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Entomol. exp. appl. 61: 117-122, 1991. �9 1991 Kluwer Academic Publishers, Printed m Belgium. 117

Effects on German cockroach nymphs of contact exposure to IGRs, singly and in combination

M. H. Ross & D. G. Cochran Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A.

Accepted: May 26, 1991

Key words." Cockroach, insect growth regulators, Blattella germanica

Abstract

Late instar German cockroach male and female nymphs were exposed continuously for two weeks to surfaces treated with fenoxycarb, diflubenzuron, and pyriproxyfen, singly and in combination. Concen- trations were determined that eliminated or nearly eliminated reproduction in matings with untreated mates, either through mortality, effects on reproduction, or a combination of mortality and 'sterility' (no hatch). The major effect of fenoxycarb, pyriproxyfen, and pyriproxyfen plus fenoxycarb was on reproduction. The major effect of diflubenzuron was mortality. No hatch occurred in matings of females that were exposed to low concentrations of pyriproxyfen and fenoxycarb (2 ng/cm 2 and 6 ng/cm 2, respectively); sterility was incomplete when females were exposed to 600 ng/cm 2 of diflubenzuron. Mortality and sterility acted together to eliminate productive matings (matings that produced nymphs) when rel- atively high concentrations of diflubenzuron were combined with one or both of the other insect growth regulators (1GRs). In the triple combination, very small amounts of fenoxycarb and pyriproxyfen (total 1.1 ng/cm 2) combined with 200 ng/cm 2 of diflubenzuron eliminated productive matings of treated females, but similar results with treated males were found only at higher concentrations of each IGR.

Introduction

Insect growth regulators (IGRs) are gaining ac- ceptance as tools in cockroach pest management programs (Bennett etal., 1986; Brenner etal., 1988; Ogg & Gold, 1988; Reid et al., 1990). IGRs with juvenile hormone activity produce deleterious effects on reproduction associated with twisted or curly wings and intermediates between nymphs and adults (supernumerary nymphs, adultoids or nymphoids) (Brenner etal., 1988; Das & Gupta, 1977; King & Bennett, 1989; Staal, 1975; Riddiford et al., 1975). The most sensitive

period is during the last nymphal stadium (Staal, 1975). Other IGRs are chitin synthesis inhibitors, which disrupt molting in immature insects (De- Mark & Bennett, 1989; Koehler & Patterson, 1989).

In field situations, cockroaches may be exposed to IGRs by contact with a treated surface or by feeding. We have conducted laboratory experiments using both treatment methods and assessed for the first time the relative effectiveness of single versus combination of IGRs. The feeding experiments were described earlier (Ross & Co- chran, 1990). The most significant findings from

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the contact experiments are described here. One of the IGRs, diflubenzuron, is a chitin synthesis inhibitor; the other two (fenoxycarb and pyriproxyfen) are juvenoids. Concentrations tested ranged from those causing little or no effect to those with extremely severe effects. The changes in phenotype and the increases in sterility and mortality that resulted from exposure to progressively higher dosages were much like those in the feeding experiments. Rather than repeat detailed de- scriptions of effects that occurred at concentrations below those of most importance, we focus this report on the lowest dosages at which most or all effective matings were eliminated.

Materials and methods

Technical grade fenoxycarb, pyriproxyfen and diflubenzuron were provided by Whitmire Research Laboratories, Inc., St. Louis, Mo. U.S.A. The particle size of diflubenzuron was 3 microns.

Fifteen male and 15 female late instar nymphs were drawn from a group of VPI strain (standard laboratory susceptible strain) nymphs that would normally mature within 2-21/2 week after the experiments were begun. Most nymphs were exposed to IGRs during part of the fifth and all of the sixth stadium (2 week exposure period). An exception was made in the experiments with fenoxycarb and in the experiment with fenoxycarb combined with diflubenzuron. Sixth instar females were used at high concentrations (>6 .0ng /cm 2) because preliminary tests indicated delayed maturation of fifth instar females resulted in their removal from the treated jar before reaching an age critical to effect on reproduction, i.e., the sixth stadium (Staal, 1975). The nymphs were exposed continuously for 2 weeks in glass jars (0.946 1). Jars were prepared by plac- ing a known amount of the IGR(s), dissolved in 4 ml of acetone, in each jar and rotating it evenly until the acetone evaporated. The amount of IGR per cm 2 was calculated on the basis of the inside surface area of the jar.

