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Relation of blood composition to age in the larval female house cricket, Acheta domesticus
J. IJI.S~CYPhy,io/.,
1971.Vol.
13, pp. 559 to 561. Peryamon
Press. PrinteJ
in Greut
Britairi
RELATION OF BLOOD COMPOSITION TO AGE IN THE LARVAL FEMALE HOUSE CRICKET, ACHETA DOMESTICUS J. P. WOODRING. C. W. CLIFFORD, R. M. ROE, and R. R. MERCIER Department of Zoology and Physiology. Louisiana State University, Baton Rouge, LA 70803. U.S.A. (Receiver/ 19
Norember
1976: recked
8 December
1976)
Abstract-The blood volume. osmolality. and sugar concentration increase: the blood protein and lipid concentration decrease; and the blood ions and NPS concentration do not change during apolysis from the 7th to 8th instar of larval Acheta domesticus. The patterns of change in all blood constituents are basically identical in both of the last two larval instars. Blood volume increase is directly related to growth, but not directly to total body water. Blood protein concentration increases in exact relation to growth, resulting in a doubling of concentration by midinstar and a small decrease over the last 2 to 3 days. The concentration of clottable protein remains constant; the soluble protein concentration increase probably providing cuticular proteins prior to and following ecdysis. Blood lipid concentration increases steadily throughout both instars, even during the non-feeding phase. Total blood carbohydrate concentration decreases greatly in the first 2 days of an instar. then remains relatively constant for the remainder of the instar. Blood trehalose concentration is 85”,, of the total carbohydrate concenincrease in ‘other sugars‘ with tration throughout the instar, but drops to 65”,, with a corresponding the onset of apolysis on the last day. Growth demands probably exceed dietary supply of amino acids resulting in a 50”,, decrease in NPS concentration in the first half of the instar. then NPS concentration increases in spite of a declining feeding rate because growth demands cease. Within the normal blood ion lability range. the principle blood ions, sodium and chloride. vary in an inverse proportion to NPS and sugar concentration at least in the first two-thirds of the instar. Thus blood osmolality is constant except for an unexplained drop in the last 1 days of an instar.
INTRODUCTION
AGRELL and LUNDQUIST(1973) point out that analyses of vertebrate blood are of practical value because the physiological background for the measured blood variations has been worked out. However, the vast accumulated data on insect blood is of little value because this information has not been related to the biology or physiology of the insect. More recent work shows that temperature, starvation, exercise, anesthesia. etc.. profoundly affect the concentration of insect blood constituents (WALL, 1970; DAHLMAN, 1975; JUTSUM et al., 1975; MATTHEWSet d.. 1976), but relatively few (HILL and GOLDSWORTHY, 1968) have related blood variations to growth, feeding, or metabolism in the cycle of growth and apolysis-ecdysis during development. Our purpose then is to relate blood variations during the developmental cycle to the biology and physiology. and we used the house cricket because of its predictable life cycle and ease of culturing. In addition, the knowledge of these normat blood variations should provide valuable clues regarding the timing of hormonal and nervous regulation of insect biochemical and physiological processes. Both the nervous system (VERRETTand MILLS, 1976; CLARK and ANSTEE, 1971) and the endocrine system (MORDUE. 1970: WALKER and BAILEY, 1971: 559
GOLLXWORTHY,1970: HILL and 17~7‘~. 1974; SROKA and BARTH. 1976) directly affect the concentration of most blood constituents, and the normal patterns of change in the blood during the developmental cycle should reflect the release and interactions of the neuroendocrine system. MATERIALS
AND
METHODS
Our culture procedures (CLIFFORD et ~1.. 1977) for domesticus permitted selection of 7th (next to last) and 8th (last) larval instars of known age kO.5 days. Larvae of known age were further selected to within k15 mg of an average weight, which was based on a growth curve calculated from over 5000 individually weighed crickets over a 3 yr period. The artificial, dry diet contained 20”” protein. 4.8”” lipid. 48”,, carbohydrate, 124,, water, and 6”,, ash. Water for the last two instars was provided in slot-edged plastic vials inverted into small, open pans to insure ud lib drinking. This is critical because we found that crickets did not drink to repletion from paper or cloth watering wicks, The last two instars were kept in groups of 10 to 12 in 4 liter cylindrical. cardboard cartons. Crickets were anesthetized individually by a IO to 2Osec exposure to COz, immediately bled, then disA&eta
560
J. P. W(X)DRING.C. W. CLIFFORD, R. M. Ror!, AND R. R. MERCIES
carded. Whole blood (haemolymph) was collected by touching a capillary tube to the clipped end of the cerci and applying gentle finger pressure on the body; this procedure produced 5 to 12jtl from 7th instar larvae and 8 to 20~1 from 8th instar larvae. Clot formation is relatively slow. about 5 min, so that whole blood can be dispensed and quickly diluted. Whole blood was used for the total solids, proteins. lipid. and osmolality determinations. Plasma. or cell free blood. was not used. Blood serum is the clear fluid obtained by centtifugation of whole blood. which we allowed to tirmly clot for ,lO to 15 min in the capillaries. Serum was used for the blood volume. ion. and sugar determinations. Deproteinized serum. for the NPS determination. was obtained by adding 3 x the blood volume of 5”,, TCA in the capillary tube (but keeping the two separate). and then centrifuging the clot through the TCA. One larva provided sufficient blood for any specific analysis, and pooled blood was used only for the gravimetric lipid analysis. Microscopic examination of the collected blood revealed only haemocytes and no nonblood cell types or tissues. Although virtually all the blood cells ruptured during the clotting process. the contribution of cellular contents to total blood concentrations is small. With a haemocylometer we determined the average cell count to be 2.5 x 10’ blood cells//l1 blood. The average cell radius is 4klrn, giving a 10nl cell volumej$ of blood or a cell volume less than I”,, of the whole blood. Thus. ruptured haemocytea do not make a significant contribution to blood protein. lipid. or carbohydrate concentration. NaCl is normally less concentrated. free amino acids are probably slightly more concentrated. and K is normally 10 times more concentrated in cells than in plasma. Therefore. only in the analyses of serum K could the ruptured blood cells cause an error approaching lo”,,. A similar conclusion was reached by BRAI)Y (1967) for several other insects. Blood proteins were determined by the Biuret Method (CORNAL.Lrt trl., 1949) scaled down to a Z-ml reaction volume. The blood was rinsed into 1 M NaOH and warmed 15min at 50 ‘C to dissolve the blood proteins. The difference in whole blood and serum protein concentrations gave an estimate of clottable protein concentration. Total blood lipid for the 8th instar was determined gravimetrically using SO/tl of pooled blood per sample. Our modification of the BLIGH and DYER (1959) extraction method assured one step extraction of r 98”,, of total blood lipid if the resultant total lipid was below 4.1 mgjlOOml of the final biphasic chloroform-methanol water mixture. This was confirmed by finding no additional lipids upon re-extraclion. The total blood lipid of the 7th instar was determined using 5 ,tl samples by the Sulfophosphovanillin Method (BARNESand BLACKSTOCK.1973). By analysis of the 8th instar we found the sulfophovanillin method requires a 1.5 x factor to duplicate the gravimetric method in larval crickets. This is an empirical
