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Effects of Contortylenchus reversus (Nematoda: Sphaerulariidae) on hemolymph composition and oocyte development in the beetle Dendroctonus pseudotsugae (Coleoptera: Scolytidae)
JOURNAL OF INVERTEBRATE PATHOLOGY
26, 91-98 (1975)
Effects of Contortylenchus reversus (Nematoda: Sphaerulariidae) on Hemolymph Composition and Oocyte Development in the Beetle Dendroctonus pseudotsugae (Coleoptera: Scolytidae) CYRIL H. S. THONG AND JOHN M. WEBSTER
Pestology Centre . Department of Biological Scien ces, Simon Fraser University, Burnaby, Vancouver , B.C. V5A lS6, Canada Received November 6, 1974 Hemolymph of health y and Contortylenchus reversus infected Douglas fir beetles, Dendroctonus pseudotsugae, was analyzed to determine carbohydr ate. protein, and amino acid content and the effects of parasitism upon these entities was studied. The normal levels of these components in healthy beetle s are discussed in connection with those found in related insects. Parasitism does not affect hemolymph trehalose levels in D. pseudotsugae. Total protein levels decrease with parasitism during the maturation of female beetles from the callow adult state. This decrease in protein levels is proportionate for all protein fractions as shown by acrylamide gel disc electrophore sis. A consequence of protein depleti on is a 20% reduction in the size of the oocytes in infected female beetles . In male beetles, tot al hemolymph protein levels do not change during par asitism but in the callow males, two protein fractions increase significantly in percentage composition over the controls. Thi s difference is not detected in mature male beetles . No significant differences could be dete cted in tot al amin o acid levels between control and infected groups of all four host categories.
INTRODUCTION The bark beetle Dendroctonus pseudotsugae is a serious pest of the Douglas fir tree, Pseudotsuga menziesii, in the Pacific northwest. A number of nematode species are associated with this beetle externally and in the gut. The main nematode parasite in the hemocoel is Contortylenchu s reversus. Gravid females, eggs, and four larval stages of this nematode may be found simultaneously in the hemocoel of larvae, pupae, and adult beetles. The fourth stage nematode larvae leave the beetle with the feces and develop quickly into adult nematodes in the gallery frass (Thong and Webster, 1973). After mating, the males die but the females infect larvae of the next generation of host. Within the host hemocoel the female nematodes increase fourfold in size and gonad maturation and egg production commences. Over a thousand nematode eggs and larvae may be found in a single infected beetle at anyone time (Thong , 1973).
Very little is known of the effects of bark beetle nematodes on their host physiology. Riihm (1956) stated that larval and adult hemocoel parasites obtain nutrients from the fat body of bark beetles. It has also been reported that Contortylenchus elongatus reduces the number of fat cells in Ips paraconfusus (Nickle, 1963), and that the growth and function of organs in D. pseudotsugae are affected by Sphaerulariopsis dendroctoni (Khan, 1957). Nematodes are also known to reduce bark beetle fecundity (Massey, 1956, 1962; Ashraf and Berryman, 1970). The present study was undertaken to elucidate some of the effects the nematode C. reversus has on its host D. pseudotsugae with respect to host hemolymph composition and oocyte development. MATERIALS AND METHODS Naturally infected Douglas fir beetles were collected from the area around Williams Lake , British Columbia, Canada. 91
Cop yright © 1975 by Academi c Pre ss, Inc. All rights of reproduction in any for m reser ved.
92
EFFECTS OF NEMATODA ON BARK
Infected beetles containing at least 50 nematodes/insect in the hemocoel were used in the experiments. Hemolymph analysis was performed on the hemolymph of both sexes of callow unemerged and mature adult beetles prior to the first oviposition. Hemolymph was drawn from beetles through a wound made by the removal of a metathoracic leg. The body of the beetle was squeezed gently with a pair of forceps to eject hemolymph through the wound. Withdrawal of hemolymph and the measurement of blood volume was done using I ILl disposable micropipettes (Drummond "Microcaps"). Hemolymph not used immediately was stored in vials at - 30· C with a crystal of phenylthiourea. Hemolymph trehalose assay was performed by thin-layer chromatography using an adsorbent mixture of 24 g MN Kieselguhr and 6 g of Celite analytical filter aid (Johns-Manville, USA). This was slurried with 80 ml of 0.02 M sodium acetate solution and layered to a thickness of 250 ILm on 20 x 20 em glass plates. Hemolymph from individual beetles and two serial dilutions of a trehalose standard solution were spotted on each tlc plate 2 cm apart. The plates were developed to a distance of 15 em using a mixture of 60 ml ethyl acetate and 40 ml isopropanol/water (2:1) as solvent. The compounds separated out were detected using a spray reagent consisting of 50 ml glacial acetic acid, 0.5 ml anisaldehyde, and 1 ml concentrated H 2S04 made up fresh before application. The plates were then heated at 11O·C in an oven till the spots attained maximum intensity (ca. 20 min). The plates were then cooled for 30 min at room temperature. Quantification was done using a Zeiss chromoscan spectrophotometer for tic using a wavelength of 370 nm. Readings were calibrated against a trehalose standard curve. Total hemolymph protein was analysed using the method of Bramhall et al. (1969) with xylene brilliant cyanin G (Michrome 1224) (Esbe Laboratory Supplies, Toronto, Canada) as a stain. Samples were read in a spectrophotometer at 620 nm wavelength
and calibrated against a bovine serum albumin standard curve. Individual insects were used in this assay. Hemolymph proteins were separated out by polyacrylamide disc electrophoresis in a standard model Polyanalyst (Buchler Instruments Division, Nuclear Chicago Corporation, USA) apparatus but using longer gel tubes (ca. 12 em) to increase resolution of the protein bands. The technique employed was essentially similar to that of Davis (1964) using a 7.0% running gel in conjunction with 2.5% spacer and sample gels. Hemolymph from eight insects was pooled and 5 ILl used for each sample run. A pH 8.9 Tris-HCl buffer was used and bromophenol blue was used as a tracking dye. A current of 5 rnA/tube was applied. Gels were subsequently fixed in 10% trichloroacetic acid (TCA) at 37"C for 30 min and stained for 1 hr at the latter temperature with a fixative/ stain solution consisting of 19 parts 10% TCA and 1 part 1% Coomassie blue RL (Michrome 100). Destaining was carried out nonelectrophoretically with several changes of 7% acetic acid (5 days). Quantification was carried out with a Joyce Loebl Chromoscan and overlapping electropherogram peaks were resolved with a Dupont 310 Curve Resolver. Quantification in arbitrary units was achieved by measuring the areas below the respective peaks with a planimeter. Total hemolymph amino acid assay was done on individual insects by the method of Rosen (1957) but the volumes of reagents used were decreased proportionally to compensate for the small volume of hemolymph obtainable from each insect. Samples were quantified in a spectrophotometer at a wavelength of 570 nm and standardized against a glycine standard curve. The rate of development of oocytes was estimated by measuring the length of the largest terminal oocyte of 13 uninfected and 13 infected mature female adult beetles from the same colony prior to the first oviposition (Thomsen, 1948; Thomsen and Moller, 1959a,b, 1963). All data were analyzed using Student's
93
THONG AND WEBSTER
t test. Significance was set at the 95% confidence limit for all data analysed.
