<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article  PUBLIC "-//NLM//DTD Journal Publishing DTD v3.0 20080202//EN" "http://dtd.nlm.nih.gov/publishing/3.0/journalpublishing3.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" dtd-version="3.0" xml:lang="en" article-type="research article"><front><journal-meta><journal-id journal-id-type="publisher-id">ACES</journal-id><journal-title-group><journal-title>Advances in Chemical Engineering and Science</journal-title></journal-title-group><issn pub-type="epub">2160-0392</issn><publisher><publisher-name>Scientific Research Publishing</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.4236/aces.2015.53038</article-id><article-id pub-id-type="publisher-id">ACES-58104</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Chemistry&amp;Materials Science</subject></subj-group></article-categories><title-group><article-title>
 
 
  Effect of Insecticides on &lt;i&gt;Trichogramma&lt;/i&gt; Parasitoids Used in Biological Control against &lt;i&gt;Prays oleae&lt;/i&gt; Insect Pest
 
</article-title></title-group><contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>men</surname><given-names>Blibech</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Mohieddine</surname><given-names>Ksantini</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Taieb</surname><given-names>Jardak</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Mohamed</surname><given-names>Bouaziz</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>Laboratory of Genetic Resources Improvement and Protection of Olive Tree, Olive Tree Institute, 
Sfax, Tunisia</addr-line></aff><aff id="aff2"><addr-line>Electrochemistry and Environment Laboratory, National School of Engineers of Sfax, University of Sfax, 
Sfax, Tunisia</addr-line></aff><author-notes><corresp id="cor1">* E-mail:<email>Imenblibech@gmail.com(MB)</email>;</corresp></author-notes><pub-date pub-type="epub"><day>19</day><month>06</month><year>2015</year></pub-date><volume>05</volume><issue>03</issue><fpage>362</fpage><lpage>372</lpage><history><date date-type="received"><day>1</day>	<month>June</month>	<year>2015</year></date><date date-type="rev-recd"><day>accepted</day>	<month>17</month>	<year>July</year>	</date><date date-type="accepted"><day>20</day>	<month>July</month>	<year>2015</year></date></history><permissions><copyright-statement>&#169; Copyright  2014 by authors and Scientific Research Publishing Inc. </copyright-statement><copyright-year>2014</copyright-year><license><license-p>This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/</license-p></license></permissions><abstract><p>
 
 
  This study investigated the direct effect of the insecticides deltamethrin and spinosad on three egg parasitoids species: 
  <em>Trichogramma</em> oleae, 
  <em>T. cacoeciae</em> and 
  <em>T. bourarachae</em>. The parasitoid pupae were exposed to pesticide residues on fresh olive tree leaves at recommended concentrations (RC) at different time intervals: 3, 10, 17, 24 and 31 days after pesticide applications. Parasitism viability (% emergence from parasitized eggs) and adult emergence time (developmental time from pupa to adult emergence) were evaluated. Regarding to the International Organization of Biological Control (IOBC) guidelines, results of toxicity effects of insecticides show that: Deltamethrin was moderately harmful to all 
  <em>Trichogramma</em> species at RC (Decis
  <sup>&amp;reg;</sup> 100 milliliters&#183;ha
  <sup>&amp;minus;1</sup>), however, spinosad was harmless to moderately harmful at RC (Tracer
  <sup>&amp;reg;</sup> 20 milliliters&#183;ha
  <sup>&amp;minus;1</sup>). 
  <em>Trichogramma</em> species revealed differences with regard to adult emergence time and exhibited significant changes in parasitism viability with increasing time after pesticide treatment. While deltamethrin residues affected parasitism viability 31 days after the product application, spinosad displayed similar viability for almost species 24 days after the application. The usefulness of 
  <em>Trichogramma</em> parasitoids used as biological control agents, in olive tree ecosystem, was discussed in integrated pest management programs for 
  <em>Prays oleae</em> control when parasitoid species were exposed during pupal stage to the insecticide residue.