correction factor and may vary with insect species. Total blood carbohydrates were determined by the phenol~sulfuric acid method (MONTGOMERY. 1957). modified only by purging the acid produced vapors with N2. The quantitation of trehalose was based on its resistance to acid hydrolysis (WYATT and KALF, 1957). Ten minutes at 97 C in 0.1 N H,SO, hydrolyzes all tri-disaccharides except trchalose. Reheating with 6 N NaOH purged v, ith NI (using aquarium tloss plugs in the test tubes) destroys all monosaccharides. A phenol- sulfuric acid analysis of the final solution gives trehalose concentration. and the difierence between serum total carbohydrates and trehalose gives ‘other sugars’. The NL purging was essential for consistent results. Total ninhydrin positive substance (NPS) was determined by a micro-modification of ROSEN’S(1957) method following in-capillary protein precipitation with TCA. The leucine standard is only stable when refrigerated and stored in a dark bottle. Cricket blood contains up to 4mM NH, and 5 mM urea (WANG and PATTON. 1969a). which taken together at these concentrations could cause a maximum error of 8”,, (ROSEN. 1957). Over 90”,, of our NPS values are probably due to free amino acids, but because we did not specifically analyze for amino acids. we use the term NPS. Blood volume was determined by the “C-inulin dilution method with a scintillation counter. Blood osmolality was determined with a Wescor Vapor Pressure Osmometer using 5 to 10~~1 of blood collected directly from the clipped cerci. Serum chlorides were determined with a Buchler-Cotlove automatic titrator. Strum sodium and potassium were determined with a flame photometer. RESULTS An overview of the results is given in Figs. I and ’ in which the change in concentration of blood con-. stituents through the last 2 larval instars is clearly illustrated. Note that the pattern and magnitude of change are similar in both instars for all constituents measured. The determined average concentration +SE, of each constituent at each age is tabulated in Tables I and 2. The amounts. given as derived pg or mg, are calculated on the basis of average blood volume for each age. For example, the blood protein concentration of a 1 day old 8th instar larva is 3.3 mg/lOO~l of blood (Table 1) and the average blood volume of a I day old larva is 39 ~1 (Table 11, therefore the amount of blood protein per 1 day old cricket is 3.3 x 391100 = 1.3 mg. The change in amount (Amg-“,) and the change in concentration (A cone-“,,) for each blood constituent is expressed as the maximum difference that occurs in an instar. and these values are given in the last two lines of Tables I and 2. The per cent maximum change of concentration and amount is determined as [(high value-low value)/low value] x 100. For example. the maximum
Age
variation
in cricket
blood
561
60 50 g
r
Age
40
t
30
:
20
0 s
(days)
Fig. 1. The relation of blood protein (H). lipid (A A), and serum carbohydrate (04) concentration and blood volume (V---Y) to age in larval A. dornesticus. Open symbols denote the 7th instar and closed symbols denote the 8th instar.
difference in amount of blood protein for the 8th instar is 4.2 - 1.3 = 2.9 mg (Table 1) and the per cent change is (2.9/1.3) 100 = 223”)“. The per cent change in concentration is probably more important than the per cent change in amount because physiological systems can only respond to concentration change. The maximum changes in concentration and amount are statistically significant (P < 0.05) for all constituents.
5 I =
15,
a E
IO-
E 0”
5. o*
\
U---U
/
“B-U
1
Treh n
WK
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.
.
.
.
.
Age (days) Fig. 2. The relation of serum chloride (M). NPS (A--A). trehalose (m). sodium (a), and potassium (V) to age in larval Achera domesticus. Open symbols denote the 7th instar and closed symbols denote the 8th instar.
Before proceeding to each blood constituent, two points should be made. If the concentration remained constant throughout an instar, then the least amount would be on the first day and the greatest amount on the last day and the quantity would parallel the blood volume. This does not occur for any blood constituents examined. If the concentration decreased during an instar solely due to dilution, resulting from the increasing blood volume, then the amounts would remain constant. This likewise does not occur for any blood constituent examined. Both the concentration (Fig. 1) and the total amount (Table 1) of blood protein increase rapidly up to just past mid-instar, basically doubling. then it plateaus and decreases slightly on the last I to 2 days. The change in serum protein parallels that of blood protein (Table 1). A distinct drop in blood protein concentration occurs during apolysis from the 7th to 8th instar. The concentration of blood lipid doubles and the amount triples in each instar (Table 1). The rate of lipid increase is uniform throughout each instar with a distinct drop during apolysis from the 7th to 8th instar (Fig. 1). The general trend of blood carbohydrate concentration is a rapid decrease in the first 2 days of both instars followed by a slight decrease over the remainder of the instar (Table 1). The maximum change in amount and concentration of blood carbohydrate is small compared to that of protein and lipid. As a generalization, blood protein and lipid concentrations greatly increase and the total carbohydrate concentration decreases through each instar. but during apolysis significant amounts of protein and lipid are removed from the blood and carbohydrates are added (Fig. 1). The serum NPS concentration decreases from day 1 to mid-instar then increases back to first day levels
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2. Relation
osmolality
7th
10 - x.h”,,
?7 - 7.X”
and
8th
Blood osmolnhly mOsM!Kgtn)
of blood
7th
.l 7.pg - 5?“,, 14.x - 54””
I OXpg - h’)“,, 143 i Oy’,,
Swum NPS mM;i I SE, (nl derived /I$
I JY pp - Xh”,, 38 - ??“,,
Y I /,g - 22X”,, 152 - l90”.
change.
to age
. I3 pg . SIV’,, -. Ih , I”,,
8th
chloride
units.
and
49 pp - ?7”,, 30 - 3”
7th
potassium.
for mg!lOO /tl cont.
Xth
Xth
other sugars, sodium. of A. dnuwstic~rs
pg = (mg/lOO~d) x (BVol/lOO~tl)
101 ,cg G’,, 352 - xv..
8th
ninhydrin positive substances (NPS). trehalose. to last (7th) and last (8th) female larval instars
Xlh
serum
/cg - (MW x mM x BVol)/lOOO for mM cont. units. The derived and lo”, variable other carbohydrates (see text). 3. The maximum change in {cg and the percent change in pg (see text). 4. The maximum change in concentration and the per cent of that concentration
2. go”,, glucose
I. The derived
Awe-“0’
Am’,.’