RESULTS
TABLE 2 Total Hemolymph Protein Levels in Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae (Mean ± 1 SE)
Carbohydrates A preliminary experiment showed the level of glucose in the hemolymph of D pseudotsugae to be extremely low and often undetectable by tlc, Trehalose appeared to be the main blood sugar making up more than 90% of the total hemolymph carbohydrates. The normal physiological level of hemolymph trehalose in D. pseudotsugae did not change significantly in the maturation of the callow female adults, but mature male adults had 25% more trehalose in the hemolymph than did callow male adults (P < 0.02) (Table I). Mature male adults also had significantly higher hemolymph trehalose levels than did mature female adults (P < 0.02). No significant differences, however, were detected in the level of hemolymph trehalose between control and infected groups in each adult category. Total Hemolymph Protein The total hemolymph protein level in normal D. pseudotsugae decreases significantly during the maturation of both sexes of callow adults (Table 2). There was no significant difference, however, between total hemolymph protein levels in male and female beetles. No significant difference in total hemolymph protein was detected between control and infected callow females, but infected mature females had 20% less hemolymph protein (P < 0.05) than control TABLE 1 Hemolymph Trehalose Levels in Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae (Mean ± 1 SE) Trehalose concentration"
Host stage and sex
n
Callow female Mature female Callow male Mature male
6 8 6 8
O..g
Control 10.7 10.6 11.0 14.9
trehalose/..l hemolymph.
± 0.9 ± 1.0 ± 0.7 ± 1.2
Infected 10.1 ± 0.9 11.3±I.4 9.7 ± 1.2 14.5 ± 1.0
Total protein concentration''
Host stage and sex
n
Callow female Mature female Callow male Mature male
6 8 6 6
Control 19.8± 1.2 14.3 ± 1.2* 21.2 ± 2.1 14.4 ± 1.6
Infected 19.2± 1.6 11.0 ± 0.7* 17.6±1.3 16.1 ± 2.3
°lLg Bovine Serum· Albumin equivalents/ILl hemolymph. *Significantly different from each other atP < 0.05.
insects of the same group. No significant differences between total protein levels of control and infected callow and control and infected mature males were detected. Individual Proteins Nine protein fractions were separated from the hemolymph of callow female beetles, with fractions I and VIII being the major fractions (Table 3; Fig. 1). No significant differences between control and infected beetles were observed with respect to the percentage of these nine fractions in the blood. Fraction IV, which was always present in the hemolymph of mature females was undetectable in callow females, and fractions VIIIB and IX that were sometimes present in mature females were totally absent in all the callow females. In mature females, a total of 12 fractions were detected in the hemolymph but fractions VIIIB, IX, and X were absent from some of the samples analysed (Table 3). There was a lower concentration of fractions V and VI and a higher concentration of fraction III in mature as compared to callow females. Despite there being no significant differences in percentage composition of protein fractions recorded between control and infected groups of mature females, the total level of protein (according to the intensity of the stain retained by the respective gels) was higher in control groups than in infected groups (Fig. 2), which supports the results obtained in the total protein analysis. Parasitism, therefore, caused a proportional
\0 ~
TABLE 3 Percentage Concentration of Protein Fractions in the Hemolymph of Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae. Proteins Separated by Acrylamide Gel Disc Electrophoresis. Concentrations Presented as Mean ± 1 SE
Fraction no. I
II III IV V VI VII VIII VIIIB IX X XI
Callow female (n = 5)
Mature female (n = 5)
R/
Control
Infected
Control
Infected
0.05-0.10 0.09-0.17 0.14-0.21 0.20-0.24 0.24-0.30 0.30-0.38 0.36-0.47 0.49-0.57 0.64-0.70 0.78-0.86 0.90-0.95 1.0
27.7 ± 1.4 3.3 ± 0.5 2.9 ± 1.1
23.7 ± 1.9 3.9 ± 0.8 4.0 ± 0.8
-
-
27.5 ± 1.0 1.3 ± 0.2 8.8 ± 2.2 3.3 ± 1.0 6.2:!; 2.1 6.4 ± 0.5 11.0 ± 1.3 28.3 ± 2.4 2.3 ± 0.1 0 0.8 ± O.~ 2.2 ± 0.30 3.7 ± 0.5
28.5 ± 1.6 2.5 ± 1.2 7.4 ± 1.0 3.8 ± 1.3 5.3 ± 1.3 6.5 ± 0.8 l1.0:!; 1.5 27.1 ± 2.5 1.9 ± 0.2 0 1.8±0.7° 1.9 ± 0.20 4.2 ± 0.4
Callow male (n = 6) Control
Mature male (n = 5)
Infected
Control
Infected
26.3± 1.9 1.3 ± 0.5 6.8 ± 1.6 5.7 ± 0.5 8.8 ± 1.0 6.9 ± 0.7 10.6 ± 1.3 24.0 ± 1.6 2.1 ± 0.3 1.50 3.5 ± 0.5 0 4.2 ± 1.2
23.2 ± 2.3 1.3 ± 0.4 7.9 ± 2.1 6.5 ± 1.2 10.6 ± 1.3 7.0 ± 0.6 11.7 ± 1.3 25.1 ± 1.5 1.5 ± 0.7 0 0.60 2.3 ± o.s« 3.8 ± 1.2
tn
'" o'" tn
-l tI>
11.7±2.4 10.9 ± 0.7 13.2 ± 2.3 23.7 ± 0.8 -
2.0 ± 0.8 4.9 ± 0.8
14.7 10.5 9.1 25.8
± 2.4 ± 1.8 ± 1.8 ± 2.0 -
1.6 ± 0.2 6.1 ± 0.3
aFraction not present in all samples. *Significantly different from each other at P < 0.05. **Significantly different from each other atP < 0.01.
22.4 2.7 8.0 14.2 4.8 14.0 9.4 22.9
± 2.1 ± 0.9 ± 1.7
± 1.9 ± 1.2* ± 1.8 ± 0.9** ± 2.3
0.6 ± 0.3 0 2.4 ± 0.8 3.6 ± 1.2
20.2 2.0 5.9 8.0 8.9 9.6 15.9 24.4
± 1.9 ± 0.4 ± 2.0 ± 3.8 ± 0.7* ± 1.5 ± 1.2** ± 1.3
1.2 ± 0.30 3.4 ± 0.7 3.6 ± 0.7
0
'"Z tn
a:::
> -l 0 0
> 0
Z
= > :Ill
i":
95
THONG AND WEBSTER
XI
m CO NTROL
x
XI ;l INFECTED
i\ f ...........-
. o
0 .5
I. D
XI
m~ 1
INFECTED
jj
11 . .. I. " 1lIl " i···. ......../ .\ "~,,,," ••...•/ ..~ !
"
.._
11 m ! ' .1\,llJ...... .. ./" .-F/>! All! !