 
</p></abstract><kwd-group><kwd>Insecticide Effect</kwd><kwd> &lt;i&gt;Trichogramma&lt;/i&gt; Species</kwd><kwd> Olive Tree</kwd><kwd> Parasitism Viability</kwd><kwd> Emergence Time</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>1. Introduction</title><p>Integrated Pest Management (IPM) is an effective and environmentally approach to pest management that relies on a combination of chemical and biological control methods. Of particular interest, parasitoids of the genus Trichogramma, which are egg-parasitoids in the family Trichogrammatidae, have been reported to be efficient in regulating populations of lepidopteran pests in agricultural cropping systems [<xref ref-type="bibr" rid="scirp.58104-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.58104-ref2">2</xref>] . Among main lepidopteran insect pest of olive tree, the olive moth Prays oleae Bern (Lepidoptera, Hyponomeutidae) is the most abundant throughout the Mediterranean and the Black Sea, the Middle East and the Canary Islands [<xref ref-type="bibr" rid="scirp.58104-ref3">3</xref>] . In Tunisia, the damage caused by P. oleae can reduce olive production by 50% [<xref ref-type="bibr" rid="scirp.58104-ref4">4</xref>] . As a consequence, efforts to develop biological control methods in IPM programs were initiated through the release of egg parasitoids and use of pheromones. However, release and integration issues that are considered important for the development of a successful IPM include important factors such as the pesticide choice and the timing of application [<xref ref-type="bibr" rid="scirp.58104-ref5">5</xref>] . In fact, Trichogramma is generally sensitive to pesticides [<xref ref-type="bibr" rid="scirp.58104-ref6">6</xref>] and most chemical products can be immediately toxic (contact toxicity) or it can persist (toxicity of dried residue) [<xref ref-type="bibr" rid="scirp.58104-ref7">7</xref>] [<xref ref-type="bibr" rid="scirp.58104-ref8">8</xref>] . Moreover, contact with pesticides at the less susceptible life stage (parasitoids within hosts) can cause prolonged development time of immature stages and reduced emergence from parasitized eggs [<xref ref-type="bibr" rid="scirp.58104-ref9">9</xref>] . Accordingly, this is important to determine the compatibility of parasitoids with pesticides for IPM programs.</p><p>Saccharopolyspora spinosa [<xref ref-type="bibr" rid="scirp.58104-ref10">10</xref>] or spinosad is a product classified as an environmentally and toxicologically reduced risk material [<xref ref-type="bibr" rid="scirp.58104-ref11">11</xref>] . Spinosad-based products have been registered in more than 30 countries for control of pest: Lepidoptera, Diptera, some Coleoptera, ants and thrips [<xref ref-type="bibr" rid="scirp.58104-ref12">12</xref>] . As a biorational pesticide, spinosad now represents an important option for pest control in a growing number crops produced under systems of integrated pest management (IPM) [<xref ref-type="bibr" rid="scirp.58104-ref13">13</xref>] . A recent review of predator and parasitoid susceptibility to spinosad concluded that this product represented one of the most judicious insecticides available for the conservation of predator populations [<xref ref-type="bibr" rid="scirp.58104-ref14">14</xref>] . However, the majority of laboratory and field studies report moderately harmful or harmful effects on populations of hymenopteran parasitoids [<xref ref-type="bibr" rid="scirp.58104-ref15">15</xref>] - [<xref ref-type="bibr" rid="scirp.58104-ref17">17</xref>] . Spinosad is commercially available under the name of Tracer 240<sup>&#174;</sup> and is used against the first and second generations of P. oleae in most olive groves in Tunisia. However larval control of the first and the second generation of P. oleae usually rely on insecticides when the damage threshold reaches 5% for flowers and 20% for fruits. Pesticide persistence and mobility are influenced by the properties of the pesticide as well as by the soil environment, site conditions, weather and application methods. Therefore, information on degradation rate also helps to assess and predict the environmental behaviour of the pesticide.</p><p>Primary insecticide used is the pyrethroid, deltamethrin (Decis<sup>&#174;</sup>), which is valued for its residual activity, persistence and high toxicity to terrestrial invertebrates [<xref ref-type="bibr" rid="scirp.58104-ref18">18</xref>] . Generally, the second generation is difficult to manage with insecticides because larvae bore into the fruit immediately after emerging from the eggs [<xref ref-type="bibr" rid="scirp.58104-ref19">19</xref>] . Other control measures are than needed and a potential alternative strategy is the release of Trichogramma species. In fact, this is a way to inhibit first larval stage of P. oleae on fruits [<xref ref-type="bibr" rid="scirp.58104-ref5">5</xref>] . However, one factor that may inhibit the ability of Trichogramma species to regulate pest population is the toxic insecticide residues.