L. M. Roth.
Control of Reproduction in Female Cockroaches with Special Reference to Nauphoeta cinera. II. Gestation and Postparturition.
Psyche 71:198-?, 1964.

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CONTROL OF REPRODUCTION IN FEMALE
COCKROACHES WITH SPECIAL REFERENCE TO
NAUPHOE TA CINEREA.
11. GESTATION AND POSTPARTURITION.
BY Louis M. ROTH
Pioneering Research Division
U. S. Army Natick Laboratories, Natick, Massachusetts In Part I (Roth, 1964) of this paper I presented the results of a study on the reproductive behavior during the first preoviposition period of 4 species of cockroaches that incubate their eggs internally. Nauphoeta cinerea (Olivier) was the principal species used, but some experiments were conducted on Lwcophaea maderae (Fabricius), Pycnofcelus surinamensis (Linnaeus), and Diploptera punctata (Eschsch~oltz) ,
In both N. cinerea and L. maderae receptivity can be correlated with the beginning of yolk deposition in the Gcytes. Yolk deposition in cockroaches is dependent upon corpus allatum hormone (Scharrer, 1946; Engelmann, 1957a, 1959; Roth and Stay, 1961, 1962b). Since allatectomized females mate (Roth and Earth, 1964), recep- tivity is determined by some event, presumably in the brain, which occurs at about the same time as onset of activity of the corpora dlata; it was suggested that the neuroseaetory system is involved in acceptance of the male by the female. Females of N. cinerea become sexually receptive and mate at an average age of 4 days; females that are starved from emergence mate at the same age as fed individuals. Females of L. maderae also undergo a precopulatory period before they become receptive, and mate at an average age of g days. In this species more than 50% of the females that are starved from emergence do not mate (Roth, 1964). Thus, in virgin females starvation affects receptivity in L. maderae but not in N. cinerea (Roth, 1964). Once a virgin N. cinerea mates, she becomes unreceptive and will not mate again during the first preoviposition period. Mechanical stimulation caused by the firm insertion of the spermatop'hore in the bursa copulatrix inhibits the female's sexual feeding response on the male's tergum (Roth, 1962, 1964). Whereas insertion of the sper- matophore renders the female unreceptive it increases the activity of the corpora allata resulting in rapid development of the Gcytes. There appears to be a synergistic action of nutrition and mating



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19641 Roth - Reproduction in Cockroaches I99 stimuli in controlling the rate of o~cyte development because both stimuli are usually required in N. cinerea and L. maderae for activating the corpora allata to their fullest extent so that the o6cytes mature at their maximum rate (Roth, 1964). In N. cinerea, the growth of the oocytes remain inhibited during most of the gestation period (Roth and Stay, 1962b). The female remains sexually unreceptive during gestation and will not mate. She may or may not mate again after parturition (Roth, 1962). Obviously, a pregnant female, or one that has given birth, differs physiologically from a virgin female or one that has mated but not yet oviposited. This report concerns the control of, and factors affecting sexual receptivity and oocyte development during gestation and after parturition.
MATERIALS AND METHODS
Nauphoeta cinerea was the principal species used, but several experiments were also performed on L. maderae, and the bisexual and
strains of Pycnoscelus surinamensis (Roth and Willis, 1961 ) . The insects were reared and maintained on Purina dog chow or laboratory chow. Methods for testing receptivity, determining corpus allatum activity, operating on females, and laboratory conditions are given in Roth (1964). To determine the effect of uterine pressure on oocyte development, artificial oothecae made of glass rods or beads were inserted in the brood sacs after the Gthecae were removed manually. To reduce the size of the ootheca, various numbers of eggs were removed from the females of I?. cinerea as oothecae were being formed (Roth and Hahn, 1964). Other techniques are described in their appropriate sections in the text. Numbers following å are standard errors of mean values. Unless otherwise indicated, parturition refers only to birth of the first litter.
RESULTS AND CONCLUSIONS
Mechanism of inhibition of the ob'cytes during gestation The growth of the Gcytes during gestation in N. cinerea is shown in figure I. Gestation may last 35-50 days. After 35 days of pregnancy, the oocytes in different individuals may vary considerably in length. Many females may have oocytes that contain large amounts of yolk prior to or at the time of parturitition. When the oothecae are removed from pregnant females, the o6cytes mature prematurely, indicating that an inhibitory influence