~.'"~
l~
"'-' '...~•..,:
.
......-----.--- - ...,...._. ..
--- ,....- - - ~ . -.----
~5
RElATIVE MOBilIT Y CRn
0
RELA TIVE MOBILITY IRf! FIG. 2
FIG. 1
FIGS . I AND 2. Representative chromo scan tracings of hemolymph proteins in control and Contortylenchus
reversus infected callow female (I) and mature female (2) adult Dendroctonus pseu dotsugae.
decrease in all protein fractions in mature female beetles. In the callow males, 11 fractions were separated. Fraction VIIIB was absent from all samples and fraction IX was detectable in only some samples. Fraction IV, which was not found in the hemolymph of callow females, accounted for about 10% of the total protein in callow males. The two fractions V and VII increased significantly in infected callow males (Table 3; Fig. 3) by approximately 4 and 6%, respectively, in percentage of total protein, which was a 3040 % increase over the controls. Fractions IV and VI decreased slightly in percentage composition but these changes were not
significant. In the mature male beetles, 12 fractions were recognized (Fig. 4), but, as in the mature females, fractions VIIIB, IX, and X were absent from some samples. No significant differences were detected in the level of the protein fractions between control and infected mature male beetles (Table 3; Fig. 4). Notably, the higher level of fractions V and VII that occurred in infected than control callow males does not occur in the mature males. Fractions IV and VI were significantly lower in control groups of mature males than in control groups of callow males. Fraction VIIIB, which was detected in some samples of hemolymph from mature males, was not detected in callow
m
~
I\ x
.J\ /\.~. l. 0
INFECTED /
.
! .n, H
\)!\._~ }JJ\ 1 vn::
0 .5 RE LATIVE MOBILITY IRI I FIG. 3
.•
o
l.0
.
0.5
a
RELATIVE MOB ILITY (Rl/ FIG. 4
FIGS . 3 AND 4. Representative chr om oscan tr acings of hemol ymph protein s in cont rol and Cont ort ylen chus reversus infected ca llow male (3) and mature male (4) adult Dendroct onu s pseudotsugae.
96
EFFECTS OF NEMATODA ON BARK
males. As in all other groups analysed, fractions I and VIII were the two major fractions in the hemolymph.
Total Hemolymph Amino Acids The level of amino acids in the hemolymph was approximately the same in callow and mature female beetles, and in callow and mature male beetles (Table 4) and no significant differences in amino acid levels were detected between control and infected members of each of the four categories studied. Oocyte Development Infected females of D. pseudotsugae had significantly smaller, i.e., 20% smaller, oocytes than did uninfected ones (Table 5). DISCUSSION The level of trehalose in D. pseudotsugae (10.6 Ilg/ III in mature females and 14.9 ug] III in mature males) is high when compared to that of other Coleoptera previously studied, e.g ., 5-7 ug] III in adult Dytiscus marginalis and 3-5 Ilg/ III in adult Hydrophilus piceus (Duchateau and Florkin, 1959). It is comparable, however, with the levels found in the hemolymph of more active fliers, e.g., 6-12 Ilg/ III in adult Apis mellifera (Duchateau and Florkin, 1959) and 14 Ilg/Ill in adult Periplaneta americana (Treherne, 1960) and adult Melanoplus differentialis (Randall and Derr, 1965). Notably, D. pseudotsugae is also considered as an active flier (Atkins, 1961; Bennett and Borden, 1971). Although no significant change was detected in the TABLE 4 Total Amino Acid Levels in the Hemolymph of Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae (Mean 1 I SE) Total amino acid concentration'[
Host stage and sex
n
Control
Infected
Callow female Mature female Callow male Mature male
7 7 7 7
10.4 ± 0.7 9.6 ± 0.6 11.5 ·10.7 10.9 ± 0.7
10.3 ± 0.7 10.5 ± 0.8 12.0 ± 1.3 11.7 ± 1.4
Q#-!g glycine equivalentsjul hemolymph.
TABLE 5 The Effect of Contortylenchus reversus on the Size of the Terminal Oocyte in Mature Female Adult Dendroctonus pseudotsugae (Mean ± I SE) Group
n
Length of largest terminal oocyte (mm)
Control Infected
13 13
0.54 ± 0.02 * 0.44 ± 0.02*
*Significantly different from each other at P < 0.01.
level of hemolymph trehalose due to C. reversus infection, it is possible that host glucose from the catabolism of trehalose may be removed by the nematode and the consequent tendency to lower the insect hemolymph trehalose level would be compensated for by the homeostatic mechanism releasing trehalose from the fat body. Other nematode species are capable of glucose uptake from the surrounding medium, e.g., Mermis nigrescens (Gordon and Webster, 1972; Rutherford and Webster, 1974) and Ascaris lumbricoides (Entner and Gonzalez, 1959; Castro and Fairbairn, 1969). Mermis nigrescens also alters carbohydrate metabolism in the fat body of the desert locust (Gordon and Webster, 1971; Gordon et al., 1971). However, the biochemistry of the fat body of D. pseudotsugae was not studied and so it is not known whether this is affected by C. reversus parasitism. Florkin and Jeuniaux (1964) stated that the average protein level in the hemolymph of Coleoptera is 30-40 Ilg/ Ill. The 14-20 Ilg/ III level found in adult female D. pseudotsugae is low when compared to this, but agrees with that found by Sahota (1970) for the same insect. The protein depletion caused by C. reversus in the beetle hemolymph detected in mature but not in callow adult females suggests that during the maturation of the latter, the normal active incorporation of hemolymph proteins into ovarial proteins occurs concurrently with nematode withdrawal of host hemolymph protein, thus upsetting the homeostatic mechanism for the maintenance of hemolymph protein levels. Nematode utilization of host protein probably also occurs in
97
THONG AND WEBSTER
the callow female, but the homeostatic control of hemolymph protein levels is able to cope with the rate of depletion, without the developing eggs drawing from the available proteins. 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 facefty (Stoffolano, 1967). 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, does not affect total hemolymph protein levels in desert locusts but depletes both fat body protein and amino acids (Gordon and Webster, 1971). The 20% reduction in size of the oocytes in infected female D. pseudotsugae is either the result of the depletion of hemolymph protein or nematode effects on the host hormonal control system for vitellogenesis. Electrophoretically, the depletion in total protein in infected mature females is shown as a proportionate decrease in all protein fractions, indicating a nonselective effect of C. reversus parasitism . However, despite the fact that parasitism does not affect total hemolymph protein levels in callow males, a significant increase was detected in the percentage of two protein fractions in the hemolymph of infected callow males over that of the controls, due either to a disproportionate uptake of specific amino acids by the parasite or to a direct or indirect effect on protein catabolism in the fat body of the beetle causing a disproportionate release of the various amino acids into the hemolymph. Mermis nigrescens, for example, depletes some protein fractions in the hemolymph of desert locusts while causing an increase in others in the insect fat body (Gordon et al., 1973). Despite there being no significant changes detected in total amino acid levels in the hemolymph of infected beetles , it is still possible that the parasite is taking up host