</p><p>Today, more than studying the toxic effect of pesticides on pests, there is a trend to evaluate their innocuousness towards natural enemies; These residues may be found in the soil, groundwater or the sediment of waterways and may also be found in vertebrates/invertebrates associated with the treated area, through direct contact or food chain effects [<xref ref-type="bibr" rid="scirp.58104-ref20">20</xref>] .</p><p>The selection of the most adequate parasitoid species is vital for any program of biological control [<xref ref-type="bibr" rid="scirp.58104-ref21">21</xref>] . It is important to select the appropriate Trichogramma species that are most adapted to the local field conditions for biolo- gical control programs. In fact, according to [<xref ref-type="bibr" rid="scirp.58104-ref22">22</xref>] , native Trichogramma species, which are generally adapted to environment, are better biological control agents. Moreover, quality measures that can be associated with the effectiveness of Trichogramma species to control the target pest are attributed mainly to the parasitism viability and to the developmental time from egg to adult emergence [<xref ref-type="bibr" rid="scirp.58104-ref23">23</xref>] . Therefore, the present study was designed to determine the side-effects of two insecticides deltamethrin (Decis<sup>&#174;</sup>) and spinosad (Tracer 240<sup>&#174;</sup>) on three native Trichogramma species T. oleae, T. cacoeciae and T. bourarachae [<xref ref-type="bibr" rid="scirp.58104-ref24">24</xref>] and to discuss their possible use in control programs against the second generation of P. oleae in olive grove areas where biological control has limited use.</p></sec><sec id="s2"><title>2. Materials and Methods</title><sec id="s2_1"><title>2.1. Rearing of Trichogramma Parasitoid</title><p>Trichogramma species: T. oleae, T. cacoeciae and T. bourarachae used for the experiment were reared under laboratory conditions on Ephestia kuehniella eggs. Glued cardboard squares (1 cm<sup>2</sup>), each with approximately 250 E. kuehniella eggs, were presented for 24 hours to recently emerged Trichogramma females (20 females per card) in glass tubes ( 6 cm &#215; 1.5 cm ) at 20˚C, 60% RH and 16L: 8D. The cards with a parasitism rate close to 100% were than transferred to other glass tubes and maintained until pupation (192 hours).</p></sec><sec id="s2_2"><title>2.2. Treatment of Olive Trees with Insecticide</title><p>Two years young olive trees maintained in greenhouse at 25˚C, 70% RH were used for the sampling. Five olive trees per insecticide were sprayed till run-off with a hand sprayer at the same day ( 3 litres of solution per tree) and an eleventh tree sprayed with water for the control. Treatments were applied according to the recommended label rate for control of P. oleae. The insecticides tested were: deltamethrin at 3.75 grams∙ha<sup>−</sup><sup>1</sup> (Decis<sup>&#174;</sup> 100 milliliters∙ha<sup>−</sup><sup>1</sup>) and spinosad at 24 grams∙ha<sup>−</sup><sup>1</sup> (Tracer<sup>&#174;</sup> 20 milliliters∙ha<sup>−</sup><sup>1</sup>).</p><p>Olive leaves were collected at 3, 10, 17, 24 and 31 after olive trees spraying and used for exposure tests of parasitoid pupae at intervals after treatment.</p></sec><sec id="s2_3"><title>2.3. Exposure Tests of Parasitoid Pupae at Intervals after Treatment</title><p>In each time after the treatment (at 3, 10, 17, 24 and 3 days), a card with pupae parasitoid was placed inside transparent plastic petri dish ( 6 cm &#215; 4 cm ) with treated (using the insecticide) or with control (using water) olive leaf samples. A 2 &#215; 2 cm aeration hole (4 cm<sup>2</sup>) covered with fine cotton cloth was made on the petri dish to assure continued ventilation in the area. In total, 30 cards for each Trichogramma specie (Each card represented a single independent replicate): 15 cards (n = 15 per treatment) and 15 cards (n = 15 per control) were conducted at 25˚C &#177; 2˚C, 60% &#177; 5% RH, and 14 L: 10 D.</p></sec><sec id="s2_4"><title>2.4. Analyses of Pesticide Residues</title><sec id="s2_4_1"><title>2.4.1. Olive Leaf Sampling</title><p>Other samples of olive leaves were collected from same young olive trees maintained in greenhouse at each time after the treatment: starting from the day of spray (one hour after pesticide application) and at different time intervals (3, 10, 17, 24 and 31 days). Leaf samples were collected randomly from each tree and cut in fine pieces (about 5 mm diameter), placed immediately in a cool-box held at 4˚C - 8˚C after collection and transferred to refrigerator for analysis within 24 - 36 hours.</p></sec><sec id="s2_4_2"><title>2.4.2. Extraction</title><p>Fifty gram samples collected at each time intervals were blended in 100 ml of acetonitrile?water (80/20; v/v) for 2 min, shaken for approximately 30 min and filtered through a buchner funnel than washed with acetonitrile- water mixture (8:2, v/v). Extract was purified using silica SPE (Solid Phase Extraction) cartridge. The acetonitrile solution was eluted, collected and immediately evaporated to dryness in a rotary vacuum evaporator. The residue was reconstituted in 1 mL of methanol: acetonitrile: 2% ammonium acetate solution (1:1:1, v/v) for final HPLC analysis (HPLC, a Beckman model 322) equipped with UV detector (250 nm). The column used was C18 ( 250 mm &#215; 4.6 mm i.d). The mobile phase was acetonitrile: methanol: 2% ammonium acetate (21:21:8) at a flow rate of 2.0 mL∙min<sup>−</sup><sup>1</sup>. A 5 μL aliquot of each sample was injected each time for residue analysis. The representative retention times of spinosyn A and D were 9.5 min and 11.0 min, respectively.