Mating tests were conducted similarly to those described by Ross and Cochran (1990). In brief, each newly-matured adult was placed with a pre-

viously unmated adult of the opposite sex (a 3- 6 day-old male or a newly-matured female. It was rare that nymphs matured before removal from the treated jar but, if so, a mating test was estab- lished immediately. The number of cockroaches that died before the results of mating tests were complete, adult phenotype (wings) of treated in- dividuals, and results of mating tests (hatch or no hatch) were determined. Adult wing deformations were classified as divergent (including some with a slight curl), curly, or nymphoid. Egg cases that failed to hatch were examined for indications of embryonic development. Masses of unfertilized eggs were recognized by misshapen compart- ments and pale color (see Ross & Cochran, 1990, Fig. 2). A few whole mounts (Ross and Nyuyen- Tan, 1982) of apparently unfertilized eggs were examined and only the egg nucleus was present, i.e., eggs not fertilized.

The experiments reported on here were selected on the basis of the effects of exposure to the following concentrations (ng/cm 2) (one test per concentration): diflubenzuron - 6.0, 20.0, 60.0, 200.0, and 600.0; fenoxycarb - 0.2, 0.6, 2.0, 6.0 and 20.0; pyriproxyfen - 0.1, 0.3, 0.6, 1.0, 2.0; fenoxycarb + pyriproxyfen, respectively - 0.1 + 0.1, 0.2+0.2, 0.6+0.4, 2.0+0.8, 6.0+ 1.0, and 20.0 + 2.0; fenoxycarb + diflubenzuron, respec- t i v e l y - 0.1+6.0, 0.2+10.0, 0.6+30.0, 2.0+ 100.0, 6.0 + 300.0, 20.0 + 600.0; pyriproxyfen + diflubenzuron, respectively - 0.1 + 6.0, 0.2 + 10.0, 0.4 + 30.0, 0.8 + 100.0, 1.0 + 300.0, 2.0 + 600.0; fenoxycarb + pyriproxyfen + diflubenzuron, respectively - 0.1 + 0.1 + 20.0, 0.3 + 0.3 + 60.0, 0.6 + 0.5 + 200.0, 2.0 + 1.0 + 400.0, 6.0 + 2.0 + 600, 20.0 + 5.0 + 1200.0. As noted above, the results described here are limited to the lowest concentrations at which productive matings (egg cases hatched) were nearly or completely eliminated. A few details of interest from results at lower concentrations are included.

Experiments with single IGRs also included exposures of 30 small nymphs at the above men- tioned concentrations. The results were negative unless indicated otherwise in the text. The experiments were conducted at 25-27 ~ 14D:10L photocycle and ambient humidity.

Results

We use the term 'sterility' to indicate mating tests in which non-hatch was due to failure to mate (unfertilized egg cases), non-hatch of partially fertilized egg cases, or failure of treated females to form egg cases. Partially fertilized egg cases were typical of sterile matings of treated males and failure to form egg cases typical of treated females in which treatment had deleterious effects on ovarian development (Ross & Cochran, 1990). 'Productive' matings are those in which the female reproduced successfully. Treated nymphs are referred to as 'large nymphs' because most were exposed during part of the fifth, as well as most of the sixth stadium. The amount of IGR per surface unit (ng/cm 2) is used to indicate relative exposures of nymphs to the IGRs. Nymphal hatch was generally normal for the VPI strain (> 35 nymphs per egg case). A few exceptions are noted.

The number of sterile matings corresponded closely to the number of nymphoids plus adults with curly wings. With rare exceptions, the mortality data were from nymphs; most adults survived sufficiently long to determine whether or not a productive mating would occur, after which they were discarded. Nymphs matured within one

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week after removal from the treated jars unless noted otherwise (development delayed). Also, no offspring were produced when we used concentrations above those that first eliminated productive matings.

S&gle IGRs.. Productive matings were not eliminated entirely following exposure of large nymphs to diflubenzuron (Table 1). The lowest number of egg cases that hatched was in matings of treated females exposed to 600 ng/cm 2. Hatch was un- usually low (9 nymphs) in one of three productive matings of males exposed to 600 ng/cm 2. All except one of the survivors exposed to 600 ng/ cm 2 had curly wings. A few wing deformations also occurred following exposure to concentrations of 60 and 200 ng/cm 2, but not at lower concentrations (<60 ng/cm2). Small nymphs that survived treatment were phenotypically normal as adults. They were somewhat more susceptible to diflubenzuron than large nymphs (100~ and 80-87~o kill at 600 ng/cm 2, respectively).

Mortality of large nymphs exposed to fenoxycarb was negligible (Table 1). Sterility in mating tests of treated males was incomplete, even at a higher concentration than shown in Table 1 (20.0 ng/cm2). They were the only males in each test group with phenotypically normal wings.