amino acids since the beetles may be able to replace their amino acids by increased food intake. A selective uptake of amino acids has been demonstrated for M. nigrescens (Gordon and Webster, 1972). Alternatively, parasite induced catabolism of fat body protein may be supplying the nematode with sufficient free amino acids in the hemolymph for uptake. Contortylenchus reversus, like M . nigrescens, does not appear to feed directly on host fat body tissue. Members of the genus Contortylenchus may take up nutrients through the cuticle as has been suggested for the closely related genus Heterotylenchus (Nicholas, 1972). Ultrastructural studies of the cuticle of C. reversus may demonstrate such a mechanism for nutrient absorption. ACKNOWLEDGMENTS This research was supported by a National Research Council of Canada Grant. We thank Drs. J. H. Borden, J. P. M. Mackauer, and J. S. Barlow for their critical review of the manuscript. REFERENCES ASHRAF, M. AND BERRYMA N, A. A. 1970. Biology of Sulphuretyl enchus elongatus (Nematoda: Sphaerulariidae), and its effect on its host, Scolytus ventralis (Coleopter a: Scolytidae). Can. Enromo/.. 102, 197213. ATKINS, M. D. 1961. A study of the flight of the Douglas fir beetle, Dendroctonus pseudot sugae Hopk ., 3. Flight capacity. Can. Entomol, 93, 467474. BENNETT, R . B. AND BORDEN, J. H. 1971. Flight arre stment of tethered Dendroctonus pseudotsugae and Trypodendron lineatum (Coleoptera: Scolytidae) in response to olfactor y stimuli. Ann . Entom ol. Soc . A mer .. 64, 1273-1 286. BRAMHALL, S., NOACK, N., Wu, M., AND LOEWENBERG, J. R. 1969. A simple colorimetric method for determination of protein . A na/. Biochem .. 31, 146148. CASTRO, G. A. ANI> FAIRBAIRN, D. 1969. Comparison of cuticular and intestinal absorption of glucose by adult A scaris lumbricoides, 1. Parasitol.. 55, 13-16. DAVIS, B. J . 1964. Disc electrophoresis. II. Method and application to huma n serum proteins . A nn. N. Y . A cad. S ci.. 121,404-4 27. DUCHATEA U, G. AND FLORKIN, M. 1959. Sur la
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EFFECTS OF NEMATODA ON BARK
trehalosemie des insectes et sa signification. Arch. Int. Physiol. Biochim.. 67, 306-314. ENTNER, N. AND GONZALEZ, C. 1959. Fate of glucose in Ascaris Lumbricoides. Exp. Parasit.. 8,471-479. FLORKIN, M. AND JEUNIAUX, C. 1964. Hemolymph: Composition. In "The Physiology 0/ Insecta" (M. Rockstein, ed.), Vol. 30. Academic Press, N.Y. GORDON, R. AND WEBSTER, J. M. 1971. Mermis nigrescens: Physiological relationship with its host, the adult desert locust Schistocerca gregoria. Exp. Parasit.. 29, 66-79. GORDON, R. AND WEBSTER, J. M. 1972. Nutritional requirements for protein synthesis during the parasitic development of the entomophilic nematode Mermis nigrescens. Parasitology. 64, 161-172. GORDON, R., WEBSTER, J. M., AND HISLOP, T. G. 1973. Mermithid parasitism, protein turnover and vitellogenesis in the desert locust, Schistocerca gregaria Forskal. Compo Biochem. Physiol., 468, 575593. GORDON, R., WEBSTER, J. M., AND MEAD, D. E. 1971. Some effects of the nematode Mermis nigrescens upon carbohydrate metabolism in the fat body of its host, the desert locust Schistocerca gregaria. Can. J.
;l001..49,431-434. KHAN, M. A. 1957. Sphaerularia ungulacauda n.sp. (Nematoda: Allantonematidae) from the Douglas fir beetle, with key to Sphaerularia species. Can. J. 35,635-639. MASSEY, C. L. 1956. Nematode parasites and associates of the Engelmann spruce beetle (Dendroctonus engelmanni Hopk.). Proc. Helminth. Soc. Wash.. 23,14-24. MASSEY, C. L. 1962. Life history of Aphelenchulus elongatus Massey (Nematoda), an endoparasite of Ips con/usus LeConte, with a description of the male. J.lnsect Pathol.. 4,95-103. NICHOLAS, W. L. 1972. The fine structure of the cuticle of Heterotylenchus. Nematologlca, 18, 138-140. NICKLE, W. R. 1963. Notes on the genus Contortylenchus Ruhrn 1956, with observations on the biology and life history of C. elongatus (Massey, 1960) n. comb., a parasite of a bark beetle. Proc. Helminth. Soc. Wash.. 30, 218-223. RANDALL, D. D. AND DERR, R. F. 1965. Trehalose: occurrence and relation to egg diapause and active
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transport in the differential grasshopper, Melanoplus differentialis. J. Insect Physiol.• 1,329-335. ROSEN, H. 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys.. 67,10-15. RUHM, W. 1956. "Die Nematoden der Ipiden." Veb Gustav Fischer Verlag, Jena. RUTHERFORD, T. A. AND WEBSTER, J. M. 1974. Transcuticular uptake of glucose by the entomophilic nematode Mermis nigrescens. J. Parasitol. (In press). SAHOTA, T. S. 1970. Haemolymph and ovarial proteins in the bark beetle, Dendroctonus pseudotsugae in relation to ovarian development. Can. J. Zool .. 48, 1307-1312. STOFFOLANO, J. G., JR. 1967. The synchronization of the life cycle of diapausing faceflies, Musca autumnalis, and of the nematode Heterotylenchus autumnalis. J. Invertebr. Pathol.. 9, 395-397. THOMSEN, E. 1948. Effect of removal of neurosecretory cells in the brain of adult Calliphora erythrocephala. Nature (London). 161,439-440. THOMSEN, E. AND MOLLER, I. B. 1959a. Neurosecretion and intestinal proteinase activity in an insect, Calliphora erythrocephala. Nature lLondon], 183, 14011402. THOMSEN, E. AND MOLLER, I. B. 1959b. Further studies on the function of the neurosecretory cells of the brain of the adult. Calliphora erythrocephala female. In "The Ontogeny of Insects," pp. 121-125. Acta Symp. Evol. Ins., Praha. THOMSEN, E. AND MOLLER, I. B. 1963. Influence of the neurosecretory cells and corpus alia tum on intestinal proteinase activity in the adult blowfly Calliphora erythrocephala Meig. J. Exp. Bioi.. 40, 301-321. THONG, C. H. S. 1973. "Bark Beetle Nematodes in British Columbia with Emphasis on the Biology and Host-Parasite Relationship of Contortylenchus reversus." Ph.D. thesis, Simon Fraser University, Canada. THONG, C. H. S. AND WEBSTER, J. M. 1973. Morphology and the post-embryonic development of the bark beetle nematode Contortylenchus reversus (Sphaerulariidae). Nematologica, 19, 159-168. TREHERNE, J. E. 1960. The nutrition of the central nervous system in the cockroach, Periplaneta americana L. The exchange and metabolism of sugars. J. Exp. Bioi., 37, 513-533.
