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

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<ul><li><p>Entomol. exp. appl. 61: 117-122, 1991. 9 1991 Kluwer Academic Publishers, Printed m Belgium. 117 </p><p>Effects on German cockroach nymphs of contact exposure to IGRs, singly and in combination </p><p>M. H. Ross &amp; D. G. Cochran Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A. </p><p>Accepted: May 26, 1991 </p><p>Key words." Cockroach, insect growth regulators, Blattella germanica </p><p>Abstract </p><p>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 repro- duction. 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, respec- tively); 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 fe- males, but similar results with treated males were found only at higher concentrations of each IGR. </p><p>Introduction </p><p>Insect growth regulators (IGRs) are gaining ac- ceptance as tools in cockroach pest management programs (Bennett etal., 1986; Brenner etal., 1988; Ogg &amp; Gold, 1988; Reid et al., 1990). IGRs with juvenile hormone activity produce deleteri- ous effects on reproduction associated with twisted or curly wings and intermediates between nymphs and adults (supernumerary nymphs, adultoids or nymphoids) (Brenner etal., 1988; Das &amp; Gupta, 1977; King &amp; Bennett, 1989; Staal, 1975; Riddiford et al., 1975). The most sensitive </p><p>period is during the last nymphal stadium (Staal, 1975). Other IGRs are chitin synthesis inhibitors, which disrupt molting in immature insects (De- Mark &amp; Bennett, 1989; Koehler &amp; Patterson, 1989). </p><p>In field situations, cockroaches may be exposed to IGRs by contact with a treated surface or by feeding. We have conducted laboratory experi- ments using both treatment methods and assessed for the first time the relative effectiveness of sin- gle versus combination of IGRs. The feeding ex- periments were described earlier (Ross &amp; Co- chran, 1990). The most significant findings from </p></li><li><p>118 </p><p>the contact experiments are described here. One of the IGRs, diflubenzuron, is a chitin synthesis inhibitor; the other two (fenoxycarb and pyriprox- yfen) are juvenoids. Concentrations tested ranged from those causing little or no effect to those with extremely severe effects. The changes in pheno- type and the increases in sterility and mortality that resulted from exposure to progressively higher dosages were much like those in the feed- ing experiments. Rather than repeat detailed de- scriptions of effects that occurred at concentra- tions below those of most importance, we focus this report on the lowest dosages at which most or all effective matings were eliminated. </p><p>Materials and methods </p><p>Technical grade fenoxycarb, pyriproxyfen and di- flubenzuron were provided by Whitmire Research Laboratories, Inc., St. Louis, Mo. U.S.A. The particle size of diflubenzuron was 3 microns. </p><p>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 ex- periments were begun. Most nymphs were ex- posed 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 fenoxy- carb combined with diflubenzuron. Sixth in- star females were used at high concentrations (&gt;6.0ng/cm 2) because preliminary tests indi- cated delayed maturation of fifth instar females resulted in their removal from the treated jar be- fore reaching an age critical to effect on repro- duction, 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. </p><p>Mating tests were conducted similarly to those described by Ross and Cochran (1990). In brief, each newly-matured adult was placed with a pre- </p><p>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 &amp; 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. </p><p>The experiments reported on here were selected on the basis of the effects of exposure to the fol- lowing concentrations (ng/cm 2) (one test per con- centration): 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 ive ly - 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, re- spectively - 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 re- sults described here are limited to the lowest con- centrations at which productive matings (egg cases hatched) were nearly or completely elimi- nated. A few details of interest from results at lower concentrations are included. </p><p>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 exper- iments were conducted at 25-27 ~ 14D:10L photocycle and ambient humidity. </p></li><li><p>Results </p><p>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 &amp; Cochran, 1990). 'Productive' matings are those in which the fe- male 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 rela- tive exposures of nymphs to the IGRs. Nymphal hatch was generally normal for the VPI strain (&gt; 35 nymphs per egg case). A few exceptions are noted. </p><p>The number of sterile matings corresponded closely to the number of nymphoids plus adults with curly wings. With rare exceptions, the mor- tality data were from nymphs; most adults sur- vived sufficiently long to determine whether or not a productive mating would occur, after which they were discarded. Nymphs matured within one </p><p>119 </p><p>week after removal from the treated jars unless noted otherwise (development delayed). Also, no offspring were produced when we used concen- trations above those that first eliminated produc- tive matings. </p><p>S&amp;gle IGRs.. Productive matings were not elim- inated 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 produc- tive 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 concentra- tions of 60 and 200 ng/cm 2, but not at lower con- centrations (</p></li><li><p>120 </p><p>Likewise, the only productive mating of a female exposed to 2.0 ng/cm 2 was the sole phenotypi- cally normal female. Female sterility was com- plete at 6.0 ng/cm 2. </p><p>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. </p><p>Exposure of large nymphs to pyriproxyfen af- fected females more severely than males (Table 1). Mortality in females was higher than in males; three treated males mated effectively when ex- posed to the same concentration that eliminated productive matings of treated females (2.0 ng/ cm2); and curly wings appeared at a lower con- centration in females (0.3 ng/cm 2) than in males (0.6 ng/cm2). Small nymphs exposed to pyriprox- yfen matured into phenotypically normal adults. </p><p>Combination treatments.. Elimination of most or all productive matings when large nymphs were exposed to combinations of fenoxycarb +pyri- proxyfen was due entirely to sterility (nymphoid or curly-wing cockroaches) (Table 2). The lowest total concentration at which productive matings </p><p>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. </p><p>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 pro- ductive matings of treated males; among females, mortality somewhat outweighed the effects of ste- rility. </p><p>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 mat- ings 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 </p><p>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. </p><p>Treated male Treated female </p><p>Conc of each Mort Matings Conc of each Mort Matings IGR c (%)d productive IGR c (%)d productive </p><p>(%)~ (%)~ </p><p>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 </p><p>2.0 F+100 D 40.0 13.3 0.6 F+30 D 40.0 13.3 6.0 F+300 D 46.7 0 2.0 F+ 100 D 73.3 0 2.0 P+600 D 53.3 6.7 1.0 P+ 300 D 73.3 0 0.6 F+0.5 P+200 D 0.6 F+0.5 F+200 D 86.7 0 6.0 F+2.0 P+600 D 40.0 0 </p><p>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. </p><p>c Concentration in ng/cm 2. d Mort - mortality (almost exclusively nymphal). e Productive matings = no. of productive matings divided by 15 (no. treated). </p></li><li><p>exposure to 6.0 ng/cm 2 of fenoxycarb + 2.0 ng/ cm 2 of pyriproxyfen + 600 ng/cm 2 of diflubenzu- ron). 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 produc- tive 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). </p><p>Discussion </p><p>Reduction in the frequency of productive females rather than males is probably the key to success- ful 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. </p><p>As expected, reproduction was affected se- verely when large nymphs were exposed to fenox- ycarb 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 concen- trations of pyriproxyfen that made it necessary to initiate exposure of females to fenoxycarb in the sixth stadium. </p><p>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 pyriprox- yfen. 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- </p><p>121 </p><p>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 phe- notypically normal adults (DeMark &amp; Bennett, 1989). </p><p>The higher mortality of small comp...</p></li></ul>


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