</p></sec></sec><sec id="s2_5"><title>2.5. Data Analyses</title><p>Parasitism viability (number of emerged adults from parasitized pupae) and adult emergence time (developmental time from pupa to adult emergence) were evaluated.</p><p>The effect of the insecticides was established by comparing the controls with the treatments using the following formula:</p><disp-formula id="scirp.58104-formula359"><graphic  xlink:href="http://html.scirp.org/file/15-3700546x6.png"  xlink:type="simple"/></disp-formula><p>where E is the insecticide effect in parasitism viability (number of emerged adults from parasitized pupae) compared to the control; Vt is parasitism viability observed for each insecticide treatment and Vc is the parasitism viability observed in the control ones. The value E calculated for the insecticide treatments were classified according to the International Organization of Biological Control (IOBC): class 1―harmless (E &lt; 30%), class 2― slightly harmful (30 % &lt; E &lt; 79%), class 3―moderately harmful (80 % &lt; E &lt; 99%) and class 4―harmful (E &gt; 99%) [<xref ref-type="bibr" rid="scirp.58104-ref25">25</xref>] . Proportional data were normalized through arcsine transformation; all the other data were normalized through square root transformation.</p><p>The tests were performed using the Statistical Package for the Social Sciences (SPSS) for Windows (version 17.0). A complete randomized factorial design was used, and data were submitted to analysis of variance (ANOVA, p &lt; 0.05). Significant mean values associated with parasitism viability were compared by Dunnett’ test.</p><p>Tukey test for multiple comparisons was performed on the various groups to determine whether differences associated with adult emergence time existed between treatments and exposure periods.</p><p>In the text and figures, mean &#177; SD (standard deviation) are used as descriptive statistics for distributed data</p></sec></sec><sec id="s3"><title>3. Results</title><sec id="s3_1"><title>3.1. Insecticide Effect on Trichogramma Species</title><p>Results from the experiment are presented in <xref ref-type="table" rid="table1">Table 1</xref>. The dominant class was 2 (30% &lt; E &lt; 79%) for all treatments and parasitoid species combinations. However, the classification changed from class 2 to class 1 (E &lt; 30%) or to class 3 (80% &lt; E &lt; 99%). In fact, result from treatment using olive leaf samples on day 31 after deltamethrin spraying has show a slightly harmful effect on parasitism viability, except the case for T. oleae where the insecticide was harmless (E% = 23.6 &#177; 8.3). However, deltamethrin was moderately harmful to T. bourarachae (E% = 80.5 &#177; 2.2) on day three. Spinosad was harmless to T. oleae and T. cacoeciae at 10 days (E% = 18.8 &#177; 9.0). However, it was slightly harmful to T. bourarachae even on day 31 (E% = 32.3 &#177; 4.8).</p></sec><sec id="s3_2"><title>3.2. Comparison between Insecticide Effects on Trichogramma Species Parasitism Viability</title><p><xref ref-type="table" rid="table2">Table 2</xref> presents the pair multiple comparisons among the treatments associated with parasitism viability for the different assessment periods.</p><p>The spinosad treatment significantly reduced parasitism viability of all three Trichogramma species for all assessment periods. Difference in the mean values associated with parasitism viability between spinosad treatment and control were 30.7 for T. bourarachae, 22.0 for T. oleae and 26.0 for T. cacoeciae (P &lt; 0.05). In addition, parasitism viability of all the three Trichogramma species was less affected by spinosad than by deltamethrin (P &lt; 0.05).</p></sec><sec id="s3_3"><title>3.3. Adult Emergence Time of Trichogramma Species at the Different Exposure Periods</title><p>Adult emergence time (Pupa-adult development time) of Trichogramma reared on E. kuehniella eggs differed among the three species in their response to insecticide residues at the different exposure periods. Periods of the experiment in the control group do not affect adult emergence time of: T. oleae (<xref ref-type="table" rid="table3">Table 3</xref>, Tukey test, F = 0.62; df = 4; p = 0.854), T. bourarachae (F = 0.5; df = 4; p = 0.736) and T. cacoeciae (F = 0.71; df = 4; p = 0.780). Wasp development from pupa to adult emergence at 25˚C normally requires about 6 days. However, pupa exposed to the insecticide residues required about one to two days longer to complete development. Adult emergence time of T. oleae was significantly long when pupa were exposed to deltamethrin residues at 3 days after the treatment. More significant decrease in adult emergence time was observed at 10 and 17 days then at 24 and 31 days (<xref ref-type="table" rid="table3">Table 3</xref>, Tukey test, F = 91, 45; df = 4; p &lt; 0.05). However, adult emergence time from T. oleae pupa exposed to spinosad residues was significantly decreased at 24 days after the treatment (<xref ref-type="table" rid="table3">Table 3</xref>, Tukey test, F = 31.67; df = 4; p &lt; 0.05).