26, 91-98 (1975)
Effects of Contortylenchus reversus (Nematoda: Sphaerulariidae) on Hemolymph Composition and Oocyte Development in the Beetle Dendroctonus pseudotsugae (Coleoptera: Scolytidae) CYRIL H. S. THONG AND JOHN M. WEBSTER
Pestology Centre . Department of Biological Scien ces, Simon Fraser University, Burnaby, Vancouver , B.C. V5A lS6, Canada Received November 6, 1974 Hemolymph of health y and Contortylenchus reversus infected Douglas fir beetles, Dendroctonus pseudotsugae, was analyzed to determine carbohydr ate. protein, and amino acid content and the effects of parasitism upon these entities was studied. The normal levels of these components in healthy beetle s are discussed in connection with those found in related insects. Parasitism does not affect hemolymph trehalose levels in D. pseudotsugae. Total protein levels decrease with parasitism during the maturation of female beetles from the callow adult state. This decrease in protein levels is proportionate for all protein fractions as shown by acrylamide gel disc electrophore sis. A consequence of protein depleti on is a 20% reduction in the size of the oocytes in infected female beetles . In male beetles, tot al hemolymph protein levels do not change during par asitism but in the callow males, two protein fractions increase significantly in percentage composition over the controls. Thi s difference is not detected in mature male beetles . No significant differences could be dete cted in tot al amin o acid levels between control and infected groups of all four host categories.
INTRODUCTION The bark beetle Dendroctonus pseudotsugae is a serious pest of the Douglas fir tree, Pseudotsuga menziesii, in the Pacific northwest. A number of nematode species are associated with this beetle externally and in the gut. The main nematode parasite in the hemocoel is Contortylenchu s reversus. Gravid females, eggs, and four larval stages of this nematode may be found simultaneously in the hemocoel of larvae, pupae, and adult beetles. The fourth stage nematode larvae leave the beetle with the feces and develop quickly into adult nematodes in the gallery frass (Thong and Webster, 1973). After mating, the males die but the females infect larvae of the next generation of host. Within the host hemocoel the female nematodes increase fourfold in size and gonad maturation and egg production commences. Over a thousand nematode eggs and larvae may be found in a single infected beetle at anyone time (Thong , 1973).
Very little is known of the effects of bark beetle nematodes on their host physiology. Riihm (1956) stated that larval and adult hemocoel parasites obtain nutrients from the fat body of bark beetles. It has also been reported that Contortylenchus elongatus reduces the number of fat cells in Ips paraconfusus (Nickle, 1963), and that the growth and function of organs in D. pseudotsugae are affected by Sphaerulariopsis dendroctoni (Khan, 1957). Nematodes are also known to reduce bark beetle fecundity (Massey, 1956, 1962; Ashraf and Berryman, 1970). The present study was undertaken to elucidate some of the effects the nematode C. reversus has on its host D. pseudotsugae with respect to host hemolymph composition and oocyte development. MATERIALS AND METHODS Naturally infected Douglas fir beetles were collected from the area around Williams Lake , British Columbia, Canada. 91
Cop yright © 1975 by Academi c Pre ss, Inc. All rights of reproduction in any for m reser ved.
92
EFFECTS OF NEMATODA ON BARK
Infected beetles containing at least 50 nematodes/insect in the hemocoel were used in the experiments. Hemolymph analysis was performed on the hemolymph of both sexes of callow unemerged and mature adult beetles prior to the first oviposition. Hemolymph was drawn from beetles through a wound made by the removal of a metathoracic leg. The body of the beetle was squeezed gently with a pair of forceps to eject hemolymph through the wound. Withdrawal of hemolymph and the measurement of blood volume was done using I ILl disposable micropipettes (Drummond "Microcaps"). Hemolymph not used immediately was stored in vials at - 30· C with a crystal of phenylthiourea. Hemolymph trehalose assay was performed by thin-layer chromatography using an adsorbent mixture of 24 g MN Kieselguhr and 6 g of Celite analytical filter aid (Johns-Manville, USA). This was slurried with 80 ml of 0.02 M sodium acetate solution and layered to a thickness of 250 ILm on 20 x 20 em glass plates. Hemolymph from individual beetles and two serial dilutions of a trehalose standard solution were spotted on each tlc plate 2 cm apart. The plates were developed to a distance of 15 em using a mixture of 60 ml ethyl acetate and 40 ml isopropanol/water (2:1) as solvent. The compounds separated out were detected using a spray reagent consisting of 50 ml glacial acetic acid, 0.5 ml anisaldehyde, and 1 ml concentrated H 2S04 made up fresh before application. The plates were then heated at 11O·C in an oven till the spots attained maximum intensity (ca. 20 min). The plates were then cooled for 30 min at room temperature. Quantification was done using a Zeiss chromoscan spectrophotometer for tic using a wavelength of 370 nm. Readings were calibrated against a trehalose standard curve. Total hemolymph protein was analysed using the method of Bramhall et al. (1969) with xylene brilliant cyanin G (Michrome 1224) (Esbe Laboratory Supplies, Toronto, Canada) as a stain. Samples were read in a spectrophotometer at 620 nm wavelength
and calibrated against a bovine serum albumin standard curve. Individual insects were used in this assay. Hemolymph proteins were separated out by polyacrylamide disc electrophoresis in a standard model Polyanalyst (Buchler Instruments Division, Nuclear Chicago Corporation, USA) apparatus but using longer gel tubes (ca. 12 em) to increase resolution of the protein bands. The technique employed was essentially similar to that of Davis (1964) using a 7.0% running gel in conjunction with 2.5% spacer and sample gels. Hemolymph from eight insects was pooled and 5 ILl used for each sample run. A pH 8.9 Tris-HCl buffer was used and bromophenol blue was used as a tracking dye. A current of 5 rnA/tube was applied. Gels were subsequently fixed in 10% trichloroacetic acid (TCA) at 37"C for 30 min and stained for 1 hr at the latter temperature with a fixative/ stain solution consisting of 19 parts 10% TCA and 1 part 1% Coomassie blue RL (Michrome 100). Destaining was carried out nonelectrophoretically with several changes of 7% acetic acid (5 days). Quantification was carried out with a Joyce Loebl Chromoscan and overlapping electropherogram peaks were resolved with a Dupont 310 Curve Resolver. Quantification in arbitrary units was achieved by measuring the areas below the respective peaks with a planimeter. Total hemolymph amino acid assay was done on individual insects by the method of Rosen (1957) but the volumes of reagents used were decreased proportionally to compensate for the small volume of hemolymph obtainable from each insect. Samples were quantified in a spectrophotometer at a wavelength of 570 nm and standardized against a glycine standard curve. The rate of development of oocytes was estimated by measuring the length of the largest terminal oocyte of 13 uninfected and 13 infected mature female adult beetles from the same colony prior to the first oviposition (Thomsen, 1948; Thomsen and Moller, 1959a,b, 1963). All data were analyzed using Student's
93
THONG AND WEBSTER
t test. Significance was set at the 95% confidence limit for all data analysed.