Table 1. Lowest concentrations at which exposure of large nymphs a to single IGRs diflubenzuron (D), fenoxycarb (F) and pyriproxyfen (P) respectively nearly b or completely ehminated productive matings

IGR Treated male Treated female

Conc c Mort Matmgs Conc ~ Mort Matmgs (Oo)r productive (Oo)d productwe

(O,o)e (Oo)e

D 600 80 20.0 600 80 13.3 F 2.0 20 13.3 2.0 0 7.6

6.0 27 13.3 6.0 20 0 P 2.0 33.3 20.0 1.0 33 7.6

2.0 60 0

Exposure initiated with fifth instar nymphs except for sixth instar females treated with fenoxycarb (F). b 'Nearly' eliminated: one (7.6,%) or two (13.3%) successful matings, except three (20~ in matings of diftubenzuron and

pyriproxyfen-treated males. c Conc - concentration in ng/cm 2. a Mort - mortality (almost exclusively nymphal). e % productive matings = no. of productive matings (egg cases hatched) divided by 15 (the number treated).

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Likewise, the only productive mating of a female exposed to 2.0 ng/cm 2 was the sole phenotypically normal female. Female sterility was complete at 6.0 ng/cm 2.

Exposure to fenoxycarb had minimal effects on small nymphs. Two or three of 30 exposed at concentrations up to 6.0cm/ng 2 died during nymphal development. On maturation, only two males and three females exposed to 0.6 ng/cm 2 had abnormal wings.

Exposure of large nymphs to pyriproxyfen affected females more severely than males (Table 1). Mortality in females was higher than in males; three treated males mated effectively when exposed to the same concentration that eliminated productive matings of treated females (2.0 ng/ cm2); and curly wings appeared at a lower concentration in females (0.3 ng/cm 2) than in males (0.6 ng/cm2). Small nymphs exposed to pyriproxyfen matured into phenotypically normal adults.

Combination treatments.. Elimination of most or all productive matings when large nymphs were exposed to combinations of fenoxycarb +pyriproxyfen was due entirely to sterility (nymphoid or curly-wing cockroaches) (Table 2). The lowest total concentration at which productive matings

of both sexes were eliminated was 2.8 ng/cm 2 (2.0 and 0.8 ng/cm 2 of fenoxycarb and pyriproxyfen, respectively). When the latter concentrations were halved, productive matings of females were nearly eliminated but four treated males (26.7 ~o) mated successfully.

Productive matings of females treated as large nymphs with combinations of diflubenzuron with either fenoxycarb or pyriproxyfen were eliminated at somewhat lower concentrations than those of treated males (Table 2). Sterility and mortality contributed about equally to the reduction in productive matings of treated males; among females, mortality somewhat outweighed the effects of sterility.

In the triple combination experiment (Table 2), complete elimination of most or all productive matings of treated males was due about equally to mortality and and sterility. High mortality (87 Yo) played a major role in eliminating productive matings of treated females with the combination of IGRs shown in Table 2, but mortality was not consistent at two higher concentrations at which productive matings continued to be eliminated (53.3 ~/o mortality from exposure to 2.0 ng/cm 2 of fenoxycarb + 2.0 ng/cm 2 of pyriproxyfen + 400 ng/cm 2 of diflubenzuron; 40.0 ~o mortality from

Table 2. Lowest concentrations of mixtures of IGRs (diflubenzuron (D), fenoxycarb (F) and pyriproxifen (P)) at which exposure of large nymphs a nearly b or completely eliminated productive matings in combination experiments.

Treated male Treated female

Conc of each Mort Matings Conc of each Mort Matings IGR c (%)d productive IGR c (%)d productive

(%)~ (%)~

2.0 F + 0 . 8 P 0 0 0.6 F + 0 . 4 P 0 13.3 2.0 F + 0 . 8 P 6.7 0

2.0 F + 1 0 0 D 40.0 13.3 0.6 F + 3 0 D 40.0 13.3 6.0 F + 3 0 0 D 46.7 0 2.0 F + 100 D 73.3 0 2.0 P + 6 0 0 D 53.3 6.7 1.0 P + 300 D 73.3 0 0.6 F+0 .5 P + 2 0 0 D 0.6 F + 0 . 5 F + 2 0 0 D 86.7 0 6.0 F + 2 . 0 P + 6 0 0 D 40.0 0

a Inititally exposed in the fifth instar except for sixth instar females in the experiment with fenoxycarb + pyriproxyfen. O/ b 'Nearly' eliminated - one (7.6/o) or two (13.3%) productive matings.

c Concentration in ng/cm 2. d Mort - mortality (almost exclusively nymphal). e Productive matings = no. of productive matings divided by 15 (no. treated).

exposure to 6.0 ng/cm 2 of fenoxycarb + 2.0 ng/ cm 2 of pyriproxyfen + 600 ng/cm 2 of diflubenzuron). At a concentration below that shown in Table 2 (2.0 + 1.0 + 400 ng/cm 2 of fenoxycarb, pyriproxyfen and diflubenzuron, respectively), nymphal hatch was low in the only two productive matings (17 and 22 nymphs per egg case compared to an average of 38.6+ 1.1 eggs/egg case for untreated cockroaches; n = 90).