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[December
GESTATION (DAYS)
Fig. 1.
Oocyte development during pregnancy in N. cinerea. All points from 0 - 34 days are mean values of 5 - 9 individuals (N = 197) ; standard errors are not indicated but were k 0.04 mm. or less for all of these measurements. Each point beyond 35 days of pregnancy represents the oocytes of one female. Open circles
females were still carrying otithecae
when dissected. Solid circles females dissected at the time of parturition or within 24 hr. after giving birth.
The horizontal line is drawn at 1 mm.
which is about the minimum size of the oocytes that contain yolk. on the corpora allata has been removed. The older the uterine eggs when removed, the less time is required to ovulate aga-'n (fig. 2). Roth and Stay (1959, 1961, 1962a, b) concluded that mechanical stimuli resulting from the ootheca in the uterus inhib't the corpora allata during pregnancy. Engelmann ( I 964) believes that in L. maderue the corpora allata are inhibited by a substance



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19 641 Roth - Reproduction in Cockroaches 201 DAYS AFTER REMOVING OOTHECA FROM UTERUS
Fig. 2.
Relationship between the age of the ootheca at the time it was removed from the uterus of N. cinerea and the time required to form a new egg case. Numbers in circles = number of insects used for each curve. Numbers in parentheses
= number of days females were pregnant when their oothecae were removed. AP = after parturition; 36-49 represents the range (days) of gestation in these females. Numbers under paren- theses mean number of days ? standard error, to form a new ootheca. The per cent ovipositing is expressed as accumulative data. released by the egg case or brood sac, acting on neurones in the nerve cord and brain.
To determine if a chemical agent or mechanical stimulation prevents the growth of oocytes in N. cinerea during pregnancy, glass tubes were inserted in the uteri of females after their oothecae were removed at 3 different periods of gestation. The results (figs. 3,4) show that with glass oothecae, the oocytes did not develop as rapidly as they did in females in which no tubes were inserted in the uteri, indicating that the corpora allata were inhibited by the artificial egg cases. When oothecae were removed about 24 hours after oviposition, females oviposited again en an average of 33 days; when oothecae were removed 10 - 11 days after oviposition, the average time taken to oviposit again was 23 days
(fig. 2). In both groups of females




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Psyche
[December
DAYS AFTER OVIPOSITION
Fig. 3. Effect of distention of the uterus on inhibition of the corpora allata during early gestation in N. cinlerea, Solid circles = Carrying oothecae (controls). Five to 18 females per point (N = 276). Open squares = oothe- cae removed <24 hr. after ovipositing; no glass tube inserted in uteri. Five to 16 females per point (N 65). Solid squares Oothecae removed <24 hr. after ovipositing, glass tube (3 - 3.5 mm. X 10 - 11 mm.) inserted in uteri. Four to 10 females per point (N = 99). Open triangles = Oothecae removed 10 - 11 days after ovipositing; no glass tube inserted in uterus. Three to 12 females per point (N 65). Solid triangles = Oothecae removed 10 - 11 days after ovipositing; glass tube (3 - 3.5 mm. X 10 - 11 mm.) inserted in uteri. Four to 10 females per point (N 42). Vertical bars are one standard error but only one-half the error is indicated where they overlapped; no bars indicate standard errors of å 0.02 mm. or less. A single curve is drawn through open triangles and open squares (see text for explanation).
the average time to oviposit again after the initial oviposition was about 33 days and for this reason in figure 3 (A and U), a single curve is drawn through the points for females whose oot'hecae were removed about 24 hours, and 10 - I I days after oviposition. Eventu- ally, inhibition of the corpora allata ceased in females with glass tubes in the uteri and the oGcytes developed. This also occurs in normal pregnant females in late gestation (fig. I). Inhibition may



