophora on phosphatases activity, 48h postinfection of 4 th instar larvae of S. littoralis.

Table 5 :
Effect of LC 50 of S. feltiae, S. riobrave and H. bacteriophora on transaminases activity, 48h postinfection of 4 th instar larvae of S. littoralis.