</p><p>Adult emergence time from T. cacoeciae pupa decreased when exposure period to deltamethrin and to spinosad residues occurred, respectively, at 31 days (<xref ref-type="table" rid="table3">Table 3</xref>, Tukey test, F=42.27; df = 4; p &lt; 0.05) and at 10 days (<xref ref-type="table" rid="table3">Table 3</xref>, Tukey test, F = 29.83; df = 4; p &lt; 0.05) after the treatment.</p><p>In contrast, adult emergence time from T. bourarachae pupa exposed to deltamethrin or to spinosad residues was similar in all the periods of exposure (<xref ref-type="table" rid="table3">Table 3</xref>, Tukey test, F = 0.72; df = 4; p = 0.624 and F = 0.52; df = 4; p = 0.736).</p><table-wrap id="table1" ><label><xref ref-type="table" rid="table1">Table 1</xref></label><caption><title> Insecticide effect (E) on parasitism viability reduction associated with T. oleae, T. cacoeciae and T. bourarachae after exposure of parasitoid pupae to residues on leaves at different times (days)</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Species</th><th align="center" valign="middle" >Treatment (grams∙ha<sup>−1</sup>)</th><th align="center" valign="middle" >Exposure time</th><th align="center" valign="middle" >E<sup>1</sup> (%)</th><th align="center" valign="middle" >Class<sup>2</sup></th></tr></thead><tr><td align="center" valign="middle" >T. oleae</td><td align="center" valign="middle" >Deltamethrin</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >62.1 &#177; 6.2</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >66.7 &#177; 8.1</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >49.6 &#177; 0.2</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >41.0 &#177; 5.8</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >23.6 &#177; 8.3</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >Spinosad</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >52.9 &#177; 1.8</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >18.8 &#177; 9.0</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >16.1 &#177; 2.6</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >14.4 &#177; 2.9</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >10.8 &#177; 4.7</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" >T. cacoeciae</td><td align="center" valign="middle" >Deltamethrin</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >65.6 &#177; 13.3</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >56.9 &#177; 4.8</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >54.4 &#177; 8.4</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >51.0 &#177; 5.5</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >51.6 &#177; 3.2</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >Spinosad</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >53.1 &#177; 12.3</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >47.5 &#177; 7.2</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >39.2 &#177; 16.6</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >23.6 &#177; 16</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >16.5 &#177; 12.8</td><td align="center" valign="middle" >1</td></tr><tr><td align="center" valign="middle" >T. bourarachae</td><td align="center" valign="middle" >Deltamethrin</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >80.5 &#177; 2.2</td><td align="center" valign="middle" >3</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >65.1 &#177; 6.0</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >48.6 &#177; 9.4</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >46.2 &#177; 7.6</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >44.2 &#177; 3.2</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >Spinosad</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >71.0 &#177; 2.0</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >45.6 &#177; 11.6</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >42.8 &#177; 13.5</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >33.5 &#177; 14.3</td><td align="center" valign="middle" >2</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >32.3 &#177; 4.8</td><td align="center" valign="middle" >2</td></tr></tbody></table></table-wrap><p><sup>1</sup>E(%) = (1 − Vt/Vc) &#215; 100, where E is the effect of the insesticide on parasitism viability reduction compared to the control, Vt is the parasitism viability for the treatments (using insecticide) and Vc is the parasitism viability for the control (using water). <sup>2</sup>Class: 1. harmless (E &lt; 30%); 2. slightly harmful (30% &lt; E &lt; 79%); 3. moderately harmful (80% &lt; E &lt; 99%); 4. harmful (E &gt; 99%).