Table
8th
in the next
b
564
J. P. WOODKIN~.C. W. CLIFFORD.R. M. Rorz. AND
by the last day in both instars (Fig. 2). Consequently there is only a slight midinstar depression in NPS amount but a net increase in amount during the instar (Table 2). There appears to be only a slight increase in serum NPS concentration during apolysis. The serum trehalose concentration (Table 2) is consistently 84 to 86”,, of the serum total carbohydrate concentration (Table I) except for the last day when trehalose drops to 64”,,. Consequently, the concentration of other sugars remains constant up to the last 1 to 2 days. at which time a large increase occurs that almost corresponds to the decrease in trehalose (Table 2). The other sugars were identified by TLC and quantified as variable but with 90”,, glucose by phenol~sulfuric analyses of TLC elutants (details to be published elsewhere). The strum chloride concentration peaks at midinstar in both instars (Fig. 2). The concentration on the last day is slightly less than the first day. and a slight increase occurs during apolysis from the 7th to 8th instar. With a decrease in chloride concentration along with a relatively constant blood volume in the last half of the instar. the amount of chloride decreases; however. there is a net increase in amount when the entire instar is considered (Table 3). The change in serum sodium concentration parallels that of chlorides (Fig. 2). The change in potassium concentration parallels that of NPS. though the magnitude is much less. The blood volume (in &cricket) increases at a constant rate up to just past mid-instar in both instars. then it levels off and remains fairly constant (Fig. 1). The I’(,-blood volume (111blood/wet body weight) decreases rapidly in the first 1 to 2 days. then it remains constant for the remainder of the instar (Table I). The blood osmolality is constant in both instars up until the last 2 days when an average drop of 20 mOsM occurs (Table 2). DISCUSSION
The patterns of concentration change in blood constituents differ from one another, and none parallel exactly the change in blood volume during the 7th and 8th larval instars. This means regulation of all blood constituents during each larval instar and implies a feedback mechanism based on changing concentrations. One important consideration is how the patterns of blood constituent concentration are related to feeding and growth during the molting cycle. A constant high level of food-water consumption occuring in the first half of the last two larval instars of Acher~ leads to a 120 to 140”; increase in dry body weight in both instars (WOODRING, et al., 1976). The food intake rapidly decreases after midinstar. falling to zero in the last 1 to 2 days. As a result, the growth curve plateaus near midinstar with a slight weight loss occurring in the last 1 to 2 days of the instar. Water consumption however continues,
R. R. MERUER
though at a reduced rate, when feeding ceases. This pattern of feeding and growth is correlated to blood constituents in the following discussions.
The “,,-blood volume range and its correlation to the growth curve in the cricket is in agreement with that reported for related insects (LEE, 1961; HILL and GOLDSWORTHY. 1968 : LOUGHTON and Tout. 1969; BERNEYSand CHAPMAN, 1974). The cricket “,-blood volume quickly drops to a constant volume by day 2 (Table I), but the I’;,-total body water requires 2 to 3 days to drop from the first day high of 76q, to a stable 68 to 70”, (WOODRINC;,er al., 1976). The amount of total body water increases by 68Y0, but the blood volume increases only by 45”,, during the 8th instar. This indicates that cell volume is increasing more rapidly than blood volume, which was also suggested to be the case in Periplaneto by VERRETT and MILLS (1976).
Body protein concentration remains constant at lY,, wet body weight throughout the last two larval instars, however, the quantity of body protein doubles by mid-instar (WOODRINGet al.. 1976). Both the concentration and the amount of blood protein approximately double by mid-instar (Table l), which indicates a strong correlation of the amount of blood and body protein to the feeding rate. The blood and body protein amounts rapidly increase during active feeding. then both level off as cricket feeding decreases. HILL and GOLDSWORTHY(1968) also concluded that the blood protein level more likely reflects the nutritional state rather than the physiological processes occurring in larval growth of locusts. Both TOBE and LOUGHTON(1969b, 1970) and HILL and GOLDSWORTHY(1968) noted a sharp decrease in blood protein concentration during apolysis of locusts. which we also find in crickets (Fig. 1). The latter authors also noted the same slight decrease in blood protein concentration on the day prior to ecdysis that we find (Table 1). TOBE and LOUGHTON (1969a) showed that labelled locust blood proteins are extensively utilized for cuticle formation during apolysis. We have indirectly confirmed this by noting that the amount of body protein does not change during apolysis. but that the amount of blood protein decreases. The clottable protein concentration is obtained by subtracting whole blood concentration from serum concentration for any age larva (Table 1). The clottable protein concentration is essentially constant (2.0”, k 0.1) throughout the 8th instar: therefore it is probably the soluble proteins that are used for the new cuticle. We suggest that the blood protein concentration is maintained at high levels. even after feeding ceases, to act as a source of new cuticle proteins. Further, that it is the nonclottable proteins that are used in order that a constant level
Age variation in cricket blood of the protective clottable proteins remain in the blood. Our determined range of serum NPS concentration (Table 2) is the same as reported by WANG and PATTON (1969a) for unaged larvae, but is somewhat less than the 600 to 900 mg/lOLl~1 found in larval locusts by HILL and GOLDSWORTHY(1968); however, the pattern of change in serum NPS in locusts was identical to what we find for crickets. Unlike blood proteins, which cannot come directly from the diet. blood amino acids may come from hydrolysis of proteins in the gut as well as from internal tissues such as the fat body. With maximal feeding in the first half of the instar, amino acid uptake from the gut must be maximal. However, in this interval serum NPS concentration declines while blood protein concentration rapidly increases. One may speculate that the demand for amino acids in the first half of an instar exceeds the dietary supply, which results in a 50 to 60”,, concentration decrease (Table 2). As growth and blood protein concentration level off just after misinstar. the concentration of blood NPS increases in spite of a declining ingestion rate because the demands of growth have ceased. The work of TOBE and LOUGHTON (1969a) confirms these assumptions. They found that C3H]-leucine injected into 1 to 8 day old larval locusts was most rapidly taken up into all tissues during the first half of the instar, and that the uptake of labelled leucine declined to a low level for the remainder of the instar. Thus, in larval crickets the blood concentration of NPS probably reflects a balance between the feeding pattern and the pattern of physiological demand within the instar.
565
and in crickets the blood lipid concentration is halved during apolysis; however, only 0.15 mg of blood lipid is removed (Table 1). which is relatively little compared to the 6 mg of the total body lipid (WXX~RING et al. 1976) lost during apolysis. Therefore. blood is not a storage site for lipids, and energy expenditure during apolysis is at the expense of body lipids. Serum carbohydrates
Our average glucose and trehalose concentrations differ from that reported by NOWOSIELSKIand PATTON (1965) for larval Achetu. which could be due to different rearing techniques and diet. DAHLMAN (1975) showed greatly different trehalose levels in hilatlduca caterpillars reared on different diets. The serum carbohydrate concentration in crickets is much higher than in Periplutwta. but much lower than in Locustu (MA~EWS et ul., 1976; HILL and GOLDS~ORTEIY, 1968). It is interesting that the serum carbohydrate concentration is higher in the 7th than the 8th instar, which DAHLMAN (1975) found also to be the case in the last 2 larval instars in Manduca. The significance of this is not clear at this time. There is a 4-fold increase in body carbohydrate content from day 1 up to mid-instar in the 7th and 8th larval instars followed by a halving of the amount of body carbohydrate during the last half of an instar (WOODRING et al., 1976). They concluded that stored carbohydrates deposited in the first half of the instar are mobilized for energy during last half of the instar when feeding declines to zero. In cricket larvae the serum total carbohydrate concentration is essentially constant after the first 2 days of an instar. a further indication that stored carbohydrates art: reduced Our determined blood lipid concentration correrather than the blood carbohydrates. This idea is supsponds to that reported by WANG and PATTON ported by the findings of HILL and ~OLI~SWORMY (1969b) for Achefa. but there is no previous work on (1970), who found that in locust larvae the total blood age variation during the developmental cycle to which carbohydrate concentration remains constant during comparisons can be made. However, a doubling of the first several days of starvation, and they conblood lipid concentration occurs in the first 10 days cluded that fat body glycogen is mobilized. of adult life in Acheta and Schistocerca (NOWOSIELSKI The elevated serum carbohydrate concentration in the first 2 days of both instars (Table 1) is due primarand PATTON, 1965; HILL and IZATT, 1974), and we find a similar doubling occurs in each of the last 2 ily to increased trehalose (Table 2). because the perlarval instars of crickets (Table 1). Total body lipid centage of trehalose of the serum total carbohydrate concentration is constant at 84 to 86:; throughout content essentially triples by mid-instar in both instars, remaining constant until the end of the instar the instar except for the last day. The elevated treha(WOOIXING et al.,1976), but the blood lipid concenlose concentration at the beginning of an instar is probably related to heightened energy demands due tration increases steadily throughout both instars. It to rapid growth, but details of the relationship are is interesting that blood lipid concentration continues to increase evenly throughout the instar (Fig. 1) even not clear. On the last day of the 8th instar the drop though food consumption essentially stops 2 to 3 days in both concentration and amount of serum trehalose corresponds to the increase in other sugars (Table before apolysis. A similar phenomenon was noted by 2). The specific trehalose molecules lost from the JUTSUM et al. (1975) who found a blood lipid concenblood are not necessarily hydrolized to the specific tration increase in adult locusts that were purposely ‘other sugars’ that appear. It is possible that glucose starved for several days. Corollary hormonal studies could increase at the beginning of apolysis (7th-8th led them to conclude that the mechanism of blood lipid increase was metabolic rather than hormonal. day of the 8th instar) because of cuticle digestion. and trehalose be taken up by other tissues. It is A decrease in blood lipid concentration during apolysis 1s well known (FLORKIN and JEUNIAUX. 1974). apparent that in crickets significant and as yet un-
566
J. P. WOOIIRI~\~~;.C. W. CLWI+KD. R. M. Ror:. ANII R. R. MERUER
known deviations in carbohydrate with the onset of apolysis.