RESULTS
TABLE 2 Total Hemolymph Protein Levels in Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae (Mean ± 1 SE)
Carbohydrates A preliminary experiment showed the level of glucose in the hemolymph of D pseudotsugae to be extremely low and often undetectable by tlc, Trehalose appeared to be the main blood sugar making up more than 90% of the total hemolymph carbohydrates. The normal physiological level of hemolymph trehalose in D. pseudotsugae did not change significantly in the maturation of the callow female adults, but mature male adults had 25% more trehalose in the hemolymph than did callow male adults (P < 0.02) (Table I). Mature male adults also had significantly higher hemolymph trehalose levels than did mature female adults (P < 0.02). No significant differences, however, were detected in the level of hemolymph trehalose between control and infected groups in each adult category. Total Hemolymph Protein The total hemolymph protein level in normal D. pseudotsugae decreases significantly during the maturation of both sexes of callow adults (Table 2). There was no significant difference, however, between total hemolymph protein levels in male and female beetles. No significant difference in total hemolymph protein was detected between control and infected callow females, but infected mature females had 20% less hemolymph protein (P < 0.05) than control TABLE 1 Hemolymph Trehalose Levels in Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae (Mean ± 1 SE) Trehalose concentration"
Host stage and sex
n
Callow female Mature female Callow male Mature male
6 8 6 8
O..g
Control 10.7 10.6 11.0 14.9
trehalose/..l hemolymph.
± 0.9 ± 1.0 ± 0.7 ± 1.2
Infected 10.1 ± 0.9 11.3±I.4 9.7 ± 1.2 14.5 ± 1.0
Total protein concentration''
Host stage and sex
n
Callow female Mature female Callow male Mature male
6 8 6 6
Control 19.8± 1.2 14.3 ± 1.2* 21.2 ± 2.1 14.4 ± 1.6
Infected 19.2± 1.6 11.0 ± 0.7* 17.6±1.3 16.1 ± 2.3
°lLg Bovine Serum· Albumin equivalents/ILl hemolymph. *Significantly different from each other atP < 0.05.
insects of the same group. No significant differences between total protein levels of control and infected callow and control and infected mature males were detected. Individual Proteins Nine protein fractions were separated from the hemolymph of callow female beetles, with fractions I and VIII being the major fractions (Table 3; Fig. 1). No significant differences between control and infected beetles were observed with respect to the percentage of these nine fractions in the blood. Fraction IV, which was always present in the hemolymph of mature females was undetectable in callow females, and fractions VIIIB and IX that were sometimes present in mature females were totally absent in all the callow females. In mature females, a total of 12 fractions were detected in the hemolymph but fractions VIIIB, IX, and X were absent from some of the samples analysed (Table 3). There was a lower concentration of fractions V and VI and a higher concentration of fraction III in mature as compared to callow females. Despite there being no significant differences in percentage composition of protein fractions recorded between control and infected groups of mature females, the total level of protein (according to the intensity of the stain retained by the respective gels) was higher in control groups than in infected groups (Fig. 2), which supports the results obtained in the total protein analysis. Parasitism, therefore, caused a proportional
\0 ~
TABLE 3 Percentage Concentration of Protein Fractions in the Hemolymph of Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae. Proteins Separated by Acrylamide Gel Disc Electrophoresis. Concentrations Presented as Mean ± 1 SE
Fraction no. I
II III IV V VI VII VIII VIIIB IX X XI
Callow female (n = 5)
Mature female (n = 5)
R/
Control
Infected
Control
Infected
0.05-0.10 0.09-0.17 0.14-0.21 0.20-0.24 0.24-0.30 0.30-0.38 0.36-0.47 0.49-0.57 0.64-0.70 0.78-0.86 0.90-0.95 1.0
27.7 ± 1.4 3.3 ± 0.5 2.9 ± 1.1
23.7 ± 1.9 3.9 ± 0.8 4.0 ± 0.8
-
-
27.5 ± 1.0 1.3 ± 0.2 8.8 ± 2.2 3.3 ± 1.0 6.2:!; 2.1 6.4 ± 0.5 11.0 ± 1.3 28.3 ± 2.4 2.3 ± 0.1 0 0.8 ± O.~ 2.2 ± 0.30 3.7 ± 0.5
28.5 ± 1.6 2.5 ± 1.2 7.4 ± 1.0 3.8 ± 1.3 5.3 ± 1.3 6.5 ± 0.8 l1.0:!; 1.5 27.1 ± 2.5 1.9 ± 0.2 0 1.8±0.7° 1.9 ± 0.20 4.2 ± 0.4
Callow male (n = 6) Control
Mature male (n = 5)
Infected
Control
Infected
26.3± 1.9 1.3 ± 0.5 6.8 ± 1.6 5.7 ± 0.5 8.8 ± 1.0 6.9 ± 0.7 10.6 ± 1.3 24.0 ± 1.6 2.1 ± 0.3 1.50 3.5 ± 0.5 0 4.2 ± 1.2
23.2 ± 2.3 1.3 ± 0.4 7.9 ± 2.1 6.5 ± 1.2 10.6 ± 1.3 7.0 ± 0.6 11.7 ± 1.3 25.1 ± 1.5 1.5 ± 0.7 0 0.60 2.3 ± o.s« 3.8 ± 1.2
tn
'" o'" tn
-l tI>
11.7±2.4 10.9 ± 0.7 13.2 ± 2.3 23.7 ± 0.8 -
2.0 ± 0.8 4.9 ± 0.8
14.7 10.5 9.1 25.8
± 2.4 ± 1.8 ± 1.8 ± 2.0 -
1.6 ± 0.2 6.1 ± 0.3
aFraction not present in all samples. *Significantly different from each other at P < 0.05. **Significantly different from each other atP < 0.01.
22.4 2.7 8.0 14.2 4.8 14.0 9.4 22.9
± 2.1 ± 0.9 ± 1.7
± 1.9 ± 1.2* ± 1.8 ± 0.9** ± 2.3
0.6 ± 0.3 0 2.4 ± 0.8 3.6 ± 1.2
20.2 2.0 5.9 8.0 8.9 9.6 15.9 24.4
± 1.9 ± 0.4 ± 2.0 ± 3.8 ± 0.7* ± 1.5 ± 1.2** ± 1.3
1.2 ± 0.30 3.4 ± 0.7 3.6 ± 0.7
0
'"Z tn
a:::
> -l 0 0
> 0
Z
= > :Ill
i":
95
THONG AND WEBSTER
XI
m CO NTROL
x
XI ;l INFECTED
i\ f ...........-
. o
0 .5
I. D
XI
m~ 1
INFECTED
jj
11 . .. I. " 1lIl " i···. ......../ .\ "~,,,," ••...•/ ..~ !
"
.._
11 m ! ' .1\,llJ...... .. ./" .-F/>! All! !
~.'"~
l~
"'-' '...~•..,:
.
......-----.--- - ...,...._. ..