</p><table-wrap id="table2" ><label><xref ref-type="table" rid="table2">Table 2</xref></label><caption><title> Comparison between the deltamethrin and spinosad treatments based on parasitism viability of Trichogramma species</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Species</th><th align="center" valign="middle" >Treatment</th><th align="center" valign="middle" >Parasitism viability Mvd<sup>*</sup> (control-treatment)</th><th align="center" valign="middle" >Signification</th></tr></thead><tr><td align="center" valign="middle" >T. oleae</td><td align="center" valign="middle" >Spinosad</td><td align="center" valign="middle" >22.071<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. cacoeciae</td><td align="center" valign="middle" >Spinosad</td><td align="center" valign="middle" >26.017<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. bourarachae</td><td align="center" valign="middle" >Spinosad</td><td align="center" valign="middle" >30.712<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. oleae</td><td align="center" valign="middle" >Deltamethrin</td><td align="center" valign="middle" >34.901<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. cacoeciae</td><td align="center" valign="middle" >Deltamethrin</td><td align="center" valign="middle" >36.007<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. bourarachae</td><td align="center" valign="middle" >Deltamethrin</td><td align="center" valign="middle" >35.002<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" ></td><td align="center" valign="middle" >(Deltamethrin-spinosad)</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >T. oleae</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >17.212<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. cacoeciae</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >19.314<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr><tr><td align="center" valign="middle" >T. bourarachae</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >23.059<sup>*</sup></td><td align="center" valign="middle" >0.000</td></tr></tbody></table></table-wrap><p>Mvd. Mean value differences <sup>*</sup>Significant difference (p &lt; 0.05, Dunnett’s test).</p><table-wrap id="table3" ><label><xref ref-type="table" rid="table3">Table 3</xref></label><caption><title> Effect of deltamethrin and spinosad treatments on the pupa-adult development time (adult emergence time) of Trichogramma species after the different exposure periods</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Trich. species period</th><th align="center" valign="middle" >Exposure</th><th align="center" valign="middle" >Adult emergence time (days) Deltamethrin</th><th align="center" valign="middle" >(&#177;SD)<sup>a</sup> Spinosad</th><th align="center" valign="middle" >Control</th></tr></thead><tr><td align="center" valign="middle" >T. oleae</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >8.10 &#177; 0.14<sup>a</sup></td><td align="center" valign="middle" >7.07 &#177; 0.23<sup>a</sup></td><td align="center" valign="middle" >6.05 &#177; 0.19<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >7.80 &#177; 0.45<sup>b</sup></td><td align="center" valign="middle" >6.93 &#177; 0.31<sup>a</sup></td><td align="center" valign="middle" >6.22 &#177; 0.22<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >7.20 &#177; 0.52<sup>b</sup></td><td align="center" valign="middle" >6.86 &#177; 0.51<sup>a</sup></td><td align="center" valign="middle" >6.35 &#177; 0.51<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >6.00 &#177; 0.41<sup>c</sup></td><td align="center" valign="middle" >6.03 &#177; 0.35<sup>b</sup></td><td align="center" valign="middle" >5.80 &#177; 0.47<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >6.41 &#177; 0.33<sup>c</sup></td><td align="center" valign="middle" >6.16 &#177; 0.51<sup>b</sup></td><td align="center" valign="middle" >6.25 &#177; 0.45<sup>a</sup></td></tr><tr><td align="center" valign="middle" >T. cacoeciae</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >7.73 &#177; 0.59<sup>a</sup></td><td align="center" valign="middle" >7.71 &#177; 0.50<sup>a</sup></td><td align="center" valign="middle" >6.56 &#177; 0.51<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >7.53 &#177; 0.51<sup>a</sup></td><td align="center" valign="middle" >7.17 &#177; 0.33<sup>b</sup></td><td align="center" valign="middle" >6.49 &#177; 0.11<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >7.50 &#177; 0.13<sup>a</sup></td><td align="center" valign="middle" >6.86 &#177; 0.81<sup>b</sup></td><td align="center" valign="middle" >6.33 &#177; 0.50<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >6.61 &#177; 0.74<sup>b</sup></td><td align="center" valign="middle" >6.29 &#177; 0.21<sup>c</sup></td><td align="center" valign="middle" >6.37 &#177; 0.54<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >06.09 &#177; 0.45<sup>b</sup></td><td align="center" valign="middle" >6.00 &#177; 0.53<sup>c</sup></td><td align="center" valign="middle" >6.13 &#177; 0.63<sup>a</sup></td></tr><tr><td align="center" valign="middle" >T. bourarachae</td><td align="center" valign="middle" >3 days</td><td align="center" valign="middle" >7.30 &#177; 0.12<sup>a</sup></td><td align="center" valign="middle" >7.06 &#177; 0.70<sup>a</sup></td><td align="center" valign="middle" >6.13 &#177; 0.74<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >10 days</td><td align="center" valign="middle" >7.28 &#177; 0.20<sup>a</sup></td><td align="center" valign="middle" >7.09 &#177; 0.79<sup>a</sup></td><td align="center" valign="middle" >6.03 &#177; 0.96<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >17 days</td><td align="center" valign="middle" >7.51 &#177; 0.02<sup>a</sup></td><td align="center" valign="middle" >6.86 &#177; 0.90<sup>a</sup></td><td align="center" valign="middle" >6.00 &#177; 0.94<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >24 days</td><td align="center" valign="middle" >7.57 &#177; 0.80<sup>a</sup></td><td align="center" valign="middle" >6.80 &#177; 0.45<sup>a</sup></td><td align="center" valign="middle" >6.06 &#177; 0.74<sup>a</sup></td></tr><tr><td align="center" valign="middle" ></td><td align="center" valign="middle" >31 days</td><td align="center" valign="middle" >07.21 &#177; 0.17<sup>a</sup></td><td align="center" valign="middle" >6.76 &#177; 0.79<sup>a</sup></td><td align="center" valign="middle" >6.00 &#177; 0.53<sup>a</sup></td></tr></tbody></table></table-wrap><p>a. Numbers in column followed by different letters are significantly different (Tukey test: p &lt; 0.05).</p></sec><sec id="s3_4"><title>3.4. Parasitism Viability of Trichogramma Species Based on the Treatment Periods</title><p>Percentage of reduction in parasitism viability of the three Trichogramma species differed between treatment and exposure time (days after spraying leaves). Figures 1-3 present percentages of parasitism viability reduction for each Trichogramma specie and corresponding exposure times. It appears that exposure time after insecticide spray had a significant effect on Trichogramma parasitism viability.</p><fig id="fig1"  position="float"><label><xref ref-type="fig" rid="fig1">Figure 1</xref></label><caption><title> Reduction in parasitism viability (%) of T. oleae at days after spraying leaves</title></caption><graphic mimetype="image"   position="float"  xlink:type="simple"  xlink:href="http://html.scirp.org/file/15-3700546x7.png"/></fig><fig id="fig2"  position="float"><label><xref ref-type="fig" rid="fig2">Figure 2</xref></label><caption><title> Reduction in parasitism viability (%) of T. cacoeciae at days after spraying leaves</title></caption><graphic mimetype="image"   position="float"  xlink:type="simple"  xlink:href="http://html.scirp.org/file/15-3700546x8.png"/></fig><fig id="fig3"  position="float"><label><xref ref-type="fig" rid="fig3">Figure 3</xref></label><caption><title> Reduction in parasitism viability (%) of T. bourarachae at days after spraying leaves</title></caption><graphic mimetype="image"   position="float"  xlink:type="simple"  xlink:href="http://html.scirp.org/file/15-3700546x9.png"/></fig><p>However, the effect seemed to differ not only from the spinosad to deltamethrin treatments but also from specie to specie. The deltamethrin treatment reduced parasitism viability of T. oleae at 50% even 17 days after parasitoid pupae exposure to residues on leaves. In fact, T. oleae displayed a parasitism viability of 80.3% in control assessment, reduced to 40.15% at 17 days after the treatment. Percentage of reduction in parasitism viability of T. oleae decreases to 36% on day 24% and to 13% on day 31 (<xref ref-type="fig" rid="fig1">Figure 1</xref>).</p><p>Spinosad exhibited low toxicity 10 days after exposure. For T. oleae, parasitism viability was similar to the control at 78.9% on day 24 and percentage of reduction in parasitism viability was 4.51% (<xref ref-type="fig" rid="fig1">Figure 1</xref>). For T. cacoeciae, deltamethrin was persisted at 31 days after the treatment showing a negative effect on parasitism viability, reduced to 44% (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Also, spinosad exhibited a negative effect on parasitism viability at 17 days after the treatment but 24 and 31 days after exposure to the treatments, reduction in parasitism viability rates decreased to 21% and 7.05% respectively (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><p>For T. bourarachae, reductions in parasitism viability curves for exposure time have the same shape for both insecticides. Indeed, reduction in parasitism viability gradually decreases with increasing time after insecticide spraying (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Deltamethrin and spinosad exhibited high reduction in parasitism viability of T. bourarachae at 3 days after treatment (72.13% and 66.70% respectively) but parasitism viability declined much more in than was the case for T. oleae or T. cacoeciae. At 31 days after parasitoid pupae exposure to residues on leaves, parasitism viability of T. bourarachae was reduced for the deltamethrin treatment at 35.5% and for the spinosad treatment at 25.5% (<xref ref-type="fig" rid="fig3">Figure 3</xref>).