metabolism
occur
7/t<, PIq%<~/oduction in L~cfr,\rlr. J. 111\ccr P/t~+ol. 17. 717 732. locusts. Because of the quick response of blood ion C’i I+~~OKI> C‘. W.. ROI R M.. and WOOlfKIM; .I. P. (1977) concentration to starvation and dehydration. it is Rearing methods for obtaining house crickets. ilcl~crct essential that excess free water and fresh food is avail&~/rrc\ri~~lr,\.of known age. \cx. and instar. 0111. UI(. Sot. able throughout any study of age variations in blood 111170. 69 74. ions. Actually, among cockroaches (WALL.. 1970: D‘\ttt.vtA\ I>. L. I 1975) Trchalosc and glucose levels in EDNEY, 1968) and crickets (unpublished) there is a hcmo)!:mph of diet-reorcd, tobacco leaf-reared. and parasttized lobacco hornworm larvae. Corn/~. Bi~chrnt. distinct limit of blood ion variation in response tco I’/lL\id 5oA. I65 167. starvation or dehydration. and beyond these limits EIN ‘r E. B. (196X) The etfect of water loss on the haemothere are effective homeostatic mechanisms that prelymph of 4rrrtitqrr and Prriplunrrrr mwricmtl. Camp. vent further concentration changes. There is an upper Bidwfr. PItr.$iol. 25, 149- 15X. and lower limit of serum ion variation in crickets. FI OKICIKM. and JFI:NIAI’\ C. (1974) Hemolymph: compowhich can be called a lability range. and the cycle \ttion. In Tltc, Phv.Go/oq~ of fmwtu (Ed. Rockstein M.) of serum ion concentration during each instar oscil5. 255 307. Academic Press. New Yorh. lates within these limits (Fig. 2). ~OI.DS\vOKTHY G. J. (1970) Hyperglycemic factors in The chloride concentration shows a mid-instar I>r. Ph~wol. 36, 61 70. first day of the 8th instar. we believe reflects the rapid HILI L. and I/ATT M. E. G. (1974) The relationships hctvvecn corpora allata and fat body and haemolymph initiation of feeding in the first day of an instar. lipids in the adult female desert locust. J. Insrct Physiol. Serum sodium concentration parallels chloride con20. 2143 2156. centration. Both potassium and NPS concentrations Ho\ I I G. (19%) Sodium kind potasstum changes occurring show an opposite pattern of change to sodium and III the h;rcmolymph of insects :tt the time of moulting potassium in the 8th instar (Fig. 7). and accounts. and thcit- phystological conscqucnces. ,V~rttrr~~.Lo~ti. 178. at least partially, for blood osmolality stability IT.JCl 1217. through most of the instar. In the first half of the Jr IM \I A. R.. AC;AR\VAL H. C.. and GOLDSWORTHY G. J. instar the decrease in NPS. sugars. and potassium (1975) Starvation and haemolymph lipids in Loc~rstc~ almost offset the increase in NaCl, but in the last migrtrtorio. .4cdtr 4, 47- 56. LIX R. M. t 1961) The vlariation of blood volume with age 2 days there occurs a drop in blood osmolality that tn the desert locust. Scllistoccrctr yrquriu. J. lnsrct Plrycannot be completely explained by counterbalancing \ird. 6, 36-~51. blood constituents LO~K~H~OP\B. G. and Tout S. S. (1969) Blood volume in .~c~,~owln/yr,t~~,~~~,~~ We thank Dr. T. DINTS for helpful the African migratory locust. Curt. J. Zoo/. 47, criticism and advjice throughout this study; Drs. M. S. Ii33 1336. BLUM and R. P. PATTON for their critical reviews of the MAI-THFU’S J. R., DOWN~K R. G. H., and MORRISON P. manuscript; and Miss P. DUROUSS~AL~for superviston of E. (19761 Estimation of glucose in the haemolymph of the production of larval crickets. the American cockroach. Puriplunutcl unwriurnc~. Camp. Biochrw. Physiol. 53A, 165~~16k. REFERENCES k’fONTGOMFKY R. (1957) Determination of glycogen. ,trcit. AC~KELI.I. P. S. and LUNIX)I:ISI A. M. ( 1973) Physiological Hiodwm Bioph~~.. 67, 378 3X6. and biochemical changes during insect development. In MOKIII I M. (1970) Evidence for the existence of diuretic
Age variation and antidiuretic hormones in locusts. J. Endow. 46, 119-120. NOWOSIELSICI J. W. and PATTON R. L. (1965) Variation in the haemolymph protein, amino acid, and lipid levels in adult house crickets. .4chetu dornesticus. of different ages. J. Insrct Pllysiol. 11, 263- 370. PK.HON Y. (1970) Ionic content of haemolymph in the cockroach. Pzriplunetu unwritma. J. exp. Biol. 53, 195 -209. ROSEN H. (1957) A modified ninhydrin calorimetric analysis for amino acids. Arcil. Bioc~hern. Biophys. 67, 1% 1S SROKA P. and BARTH R. H. (1976) Hormonal control 01 diglyceride metabolism during vitellogenesis in the cockroach. Eddulxwrs posticus. J. Insrcr Ph~~siol. 22, 951-954. TOI%ES. S. and L~LJGHTONB. G. (1969a) An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust. J. fmwr Ph~siol. 15, 1331 1346. Tonb S. S. and LO~(;HTON B. G. (1969b) An mvestlgatlon of hacmolymph protein economy during the fifth instal of Locxsftr miqrclloriu. J. /II.SM~ Physiol. 15. I659- 1673. TOIIE S. S. and LOUCIHTON B. G. (1970) Haemolymph protein metabolism during the fifth instar of Locitstrr. Crrrz. J. Zoo/. 48. 297-304. VbKar I r J. M. and MILLS R. R. (1976) Water balance dur-
in cricket
blood
567
ing vitellogenesis by the American cockroach: elfect of frontal ganglionectomy. J. Insect Physiol. 22, 251-257. WALKER P. R. and BAILEY E. (1971) Effect of allatectomy on fat body lipid metabolism of the adult male desert locust during adult development. J. Insect Ph!,sio/. 17, 813-821. WALL B. J. (1970) Effects of dehydration and rehydration on Periplmwtd arnericarw. J. Insect Physiol. 16. 1027- 1042. WANC; C. M. and PATTON R. L. (1969a) Nitrogenous compounds in the haemolymph of the cricket. /Iclretcr dorrzesficus. J. Imcct Physsioi. 15. 543-548. WANC; C. M. and PATTON R. L. (1969b) Lipids in the hatmolymph of the cricket. .4&m dowsticus, .J. III\CYI Ph~~iol. 15. X51-X60. WANG C. M. and PATTON R. L. (1969~) Carbohydrates in the haemolymph of the cricket. .¶chrtu do!tlrsticus. J. IKWI Ph~siol. 15. 861-X66. WOODRING J. P.. Rot R. M.. and CLIFFORD C. W. (1976) Relation of feeding. growth and metabolism to age m the larval. female house cricket. J. I~~.srct PhyGol 23. In press. Wy~rr G. R. and KALF G. P. (1957) The chemistry of insect haemolymph-II. Trehalose and other carbohydrates. J. pm Physiol. 40. X33-847.