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19 641 Roth -- Reproduction in Cockroaches DAYS AFTER OVIPOSITION
Fig. 4.
Effect of distention of the uterus on inhibition of the corpora ,q1lnta during late gestation in N. cinerea. Solid circles = Carrying oothecae (controls).
Ten to 18 females (N = 143) used for each point. Open circles
Oothecae removed 30 - 31 days after oviposition; no tube inserted in uteri, Ten females (N = 100) used for each point. The average time required to oviposit again, when the oothecae were removed at 31 - 32 days of pregnancy, was 12 days
(fig. 2) or 43 - 4-4 days after the initial ovi- posit'on. Solid squares
Oothecae removed from females 30 - 31 days after oviposition and glass tubes (4.1 - 4.3 mm. x 10 - 11 mm.) inserted in the
uteri.
Five to 16 females (N = 92) used for each point. Vertical bars are one standard error but only one-half the error is indicated where they overlapped; no bars indicate standard errors of k 0.02 mm. or less. cease earlier in some females with glass inserts &an in females carry- ing oothecae (fig. 3).
The effect of different degrees of uterine stretching, using glass rods, and glass beads of 3 different diameters, was investigated in



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204 Psyche [December
2 strains of P. surinamensis. The artificial oothecae were inserted into the uteri of females after their oothecae were remloved <24 hr. after ovipositing, The results are shown in figure 5 and table I. The Gcytes of some females with bead inserts developed as rapidly as in females without beads. In the bisexual strain, the largest bead, and consequently the greatest uterine streching, was more effective than the two smaller beads in inhibiting the corpora allata. In the parthe- nogenetic strain, 4-0 mm. beads were about as effective as 4.5 mm. beads (table I). The bead experiments were conducted for a maxi- mum of 56 days in the parthenogenetic strain and for 37 days in the bisexual strain. In the glass rod experiments, females were sacrificed from 27 - 100 days after the rods were inserted in the uteri. Inhi- bition was virtually complete in all females (table I), their oocytes ranging in size from 0.58 - 0.88 mm. long (x = 0.74 & 0.01 mm.). Of 68 bisexual strain females that had rods inserted in their uteri, only 3 females had well-developed oocytes when examined on the 17th) 25th, and 36th days. The other 65 females had oocytes that were virtually completely undeveloped when examined 24 - 71 days after the rods were inserted; their okytes ranged from 0.54 - 0.95 mm. and averaged 0.7 I & 0.01 mm. in length. It was of interest to determine if reduction in size of the 0,otheca would influence the size of the ovarian 06cytes at parturition. There are an average of 33 eggs in the ootheca of N. cinerea (Willis et al., 1958). The results of reducing the number of eggs in the ootheca are shtown in figure 6. The oocytes of females that had carried small oothecae were not larger than the oocytes of the control females. Fig. 5.
Effect of distention of the uterus with glass beads on inhibition of the corpora allata during gestation in two strains of P. surinamensis. Solid circles = Carrying oothecae (controls). Each point represents one female or is an average value of 2 - 5 females. Bisexual - N = 107; arth he no genetic - N
75. Open circles = Oothecae removed <24 hr. after ovipositing; no glass beads inserted in uteri. Bisexual - 9 - 16 females per point (N 189) ; parthenogenetic - 4 - 14 females per point (N = 96). The 3 remaining symbols were females whose oothecae were removed <24 hr. after oviposition and glass beads of the following approximate diameters were inserted in the uteri; open squares = 3.5 mm. Bisexual - 7 - 11 females per point
(N = 86) ; parthenogenetic- 5 - 8 females per point (N 36).
Open triangles = 4.0 mm.
Bisexual - 8 - 15 females per point (N
65) ;
parthenogenetic- 3 - 10 females Der point (N = 48) ; solid squares = 4.5 mm. Bisexual - 10 - 13 females per point (N = 44) ; parthenogenetic - 6 - 9 females per point (N = 49). Curves are drawn through solid and open circles only, For the points indicating females with bead inserts, vertical lines are one standard error of mean values; no vertical lines indicate standard errors of 2 0.02 mm. or less.