--- ,....- - - ~ . -.----
~5
RElATIVE MOBilIT Y CRn
0
RELA TIVE MOBILITY IRf! FIG. 2
FIG. 1
FIGS . I AND 2. Representative chromo scan tracings of hemolymph proteins in control and Contortylenchus
reversus infected callow female (I) and mature female (2) adult Dendroctonus pseu dotsugae.
decrease in all protein fractions in mature female beetles. In the callow males, 11 fractions were separated. Fraction VIIIB was absent from all samples and fraction IX was detectable in only some samples. Fraction IV, which was not found in the hemolymph of callow females, accounted for about 10% of the total protein in callow males. The two fractions V and VII increased significantly in infected callow males (Table 3; Fig. 3) by approximately 4 and 6%, respectively, in percentage of total protein, which was a 3040 % increase over the controls. Fractions IV and VI decreased slightly in percentage composition but these changes were not
significant. In the mature male beetles, 12 fractions were recognized (Fig. 4), but, as in the mature females, fractions VIIIB, IX, and X were absent from some samples. No significant differences were detected in the level of the protein fractions between control and infected mature male beetles (Table 3; Fig. 4). Notably, the higher level of fractions V and VII that occurred in infected than control callow males does not occur in the mature males. Fractions IV and VI were significantly lower in control groups of mature males than in control groups of callow males. Fraction VIIIB, which was detected in some samples of hemolymph from mature males, was not detected in callow
m
~
I\ x
.J\ /\.~. l. 0
INFECTED /
.
! .n, H
\)!\._~ }JJ\ 1 vn::
0 .5 RE LATIVE MOBILITY IRI I FIG. 3
.•
o
l.0
.
0.5
a
RELATIVE MOB ILITY (Rl/ FIG. 4
FIGS . 3 AND 4. Representative chr om oscan tr acings of hemol ymph protein s in cont rol and Cont ort ylen chus reversus infected ca llow male (3) and mature male (4) adult Dendroct onu s pseudotsugae.
96
EFFECTS OF NEMATODA ON BARK
males. As in all other groups analysed, fractions I and VIII were the two major fractions in the hemolymph.
Total Hemolymph Amino Acids The level of amino acids in the hemolymph was approximately the same in callow and mature female beetles, and in callow and mature male beetles (Table 4) and no significant differences in amino acid levels were detected between control and infected members of each of the four categories studied. Oocyte Development Infected females of D. pseudotsugae had significantly smaller, i.e., 20% smaller, oocytes than did uninfected ones (Table 5). DISCUSSION The level of trehalose in D. pseudotsugae (10.6 Ilg/ III in mature females and 14.9 ug] III in mature males) is high when compared to that of other Coleoptera previously studied, e.g ., 5-7 ug] III in adult Dytiscus marginalis and 3-5 Ilg/ III in adult Hydrophilus piceus (Duchateau and Florkin, 1959). It is comparable, however, with the levels found in the hemolymph of more active fliers, e.g., 6-12 Ilg/ III in adult Apis mellifera (Duchateau and Florkin, 1959) and 14 Ilg/Ill in adult Periplaneta americana (Treherne, 1960) and adult Melanoplus differentialis (Randall and Derr, 1965). Notably, D. pseudotsugae is also considered as an active flier (Atkins, 1961; Bennett and Borden, 1971). Although no significant change was detected in the TABLE 4 Total Amino Acid Levels in the Hemolymph of Control and Contortylenchus reversus Infected Adult Dendroctonus pseudotsugae (Mean 1 I SE) Total amino acid concentration'[
Host stage and sex
n
Control
Infected
Callow female Mature female Callow male Mature male
7 7 7 7
10.4 ± 0.7 9.6 ± 0.6 11.5 ·10.7 10.9 ± 0.7
10.3 ± 0.7 10.5 ± 0.8 12.0 ± 1.3 11.7 ± 1.4
Q#-!g glycine equivalentsjul hemolymph.
TABLE 5 The Effect of Contortylenchus reversus on the Size of the Terminal Oocyte in Mature Female Adult Dendroctonus pseudotsugae (Mean ± I SE) Group
n
Length of largest terminal oocyte (mm)
Control Infected
13 13
0.54 ± 0.02 * 0.44 ± 0.02*
*Significantly different from each other at P < 0.01.
level of hemolymph trehalose due to C. reversus infection, it is possible that host glucose from the catabolism of trehalose may be removed by the nematode and the consequent tendency to lower the insect hemolymph trehalose level would be compensated for by the homeostatic mechanism releasing trehalose from the fat body. Other nematode species are capable of glucose uptake from the surrounding medium, e.g., Mermis nigrescens (Gordon and Webster, 1972; Rutherford and Webster, 1974) and Ascaris lumbricoides (Entner and Gonzalez, 1959; Castro and Fairbairn, 1969). Mermis nigrescens also alters carbohydrate metabolism in the fat body of the desert locust (Gordon and Webster, 1971; Gordon et al., 1971). However, the biochemistry of the fat body of D. pseudotsugae was not studied and so it is not known whether this is affected by C. reversus parasitism. Florkin and Jeuniaux (1964) stated that the average protein level in the hemolymph of Coleoptera is 30-40 Ilg/ Ill. The 14-20 Ilg/ III level found in adult female D. pseudotsugae is low when compared to this, but agrees with that found by Sahota (1970) for the same insect. The protein depletion caused by C. reversus in the beetle hemolymph detected in mature but not in callow adult females suggests that during the maturation of the latter, the normal active incorporation of hemolymph proteins into ovarial proteins occurs concurrently with nematode withdrawal of host hemolymph protein, thus upsetting the homeostatic mechanism for the maintenance of hemolymph protein levels. Nematode utilization of host protein probably also occurs in
97
THONG AND WEBSTER
the callow female, but the homeostatic control of hemolymph protein levels is able to cope with the rate of depletion, without the developing eggs drawing from the available proteins. 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 facefty (Stoffolano, 1967). 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, does not affect total hemolymph protein levels in desert locusts but depletes both fat body protein and amino acids (Gordon and Webster, 1971). The 20% reduction in size of the oocytes in infected female D. pseudotsugae is either the result of the depletion of hemolymph protein or nematode effects on the host hormonal control system for vitellogenesis. Electrophoretically, the depletion in total protein in infected mature females is shown as a proportionate decrease in all protein fractions, indicating a nonselective effect of C. reversus parasitism . However, despite the fact that parasitism does not affect total hemolymph protein levels in callow males, a significant increase was detected in the percentage of two protein fractions in the hemolymph of infected callow males over that of the controls, due either to a disproportionate uptake of specific amino acids by the parasite or to a direct or indirect effect on protein catabolism in the fat body of the beetle causing a disproportionate release of the various amino acids into the hemolymph. Mermis nigrescens, for example, depletes some protein fractions in the hemolymph of desert locusts while causing an increase in others in the insect fat body (Gordon et al., 1973). Despite there being no significant changes detected in total amino acid levels in the hemolymph of infected beetles , it is still possible that the parasite is taking up host