1971.Vol.
13, pp. 559 to 561. Peryamon
Press. PrinteJ
in Greut
Britairi
RELATION OF BLOOD COMPOSITION TO AGE IN THE LARVAL FEMALE HOUSE CRICKET, ACHETA DOMESTICUS J. P. WOODRING. C. W. CLIFFORD, R. M. ROE, and R. R. MERCIER Department of Zoology and Physiology. Louisiana State University, Baton Rouge, LA 70803. U.S.A. (Receiver/ 19
Norember
1976: recked
8 December
1976)
Abstract-The blood volume. osmolality. and sugar concentration increase: the blood protein and lipid concentration decrease; and the blood ions and NPS concentration do not change during apolysis from the 7th to 8th instar of larval Acheta domesticus. The patterns of change in all blood constituents are basically identical in both of the last two larval instars. Blood volume increase is directly related to growth, but not directly to total body water. Blood protein concentration increases in exact relation to growth, resulting in a doubling of concentration by midinstar and a small decrease over the last 2 to 3 days. The concentration of clottable protein remains constant; the soluble protein concentration increase probably providing cuticular proteins prior to and following ecdysis. Blood lipid concentration increases steadily throughout both instars, even during the non-feeding phase. Total blood carbohydrate concentration decreases greatly in the first 2 days of an instar. then remains relatively constant for the remainder of the instar. Blood trehalose concentration is 85”,, of the total carbohydrate concenincrease in ‘other sugars‘ with tration throughout the instar, but drops to 65”,, with a corresponding the onset of apolysis on the last day. Growth demands probably exceed dietary supply of amino acids resulting in a 50”,, decrease in NPS concentration in the first half of the instar. then NPS concentration increases in spite of a declining feeding rate because growth demands cease. Within the normal blood ion lability range. the principle blood ions, sodium and chloride. vary in an inverse proportion to NPS and sugar concentration at least in the first two-thirds of the instar. Thus blood osmolality is constant except for an unexplained drop in the last 1 days of an instar.
INTRODUCTION
AGRELL and LUNDQUIST(1973) point out that analyses of vertebrate blood are of practical value because the physiological background for the measured blood variations has been worked out. However, the vast accumulated data on insect blood is of little value because this information has not been related to the biology or physiology of the insect. More recent work shows that temperature, starvation, exercise, anesthesia. etc.. profoundly affect the concentration of insect blood constituents (WALL, 1970; DAHLMAN, 1975; JUTSUM et al., 1975; MATTHEWSet d.. 1976), but relatively few (HILL and GOLDSWORTHY, 1968) have related blood variations to growth, feeding, or metabolism in the cycle of growth and apolysis-ecdysis during development. Our purpose then is to relate blood variations during the developmental cycle to the biology and physiology. and we used the house cricket because of its predictable life cycle and ease of culturing. In addition, the knowledge of these normat blood variations should provide valuable clues regarding the timing of hormonal and nervous regulation of insect biochemical and physiological processes. Both the nervous system (VERRETTand MILLS, 1976; CLARK and ANSTEE, 1971) and the endocrine system (MORDUE. 1970: WALKER and BAILEY, 1971: 559
GOLLXWORTHY,1970: HILL and 17~7‘~. 1974; SROKA and BARTH. 1976) directly affect the concentration of most blood constituents, and the normal patterns of change in the blood during the developmental cycle should reflect the release and interactions of the neuroendocrine system. MATERIALS
AND
METHODS
Our culture procedures (CLIFFORD et ~1.. 1977) for domesticus permitted selection of 7th (next to last) and 8th (last) larval instars of known age kO.5 days. Larvae of known age were further selected to within k15 mg of an average weight, which was based on a growth curve calculated from over 5000 individually weighed crickets over a 3 yr period. The artificial, dry diet contained 20”” protein. 4.8”” lipid. 48”,, carbohydrate, 124,, water, and 6”,, ash. Water for the last two instars was provided in slot-edged plastic vials inverted into small, open pans to insure ud lib drinking. This is critical because we found that crickets did not drink to repletion from paper or cloth watering wicks, The last two instars were kept in groups of 10 to 12 in 4 liter cylindrical. cardboard cartons. Crickets were anesthetized individually by a IO to 2Osec exposure to COz, immediately bled, then disA&eta
560
J. P. W(X)DRING.C. W. CLIFFORD, R. M. Ror!, AND R. R. MERCIES
carded. Whole blood (haemolymph) was collected by touching a capillary tube to the clipped end of the cerci and applying gentle finger pressure on the body; this procedure produced 5 to 12jtl from 7th instar larvae and 8 to 20~1 from 8th instar larvae. Clot formation is relatively slow. about 5 min, so that whole blood can be dispensed and quickly diluted. Whole blood was used for the total solids, proteins. lipid. and osmolality determinations. Plasma. or cell free blood. was not used. Blood serum is the clear fluid obtained by centtifugation of whole blood. which we allowed to tirmly clot for ,lO to 15 min in the capillaries. Serum was used for the blood volume. ion. and sugar determinations. Deproteinized serum. for the NPS determination. was obtained by adding 3 x the blood volume of 5”,, TCA in the capillary tube (but keeping the two separate). and then centrifuging the clot through the TCA. One larva provided sufficient blood for any specific analysis, and pooled blood was used only for the gravimetric lipid analysis. Microscopic examination of the collected blood revealed only haemocytes and no nonblood cell types or tissues. Although virtually all the blood cells ruptured during the clotting process. the contribution of cellular contents to total blood concentrations is small. With a haemocylometer we determined the average cell count to be 2.5 x 10’ blood cells//l1 blood. The average cell radius is 4klrn, giving a 10nl cell volumej$ of blood or a cell volume less than I”,, of the whole blood. Thus. ruptured haemocytea do not make a significant contribution to blood protein. lipid. or carbohydrate concentration. NaCl is normally less concentrated. free amino acids are probably slightly more concentrated. and K is normally 10 times more concentrated in cells than in plasma. Therefore. only in the analyses of serum K could the ruptured blood cells cause an error approaching lo”,,. A similar conclusion was reached by BRAI)Y (1967) for several other insects. Blood