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19 641
Roth - Rejtv-oduction in Cockroaches
I 4
Bisexual
1
Parthenogenetic
DAYS AFTER OVIPOSITION
If small oothecae, and, consequently, less stretching of the uterus (principally lengthwise) , were less effective than normal-sized egg cases in inhibiting the corpora allata, one would expect the oikytes to be largrr at parturition. Since the small oothecae are considerably reduced in length, the results suggest that in N. cinerea: I) trans- verse I-ather than longitudinal stretching of the uterus, and 2) the gradual increase in size of the uterine eggs, even in o~thecae with



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206 Psyche [December
NUMBER OF EGGS IN OOTHECA
Fig. 6. Effect of reducing the number of eggs in the ootheca on the size of 06cytes at parturition in N. cinerea. Open circles = number of eggs in 06thecae reduced manually (N = 66). Solid circles = number of eggs in oothecae not reduced manually
(controls; N = 84). Each circle represents one female. Arrows indicate the average number of eggs in the oothecae for the 2 groups of females.
Numbers in brackets are the mean lengths ?c standard errors of the oocytes for both groups. Oocytes were measured <24 hr, after parturiti~on. Gestation period for the females whose oothecae were artificially reduced in size averaged 60 days and for the control group 58 days. (Room temperature.)
very few eggs, are important in inhibiting the corpora allata during pregnancy.
Mechanism of inhibition of receptivity during gestation Pregnant females of N. cinerea are unreceptive and do not respond to the courting male (Rloth, 1962). Eight pregnant females had their ventral nerve cords transected <24 hr. after ovipositing. They were exposed to males for I - 2 hr. trials daily to determine if they wotuld mate. Six females mated in 2 - 8 days after cord transection; one mated 18 days after transection. The spermatophores were attached to the surface of the ootheca, usually in the vestibule, because



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T,ABLE 1-Effect of inserting glass beads and rods in the uteri on oocyte development in bisexual and parthenogenetic strains of P. surinamensis Females in which growth of oocytes
was retarded or completely
Treated females whose
oocytes developed as
rapidly as in females
without glass inserts**
Approximate size (mm.)
of glass beads and rods
inserted in uteri*
inhibi
-- -
Partially
i by glass in;
Completely
inhibited$
inhibited
Totals
Bisexual Strain
N %.
3 8 $44-
22 34
6 14
Beads7
3.5
4.0
4.5
Rods
3-3.5 X 7.5-10 1 1
Parthenogenetic Strain
Beads7
3.5
4.0
4.5
Rods
3-3.5 x 7-9
Size of beads refers to diameter) and of rods to diameter and length. Based on the range in variation of oocyte development in females whose oothecae were removed and no beads were inserted.
For the bead experiments, oocytes were 0.75 mm. or less; up until the fortieth day of pregnancy the maximum oocyte length of females carrying oothecae was 0.75 mm. in the bisexual strain and 0.71 mm. in the parthenogenetic strain.
For the rod experiments) oocytes were 0.78 mm. or less until the 58th day (maximum gestation period) and later in the bisexual strain. For the parthenogenetic strain) oocytes were 0.85 mm. or less until 60 days; this was the maximum size of the oocytes at parturition. Data for the bead experiments are taken from females used in fig. 5.



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Psyche
[December
DAYS AFTER INITIAL MATING
Fig. 7. Effect of ovariectomy on subsequent return of receptivity of females of N. cinerea. Open circles = ovaiiectomized only. Solid circles =
ovariectomized; glass tube inserted in uterus on the sixth or seventh days (arrows) after mating; tubes then removed 3 - 10 days after being inserted and the females kept with males until they mated. Numbers in circles indicate the number of females used. Per cent mating is expressed as accumulative data.
the egg case blocked the bursa copulatrix. Thus in mated females, as in virgin females that oviposit (Roth, I 964)) the mechanical presence of the ootheca inhibits receptivity. Ovariectomized (ovaries removed in last nymphal stage) females of N. cinerea mate within 3 - 6 days after emergence. When kept with males continuously only about 20% of ovariectomized females mated again and this occurred over about a 2-month period (fig. 7). Thus, either the lack of ovaries or ootheca in some way prevented return of receptivity. Sixteen pregnant females had their oothecae and ovaries removed
I 2 - I 3 days after oviposition ; eleven (69%) mated 7 - 17 days after the operation, showing that the presence of the ovaries themselves were not needed for the return of receptivity. N. cinerea nymphs were ovariectomized and the resulting adults were mated 3 - 6 days after emergence. After the spermatophores were dropped (6 - 7 days after copulation), glass tubes (about 4.3 mm. X 10 mm.) were inserted into the uteri; the posterior ends of the tubes extended beyond the abdomen and were cemented to the last abdominal segment to prevent their being extruded. After re-