Discussion

Reduction in the frequency of productive females rather than males is probably the key to successful application of IGRs. In populations exposed to IGRs, males capable of mating and causing females to be nonproductive might not prevent successful matings of males that were either less adversely affected or unaffected. The latter would normally be capable of multiple matings (Ueda et al., 1969). If a female is 'sterilized' or killed she obviously will not reproduce.

As expected, reproduction was affected severely when large nymphs were exposed to fenoxycarb or pyriproxyfen. The effects of fenoxycarb on females were somewhat less severe than on females exposed to pyriproxyfen, regardless of whether exposure was initiated in the fifth or sixth stadium. We did not find the marked delay in development of females exposed to high concentrations of pyriproxyfen that made it necessary to initiate exposure of females to fenoxycarb in the sixth stadium.

Offspring were still produced in a few matings after exposure to two orders of magnitude higher concentrations of diflubenzuron than were used in the experiments with fenoxycarb and pyriproxyfen. Advantages that might partially offset the latter problems with diflubenzuron were: 1)the more immediate effects of mortality compared to those of sterility and 2)there was no critical de- velopmental period during which nymphs had to be exposed in order for this IGR to be effective. We do not known whether acetone altered the effectiveness of the 3 micron particle size of the technical grade diflubenzuron, but the experi-

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ments of Koehler and Patterson (1989) also sug- gest that contact exposure to diflubenzuron was not highly effective on the German cockroach. Our results were similar to those of experiments with other chitin synthesis inhibitors in that nearly all of the surviving nymphs matured into into phenotypically normal adults (DeMark & Bennett, 1989).

The higher mortality of small compared to large nymphs when exposed to diflubenzuron is probably attributable to a larger number of molts by the small nymphs. Diflubenzuron kills nymphs during ecdysis (Koehler & Patterson, 1989). Small nymphs would molt more frequently than large nymphs within a two-week exposure period because the interval between molts is shorter in early than in late instars (Willis et al., 1958).

The lowest total concentration of IGRs that eliminated productive matings in combination treatments was in the experiment with pyriproxyfen + fenoxycarb. Since females exposed to the most nearly similar concentration of the single IGRs were completely sterile, it does not ap- pear that control would be enhanced by combin- ing the IGRs. The delay of development when exposure of females was initiated in the fifth rather than sixth stadium was clearly an effect of fenoxycarb.

The effect on late instar nymphs of exposure to the combination treatment of diflubenzuron + pyriproxyfen were generally similar to those in the experiment with diflubenzuron + fenoxycarb. Mortality was higher in females than in males, but, in both sexes, mortality and sterility played important roles in the elimination of productive matings, thus expressing the effects expected from exposure to both a juvenoid and a chitin synthesis inhibitor. The combination of diflubenzuron with either pyriproxyfen or fenoxycarb enhanced the effectiveness of diflubenzuron, but productive matings were eliminated with a much smaller total amount of IGR in the combination of pyriproxyfen + fenoxycarb.

The concentrations required to eliminate productive matings of males in the triple combination occurred only at unexpectedly high concentrations of fenoxycarb and pyriproxyfen, as well as

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diflubenzuron. Also, mortality of male nymphs was lower than expected, as well as in the combination of diflubenzuron with either pyriproxyfen or fenoxycarb. Apparently, the lethal effects of diflubenzuron on male nymphs were diminished in the presence of either or both pyriproxyfen and fenoxycarb.

The above effects on males in the triple combination experiment widened the gap between the amounts of IGRs that eliminated productive matings of treated males and females. Female mortality was high at the lowest concentration that eliminated productive matings but sterility was the major cause of eliminating productive matings at higher concentrations. This appeared to be a partial expression in females of the diminished effects of diflubenzuron on mortality that were seen in treated males in the combination experiments. At some of the concentrations lower than reported on here, hatch was frequently reduced in matings of treated females. This was similar to the feeding experiments, where reduced numbers of nymphs per egg case were unique to the experiment that combined the three IGRs (Ross & Cochran, 1990).

If one wishes to take advantage of the lethal effects of diflubenzuron, the best choice would be to use a combination with one or both of the other IGRs. The triple combination might have an advantage over combinations of two due to a slightly higher female mortality and very small amounts of pyriproxyfen and fenoxycarb. From the point of view of eliminating productive matings with low concentrations, especially of treated females, either pyriproxyfen alone or in combination with fenoxycarb would be highly effective.

Acknowledgement

The authors thank Whitmire Research Labora- tories. Inc., for financial support and for supply- ing the IGRs. We also appreciate the technical assistance of Elizabeth Watson and Nancy Boles.

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