proteins were determined by the Biuret Method (CORNAL.Lrt trl., 1949) scaled down to a Z-ml reaction volume. The blood was rinsed into 1 M NaOH and warmed 15min at 50 ‘C to dissolve the blood proteins. The difference in whole blood and serum protein concentrations gave an estimate of clottable protein concentration. Total blood lipid for the 8th instar was determined gravimetrically using SO/tl of pooled blood per sample. Our modification of the BLIGH and DYER (1959) extraction method assured one step extraction of r 98”,, of total blood lipid if the resultant total lipid was below 4.1 mgjlOOml of the final biphasic chloroform-methanol water mixture. This was confirmed by finding no additional lipids upon re-extraclion. The total blood lipid of the 7th instar was determined using 5 ,tl samples by the Sulfophosphovanillin Method (BARNESand BLACKSTOCK.1973). By analysis of the 8th instar we found the sulfophovanillin method requires a 1.5 x factor to duplicate the gravimetric method in larval crickets. This is an empirical
correction factor and may vary with insect species. Total blood carbohydrates were determined by the phenol~sulfuric acid method (MONTGOMERY. 1957). modified only by purging the acid produced vapors with N2. The quantitation of trehalose was based on its resistance to acid hydrolysis (WYATT and KALF, 1957). Ten minutes at 97 C in 0.1 N H,SO, hydrolyzes all tri-disaccharides except trchalose. Reheating with 6 N NaOH purged v, ith NI (using aquarium tloss plugs in the test tubes) destroys all monosaccharides. A phenol- sulfuric acid analysis of the final solution gives trehalose concentration. and the difierence between serum total carbohydrates and trehalose gives ‘other sugars’. The NL purging was essential for consistent results. Total ninhydrin positive substance (NPS) was determined by a micro-modification of ROSEN’S(1957) method following in-capillary protein precipitation with TCA. The leucine standard is only stable when refrigerated and stored in a dark bottle. Cricket blood contains up to 4mM NH, and 5 mM urea (WANG and PATTON. 1969a). which taken together at these concentrations could cause a maximum error of 8”,, (ROSEN. 1957). Over 90”,, of our NPS values are probably due to free amino acids, but because we did not specifically analyze for amino acids. we use the term NPS. Blood volume was determined by the “C-inulin dilution method with a scintillation counter. Blood osmolality was determined with a Wescor Vapor Pressure Osmometer using 5 to 10~~1 of blood collected directly from the clipped cerci. Serum chlorides were determined with a Buchler-Cotlove automatic titrator. Strum sodium and potassium were determined with a flame photometer. RESULTS An overview of the results is given in Figs. I and ’ in which the change in concentration of blood con-. stituents through the last 2 larval instars is clearly illustrated. Note that the pattern and magnitude of change are similar in both instars for all constituents measured. The determined average concentration +SE, of each constituent at each age is tabulated in Tables I and 2. The amounts. given as derived pg or mg, are calculated on the basis of average blood volume for each age. For example, the blood protein concentration of a 1 day old 8th instar larva is 3.3 mg/lOO~l of blood (Table 1) and the average blood volume of a I day old larva is 39 ~1 (Table 11, therefore the amount of blood protein per 1 day old cricket is 3.3 x 391100 = 1.3 mg. The change in amount (Amg-“,) and the change in concentration (A cone-“,,) for each blood constituent is expressed as the maximum difference that occurs in an instar. and these values are given in the last two lines of Tables I and 2. The per cent maximum change of concentration and amount is determined as [(high value-low value)/low value] x 100. For example. the maximum
Age
variation
in cricket
blood
561
60 50 g
r
Age
40
t
30
:
20
0 s
(days)
Fig. 1. The relation of blood protein (H). lipid (A A), and serum carbohydrate (04) concentration and blood volume (V---Y) to age in larval A. dornesticus. Open symbols denote the 7th instar and closed symbols denote the 8th instar.
difference in amount of blood protein for the 8th instar is 4.2 - 1.3 = 2.9 mg (Table 1) and the per cent change is (2.9/1.3) 100 = 223”)“. The per cent change in concentration is probably more important than the per cent change in amount because physiological systems can only respond to concentration change. The maximum changes in concentration and amount are statistically significant (P < 0.05) for all constituents.
5 I =
15,
a E
IO-
E 0”
5. o*
\
U---U
/
“B-U
1
Treh n
WK
012345678
.
.
.
.
.
Age (days) Fig. 2. The relation of serum chloride (M). NPS (A--A). trehalose (m). sodium (a), and potassium (V) to age in larval Achera domesticus. Open symbols denote the 7th instar and closed symbols denote the 8th instar.
Before proceeding to each blood constituent, two points should be made. If the concentration remained constant throughout an instar, then the least amount would be on the first day and the greatest amount on the last day and the quantity would parallel the blood volume. This does not occur for any blood constituents examined. If the concentration decreased during an instar solely due to dilution, resulting from the increasing blood volume, then the amounts would remain constant. This likewise does not occur for any blood constituent examined. Both the concentration (Fig. 1) and the total amount (Table 1) of blood protein increase rapidly up to just past mid-instar, basically doubling. then it plateaus and decreases slightly on the last I to 2 days. The change in serum protein parallels that of blood protein (Table 1). A distinct drop in blood protein concentration occurs during apolysis from the 7th to 8th instar. The concentration of blood lipid doubles and the amount triples in each instar (Table 1). The rate of lipid increase is uniform throughout each instar with a distinct drop during apolysis from the 7th to 8th instar (Fig. 1). The general trend of blood carbohydrate concentration is a rapid decrease in the first 2 days of both instars followed by a slight decrease over the remainder of the instar (Table 1). The maximum change in amount and concentration of blood carbohydrate is small compared to that of protein and lipid. As a generalization, blood protein and lipid concentrations greatly increase and the total carbohydrate concentration decreases through each instar. but during apolysis significant amounts of protein and lipid are removed from the blood and carbohydrates are added (Fig. 1). The serum NPS concentration decreases from day 1 to mid-instar then increases back to first day levels
.1. P
Woo~xuh~;.
C.
W.
C‘LIFtorw.
R.