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19641 Roth - Reproduction in Cockroaches 209 DAYS AFTER PARTURITION
Fig. 8. Relationship between receptivity and subsequent oviposition, and the effect of mating following parturition on oviposition in N. cinerea. Top: Females that did or did not mate when exposed to males from time of, or <24 hr. after, parturition. (N 57 for each group). Bottom: Females that were not mated after parturition. (N = 181). All females had mated previously only once, as virgins, prior to the first oviposition. Gestation period for all females ranged from 36 - 48 days. Arrows and numbers indicate the mean days I!Z standard errors to oviposit for each of the three groups.
maining in the uteri for 3 - 10 days, the tubes were removed and the females were placed with males continuously. About 70% of the
females treated in this manner eventually mated and these generally became receptive earlier than ovariectomized females that had not had artificial o6thecae inserted (fig. 7). These results suggest that the prerequisites for the return of receptivity in the normal period of time in mated females are I) the presence of an ootheca (uterine stretching) for at least a short time and then 2) the absence of the ootheca (removal of stretch stimuli).
Receptivity of females after parturition, and the effect of mating on oOcyte deevlopment
Females of N. cinerea may or may not mate again after they give birth (Roth, 1962). If not mated after ~arturition, females averaged about 8 days tlo oviposit (fig, 8, bottom). The time required for



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210 Psyche [December
oviposition by females which did or did not mate when exposed to males after parturition is shown in figure 8 (top). Females which mated oviposited later than those that did not mate, indicating that the females which were receptive afer ~arturition had comparatively smaller o6cytes (see below) and therefore took longer to oviposit than the unmated females. This apparent lack of stimulation of mating differs from that produced by mating during the first pre- oviposition period where firm insertion of the spermatophore results in an increase in the rate of devel~o~ment of the oocytes (Roth, 1964)
In L. maderae, mating after parturition also does not accelerate o6cyte maturation (Engelmann, I 96oa). Leucophaea maderae differs from N. cinerea in that the time taken to oviposit after parturition in females that do or do not mate is essentially the same; 14.5 2 0.5 days and 15.4 & 0.3 for mated and unmated females respectively (table g), whereas N. cinerea females that do not mate oviposit sooner than mated females. The difference between these species apparently is due to the fact that in L. maderae the corpora allata are inhibited during the entire gestation period and the oocytes of females at parturition are more or less the same size and do not contain yolk (see fig. 7 in Roth and Stay, 19620) ; in N. cinerea the oocytes at parturition may vary considerably in size (figs. I, 6). At high (26O- 28OC.), fairly constant insectary temperatures, the number of N. cinerea which have yolk in the Gcytes at or prior to parturition was greater than females maintained at usually lower, markedly fluctuating, room temperatures (fig. 9, bottom). Females which become receptive after parturition generally mate within 24 hr. Figure 9
(top) shows the lengths of the oocytes about 24 hr. after parturition of females that did and did not mate when they had access to males. The females with comparatively small oocytes TABLE 2 - Relationship between length of gestation period and receptivity following parturition in N. cinerea
Gestation period (days)
Number used and percent mating*
N %
* Females kept with males until they mated or oviposited.



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19641 RothÌ Reproduction in Cockroaches 21 I TABLE 3 - Effect of starvation and delayed exposure to males on receptivity of L. maderae following parturition
Days to mate
Treatment after parturition1 and percent males (Mean k S. E.)