amino acids since the beetles may be able to replace their amino acids by increased food intake. A selective uptake of amino acids has been demonstrated for M. nigrescens (Gordon and Webster, 1972). Alternatively, parasite induced catabolism of fat body protein may be supplying the nematode with sufficient free amino acids in the hemolymph for uptake. Contortylenchus reversus, like M . nigrescens, does not appear to feed directly on host fat body tissue. Members of the genus Contortylenchus may take up nutrients through the cuticle as has been suggested for the closely related genus Heterotylenchus (Nicholas, 1972). Ultrastructural studies of the cuticle of C. reversus may demonstrate such a mechanism for nutrient absorption. ACKNOWLEDGMENTS This research was supported by a National Research Council of Canada Grant. We thank Drs. J. H. Borden, J. P. M. Mackauer, and J. S. Barlow for their critical review of the manuscript. REFERENCES ASHRAF, M. AND BERRYMA N, A. A. 1970. Biology of Sulphuretyl enchus elongatus (Nematoda: Sphaerulariidae), and its effect on its host, Scolytus ventralis (Coleopter a: Scolytidae). Can. Enromo/.. 102, 197213. ATKINS, M. D. 1961. A study of the flight of the Douglas fir beetle, Dendroctonus pseudot sugae Hopk ., 3. Flight capacity. Can. Entomol, 93, 467474. BENNETT, R . B. AND BORDEN, J. H. 1971. Flight arre stment of tethered Dendroctonus pseudotsugae and Trypodendron lineatum (Coleoptera: Scolytidae) in response to olfactor y stimuli. Ann . Entom ol. Soc . A mer .. 64, 1273-1 286. BRAMHALL, S., NOACK, N., Wu, M., AND LOEWENBERG, J. R. 1969. A simple colorimetric method for determination of protein . A na/. Biochem .. 31, 146148. CASTRO, G. A. ANI> FAIRBAIRN, D. 1969. Comparison of cuticular and intestinal absorption of glucose by adult A scaris lumbricoides, 1. Parasitol.. 55, 13-16. DAVIS, B. J . 1964. Disc electrophoresis. II. Method and application to huma n serum proteins . A nn. N. Y . A cad. S ci.. 121,404-4 27. DUCHATEA U, G. AND FLORKIN, M. 1959. Sur la
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EFFECTS OF NEMATODA ON BARK
trehalosemie des insectes et sa signification. Arch. Int. Physiol. Biochim.. 67, 306-314. ENTNER, N. AND GONZALEZ, C. 1959. Fate of glucose in Ascaris Lumbricoides. Exp. Parasit.. 8,471-479. FLORKIN, M. AND JEUNIAUX, C. 1964. Hemolymph: Composition. In "The Physiology 0/ Insecta" (M. Rockstein, ed.), Vol. 30. Academic Press, N.Y. GORDON, R. AND WEBSTER, J. M. 1971. Mermis nigrescens: Physiological relationship with its host, the adult desert locust Schistocerca gregoria. Exp. Parasit.. 29, 66-79. GORDON, R. AND WEBSTER, J. M. 1972. Nutritional requirements for protein synthesis during the parasitic development of the entomophilic nematode Mermis nigrescens. Parasitology. 64, 161-172. GORDON, R., WEBSTER, J. M., AND HISLOP, T. G. 1973. Mermithid parasitism, protein turnover and vitellogenesis in the desert locust, Schistocerca gregaria Forskal. Compo Biochem. Physiol., 468, 575593. GORDON, R., WEBSTER, J. M., AND MEAD, D. E. 1971. Some effects of the nematode Mermis nigrescens upon carbohydrate metabolism in the fat body of its host, the desert locust Schistocerca gregaria. Can. J.
;l001..49,431-434. KHAN, M. A. 1957. Sphaerularia ungulacauda n.sp. (Nematoda: Allantonematidae) from the Douglas fir beetle, with key to Sphaerularia species. Can. J. 35,635-639. MASSEY, C. L. 1956. Nematode parasites and associates of the Engelmann spruce beetle (Dendroctonus engelmanni Hopk.). Proc. Helminth. Soc. Wash.. 23,14-24. MASSEY, C. L. 1962. Life history of Aphelenchulus elongatus Massey (Nematoda), an endoparasite of Ips con/usus LeConte, with a description of the male. J.lnsect Pathol.. 4,95-103. NICHOLAS, W. L. 1972. The fine structure of the cuticle of Heterotylenchus. Nematologlca, 18, 138-140. NICKLE, W. R. 1963. Notes on the genus Contortylenchus Ruhrn 1956, with observations on the biology and life history of C. elongatus (Massey, 1960) n. comb., a parasite of a bark beetle. Proc. Helminth. Soc. Wash.. 30, 218-223. RANDALL, D. D. AND DERR, R. F. 1965. Trehalose: occurrence and relation to egg diapause and active
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transport in the differential grasshopper, Melanoplus differentialis. J. Insect Physiol.• 1,329-335. ROSEN, H. 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys.. 67,10-15. RUHM, W. 1956. "Die Nematoden der Ipiden." Veb Gustav Fischer Verlag, Jena. RUTHERFORD, T. A. AND WEBSTER, J. M. 1974. Transcuticular uptake of glucose by the entomophilic nematode Mermis nigrescens. J. Parasitol. (In press). SAHOTA, T. S. 1970. Haemolymph and ovarial proteins in the bark beetle, Dendroctonus pseudotsugae in relation to ovarian development. Can. J. Zool .. 48, 1307-1312. STOFFOLANO, J. G., JR. 1967. The synchronization of the life cycle of diapausing faceflies, Musca autumnalis, and of the nematode Heterotylenchus autumnalis. J. Invertebr. Pathol.. 9, 395-397. THOMSEN, E. 1948. Effect of removal of neurosecretory cells in the brain of adult Calliphora erythrocephala. Nature (London). 161,439-440. THOMSEN, E. AND MOLLER, I. B. 1959a. Neurosecretion and intestinal proteinase activity in an insect, Calliphora erythrocephala. Nature lLondon], 183, 14011402. THOMSEN, E. AND MOLLER, I. B. 1959b. Further studies on the function of the neurosecretory cells of the brain of the adult. Calliphora erythrocephala female. In "The Ontogeny of Insects," pp. 121-125. Acta Symp. Evol. Ins., Praha. THOMSEN, E. AND MOLLER, I. B. 1963. Influence of the neurosecretory cells and corpus alia tum on intestinal proteinase activity in the adult blowfly Calliphora erythrocephala Meig. J. Exp. Bioi.. 40, 301-321. THONG, C. H. S. 1973. "Bark Beetle Nematodes in British Columbia with Emphasis on the Biology and Host-Parasite Relationship of Contortylenchus reversus." Ph.D. thesis, Simon Fraser University, Canada. THONG, C. H. S. AND WEBSTER, J. M. 1973. Morphology and the post-embryonic development of the bark beetle nematode Contortylenchus reversus (Sphaerulariidae). Nematologica, 19, 159-168. TREHERNE, J. E. 1960. The nutrition of the central nervous system in the cockroach, Periplaneta americana L. The exchange and metabolism of sugars. J. Exp. Bioi., 37, 513-533.