I
_
_ -
;
,;
‘_
=
‘1
=
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,,
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--
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R. R. MERCEK
2. Relation
osmolality
7th
10 - x.h”,,
?7 - 7.X”
and
8th
Blood osmolnhly mOsM!Kgtn)
of blood
7th
.l 7.pg - 5?“,, 14.x - 54””
I OXpg - h’)“,, 143 i Oy’,,
Swum NPS mM;i I SE, (nl derived /I$
I JY pp - Xh”,, 38 - ??“,,
Y I /,g - 22X”,, 152 - l90”.
change.
to age
. I3 pg . SIV’,, -. Ih , I”,,
8th
chloride
units.
and
49 pp - ?7”,, 30 - 3”
7th
potassium.
for mg!lOO /tl cont.
Xth
Xth
other sugars, sodium. of A. dnuwstic~rs
pg = (mg/lOO~d) x (BVol/lOO~tl)
101 ,cg G’,, 352 - xv..
8th
ninhydrin positive substances (NPS). trehalose. to last (7th) and last (8th) female larval instars
Xlh
serum
/cg - (MW x mM x BVol)/lOOO for mM cont. units. The derived and lo”, variable other carbohydrates (see text). 3. The maximum change in {cg and the percent change in pg (see text). 4. The maximum change in concentration and the per cent of that concentration
2. go”,, glucose
I. The derived
Awe-“0’
Am’,.’
Table
8th
in the next
b
564
J. P. WOODKIN~.C. W. CLIFFORD.R. M. Rorz. AND
by the last day in both instars (Fig. 2). Consequently there is only a slight midinstar depression in NPS amount but a net increase in amount during the instar (Table 2). There appears to be only a slight increase in serum NPS concentration during apolysis. The serum trehalose concentration (Table 2) is consistently 84 to 86”,, of the serum total carbohydrate concentration (Table I) except for the last day when trehalose drops to 64”,,. Consequently, the concentration of other sugars remains constant up to the last 1 to 2 days. at which time a large increase occurs that almost corresponds to the decrease in trehalose (Table 2). The other sugars were identified by TLC and quantified as variable but with 90”,, glucose by phenol~sulfuric analyses of TLC elutants (details to be published elsewhere). The strum chloride concentration peaks at midinstar in both instars (Fig. 2). The concentration on the last day is slightly less than the first day. and a slight increase occurs during apolysis from the 7th to 8th instar. With a decrease in chloride concentration along with a relatively constant blood volume in the last half of the instar. the amount of chloride decreases; however. there is a net increase in amount when the entire instar is considered (Table 3). The change in serum sodium concentration parallels that of chlorides (Fig. 2). The change in potassium concentration parallels that of NPS. though the magnitude is much less. The blood volume (in &cricket) increases at a constant rate up to just past mid-instar in both instars. then it levels off and remains fairly constant (Fig. 1). The I’(,-blood volume (111blood/wet body weight) decreases rapidly in the first 1 to 2 days. then it remains constant for the remainder of the instar (Table I). The blood osmolality is constant in both instars up until the last 2 days when an average drop of 20 mOsM occurs (Table 2). DISCUSSION
The patterns of concentration change in blood constituents differ from one another, and none parallel exactly the change in blood volume during the 7th and 8th larval instars. This means regulation of all blood constituents during each larval instar and implies a feedback mechanism based on changing concentrations. One important consideration is how the patterns of blood constituent concentration are related to feeding and growth during the molting cycle. A constant high level of food-water consumption occuring in the first half of the last two larval instars of Acher~ leads to a 120 to 140”; increase in dry body weight in both instars (WOODRING, et al., 1976). The food intake rapidly decreases after midinstar. falling to zero in the last 1 to 2 days. As a result, the growth curve plateaus near midinstar with a slight weight loss occurring in the last 1 to 2 days of the instar. Water consumption however continues,
R. R. MERUER
though at a reduced rate, when feeding ceases. This pattern of feeding and growth is correlated to blood constituents in the following discussions.
The “,,-blood volume range and its correlation to the growth curve in the cricket is in agreement with that reported for related insects (LEE, 1961; HILL and GOLDSWORTHY. 1968 : LOUGHTON and Tout. 1969; BERNEYSand CHAPMAN, 1974). The cricket “,-blood volume quickly drops to a constant volume by day 2 (Table I), but the I’;,-total body water requires 2 to 3 days to drop from the first day high of 76q, to a stable 68 to 70”, (WOODRINC;,er al., 1976). The amount of total body water increases by 68Y0, but the blood volume increases only by 45”,, during the 8th instar. This indicates that cell volume is increasing more rapidly than blood volume, which was also suggested to be the case in Periplaneto by VERRETT and MILLS (1976).
Body protein concentration remains constant at lY,, wet body weight throughout the last two larval instars, however, the quantity of body protein doubles by mid-instar (WOODRINGet al.. 1976). Both the concentration and the amount of blood protein approximately double by mid-instar (Table l), which indicates a strong correlation of the amount of blood and body protein to the feeding rate. The blood and body protein amounts rapidly increase during active feeding. then both level off as cricket feeding decreases. HILL and GOLDSWORTHY(1968) also concluded that the blood protein level more likely reflects the nutritional state rather than the physiological processes occurring in larval growth of locusts. Both TOBE and LOUGHTON(1969b, 1970) and HILL and GOLDSWORTHY(1968) noted a sharp decrease in blood protein concentration during apolysis of locusts. which we also find in crickets (Fig. 1). The latter authors also noted the same slight decrease in blood protein concentration on the day prior to ecdysis that we find (Table 1). TOBE and LOUGHTON (1969a) showed that labelled locust blood proteins are extensively utilized for cuticle formation during apolysis. We have indirectly confirmed this by noting that the amount of body protein does not change during apolysis. but that the amount of blood protein decreases. The clottable protein concentration is obtained by subtracting whole blood concentration from serum concentration for any age larva (Table 1). The clottable protein concentration is essentially constant (2.0”, k 0.1) throughout the 8th instar: therefore it is probably the soluble proteins that are used for the new cuticle. We suggest that the blood protein concentration is maintained at high levels. even after feeding ceases, to act as a source of new cuticle proteins. Further, that it is the nonclottable proteins that are used in order that a constant level
Age variation in cricket blood of the protective clottable proteins remain in the blood. Our determined range of serum NPS concentration (Table 2) is the same as reported by WANG and PATTON (1969a) for unaged larvae, but is somewhat less than the 600 to 900 mg/lOLl~1 found in larval locusts by HILL and GOLDSWORTHY(1968); however, the pattern of change in serum NPS in locusts was identical to what we find for crickets. Unlike blood proteins, which cannot come directly from the diet. blood amino acids may come from hydrolysis of proteins in the gut as well as from internal tissues such as the fat body. With maximal feeding in the first half of the instar, amino acid uptake from the gut must be maximal. However, in this interval serum NPS concentration declines while blood protein concentration rapidly increases. One may speculate that the demand for amino acids in the first half of an instar exceeds the dietary supply, which results in a 50 to 60”,, concentration decrease (Table 2). As growth and blood protein concentration level off just after misinstar. the concentration of blood NPS increases in spite of a declining ingestion rate because the demands of growth have ceased. The work of TOBE and LOUGHTON (1969a) confirms these assumptions. They found that C3H]-leucine injected into 1 to 8 day old larval locusts was most rapidly taken up into all tissues during the first half of the instar, and that the uptake of labelled leucine declined to a low level for the remainder of the instar. Thus, in larval crickets the blood concentration of NPS probably reflects a balance between the feeding pattern and the pattern of physiological demand within the instar.
565
and in crickets the blood lipid concentration is halved during apolysis; however, only 0.15 mg of blood lipid is removed (Table 1). which is relatively little compared to the 6 mg of the total body lipid (WXX~RING et al. 1976) lost during apolysis. Therefore. blood is not a storage site for lipids, and energy expenditure during apolysis is at the expense of body lipids. Serum carbohydrates
Our average glucose and trehalose concentrations differ from that reported by NOWOSIELSKIand PATTON (1965) for larval Achetu. which could be due to different rearing techniques and diet. DAHLMAN (1975) showed greatly different trehalose levels in hilatlduca caterpillars reared on different diets. The serum carbohydrate concentration in crickets is much higher than in Periplutwta. but much lower than in Locustu (MA~EWS et ul., 1976; HILL and GOLDS~ORTEIY, 1968). It is interesting that the serum carbohydrate concentration is higher in the 7th than the 8th instar, which DAHLMAN (1975) found also to be the case in the last 2 larval instars in Manduca. The significance of this is not clear at this time. There is a 4-fold increase in body carbohydrate content from day 1 up to mid-instar in the 7th and 8th larval instars followed by a halving of the amount of body carbohydrate during the last half of an instar (WOODRING et al., 1976). They concluded that stored carbohydrates deposited in the first half of the instar are mobilized for energy during last half of the instar when feeding declines to zero. In cricket larvae the serum total carbohydrate concentration is essentially constant after the first 2 days of an instar. a further indication that stored carbohydrates art: reduced Our determined blood lipid concentration correrather than the blood carbohydrates. This idea is supsponds to that reported by WANG and PATTON ported by the findings of HILL and ~OLI~SWORMY (1969b) for Achefa. but there is no previous work on (1970), who found that in locust larvae the total blood age variation during the developmental cycle to which carbohydrate concentration remains constant during comparisons can be made. However, a doubling of the first several days of starvation, and they conblood lipid concentration occurs in the first 10 days cluded that fat body glycogen is mobilized. of adult life in Acheta and Schistocerca (NOWOSIELSKI The elevated serum carbohydrate concentration in the first 2 days of both instars (Table 1) is due primarand PATTON, 1965; HILL and IZATT, 1974), and we find a similar doubling occurs in each of the last 2 ily to increased trehalose (Table 2). because the perlarval instars of crickets (Table 1). Total body lipid centage of trehalose of the serum total carbohydrate concentration is constant at 84 to 86:; throughout content essentially triples by mid-instar in both instars, remaining constant until the end of the instar the instar except for the last day. The elevated treha(WOOIXING et al.,1976), but the blood lipid concenlose concentration at the beginning of an instar is probably related to heightened energy demands due tration increases steadily throughout both instars. It to rapid growth, but details of the relationship are is interesting that blood lipid concentration continues to increase evenly throughout the instar (Fig. 1) even not clear. On the last day of the 8th instar the drop though food consumption essentially stops 2 to 3 days in both concentration and amount of serum trehalose corresponds to the increase in other sugars (Table before apolysis. A similar phenomenon was noted by 2). The specific trehalose molecules lost from the JUTSUM et al. (1975) who found a blood lipid concenblood are not necessarily hydrolized to the specific tration increase in adult locusts that were purposely ‘other sugars’ that appear. It is possible that glucose starved for several days. Corollary hormonal studies could increase at the beginning of apolysis (7th-8th led them to conclude that the mechanism of blood lipid increase was metabolic rather than hormonal. day of the 8th instar) because of cuticle digestion. and trehalose be taken up by other tissues. It is A decrease in blood lipid concentration during apolysis 1s well known (FLORKIN and JEUNIAUX. 1974). apparent that in crickets significant and as yet un-
566
J. P. WOOIIRI~\~~;.C. W. CLWI+KD. R. M. Ror:. ANII R. R. MERUER
known deviations in carbohydrate with the onset of apolysis.
metabolism
occur
7/t<, PIq%<~/oduction in L~cfr,\rlr. J. 111\ccr P/t~+ol. 17. 717 732. locusts. Because of the quick response of blood ion C’i I+~~OKI> C‘. W.. ROI R M.. and WOOlfKIM; .I. P. (1977) concentration to starvation and dehydration. it is Rearing methods for obtaining house crickets. ilcl~crct essential that excess free water and fresh food is avail&~/rrc\ri~~lr,\.of known age. \cx. and instar. 0111. UI(. Sot. able throughout any study of age variations in blood 111170. 69 74. ions. Actually, among cockroaches (WALL.. 1970: D‘\ttt.vtA\ I>. L. I 1975) Trchalosc and glucose levels in EDNEY, 1968) and crickets (unpublished) there is a hcmo)!:mph of diet-reorcd, tobacco leaf-reared. and parasttized lobacco hornworm larvae. Corn/~. Bi~chrnt. distinct limit of blood ion variation in response tco I’/lL\id 5oA. I65 167. starvation or dehydration. and beyond these limits EIN ‘r E. B. (196X) The etfect of water loss on the haemothere are effective homeostatic mechanisms that prelymph of 4rrrtitqrr and Prriplunrrrr mwricmtl. Camp. vent further concentration changes. There is an upper Bidwfr. PItr.$iol. 25, 149- 15X. and lower limit of serum ion variation in crickets. FI OKICIKM. and JFI:NIAI’\ C. (1974) Hemolymph: compowhich can be called a lability range. and the cycle \ttion. In Tltc, Phv.Go/oq~ of fmwtu (Ed. Rockstein M.) of serum ion concentration during each instar oscil5. 255 307. Academic Press. New Yorh. lates within these limits (Fig. 2). ~OI.DS\vOKTHY G. J. (1970) Hyperglycemic factors in The chloride concentration shows a mid-instar I>
Age variation and antidiuretic hormones in locusts. J. Endow. 46, 119-120. NOWOSIELSICI J. W. and PATTON R. L. (1965) Variation in the haemolymph protein, amino acid, and lipid levels in adult house crickets. .4chetu dornesticus. of different ages. J. Insrct Pllysiol. 11, 263- 370. PK.HON Y. (1970) Ionic content of haemolymph in the cockroach. Pzriplunetu unwritma. J. exp. Biol. 53, 195 -209. ROSEN H. (1957) A modified ninhydrin calorimetric analysis for amino acids. Arcil. Bioc~hern. Biophys. 67, 1% 1S SROKA P. and BARTH R. H. (1976) Hormonal control 01 diglyceride metabolism during vitellogenesis in the cockroach. Eddulxwrs posticus. J. Insrcr Ph~~siol. 22, 951-954. TOI%ES. S. and L~LJGHTONB. G. (1969a) An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust. J. fmwr Ph~siol. 15, 1331 1346. Tonb S. S. and LO~(;HTON B. G. (1969b) An mvestlgatlon of hacmolymph protein economy during the fifth instal of Locxsftr miqrclloriu. J. /II.SM~ Physiol. 15. I659- 1673. TOIIE S. S. and LOUCIHTON B. G. (1970) Haemolymph protein metabolism during the fifth instar of Locitstrr. Crrrz. J. Zoo/. 48. 297-304. VbKar I r J. M. and MILLS R. R. (1976) Water balance dur-
in cricket
blood
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