</p></sec></sec><sec id="s4"><title>4. Discussion</title><p>Parasitoids in the genus Trichogramma are reported to be excellent indicator species associated with the side effects of pesticides [<xref ref-type="bibr" rid="scirp.58104-ref25">25</xref>] . It was found that insecticides affected Trichogramma emergence from host eggs when exposed at different preimaginal stages of development (larval, prepupal, or pupal) [<xref ref-type="bibr" rid="scirp.58104-ref7">7</xref>] . The present study was designed to evaluate three Trichogramma species: T. oleae, T. cacoeciae and T. bourarachae for their susceptibility to deltamethrin and spinosad when exposed at pupal stage of development. These parasitoids were candidate to be used in pest management programmes against the second generation of P. oleae, as it is especially difficult to be managed with insecticides in most olive areas in Tunisia.</p><p>The insecticide concentrations tested were equivalent to those recommended for field application. Side effect evaluation is carried out by measuring parasitism viability rates and adult emergence time of Trichogramma species exposed to residues on olive foliage at different time intervals. These measures are important in order to investigate compatibility of Trichogramma species with insecticide residues after exposure under semi-field conditions. Most laboratory and field studies have shown that Trichogramma wasps are highly susceptible to broad-spectrum insecticides [<xref ref-type="bibr" rid="scirp.58104-ref26">26</xref>] . Consequently, use of insecticides and Trichogramma has historically been considered incompatible. The insecticides from the group of Piretroids were commonly described as harmful to beneficial arthropods [<xref ref-type="bibr" rid="scirp.58104-ref27">27</xref>] . According to the IOBC standard methods, deltamethrin is classified as harmful for adults and as slightly harmful for pupae of Trichogramma [<xref ref-type="bibr" rid="scirp.58104-ref28">28</xref>] . Similarly, this study demonstrate that exposure to deltamethrin resulted in parasitism viability reduction of the three Trichogramma species and showed slightly harmful effect for the parasitoid pupae of T. oleae and T. cacoeciae (30% &lt; E &lt; 79%). However, T. bourarachae exhibited moderately harmful effect 3 days after pupae exposure to deltamethrin residues on leaves (80% &lt; E &lt; 99%). Additionally, data show that residues from the deltamethrin treatments were remaining after 24 days. This result indicates that persistence ranged widely, from three to 31 days.</p><p>Spinosad, which is obtained from a naturally occurring soil organism, is not considered without risks on beneficials such as predatory insects [<xref ref-type="bibr" rid="scirp.58104-ref29">29</xref>] . However, although spinosad was slightly harmful at the outset of this study (30% &lt; E &lt; 79%), reduction in parasitism viability decreased after a period of exposure to the treatment. This result is similar to this reported by [<xref ref-type="bibr" rid="scirp.58104-ref12">12</xref>] , showing that spinosad degrade quickly following application in the field with residues no longer toxic by 7 to 10 days post-application. However, it appears from our results that susceptibility to spinosad differed from specie to another even after a period of exposure to the treatment.</p><p>Overall, the present experiment demonstrated that spinosad was less harmful, based on effect on parasitism viability, than deltamethrin to the three Trichogramma species. At the label rates, spinosad was harmless (E &lt; 30%) to T. oleae and T. cacoeciae and slightly harmful (30% &lt; E &lt; 79%) to T. bourarachae after exposure at 31 days. Spinosad appears to be more compatible with the three Trichogramma species than deltamethrin which exhibited moderately harmful effects to T. bourarachae (80% &lt; E &lt; 99%).</p><p>Differences regarding susceptibility to the two insecticides show that T. cacoeciae was more susceptible to spinosad than T. oleae. Also, T. bourarachae was more susceptible to the insecticides than T. oleae and T. cacoeciae with a decrease in parasitism viability when compared to the control. Besides longevity and fecundity that are key attribute for the quality of commercially available Trichogramma, the result of this study is important in biocontrol programs where the choice of less susceptible specie to insecticide can be critical.</p><p>Although some studies showed that pre-imaginal stages developing within host eggs appear to be protected from many insecticides, [<xref ref-type="bibr" rid="scirp.58104-ref26">26</xref>] reported that spinosad and other piretroids adversely affected Trichogramma emergence from host eggs when exposed at different pre-imaginal stages of development (larval, pre-pupal, or pupal). In addition, our results indicate that Trichogramma pupa is susceptible to residues of spinosad as well deltamethrin weathered on olive leaves for up to 3 at 31 days.</p><p>Adult emergence time (Pupa-adult development time) appeared to be increased for 1 to 2 days when Trichogramma pupa were exposed to insecticide residues at the shortest period after treatment, whereas, exposure at longer periods after the treatment seemed to eliminate this effect, especially, for T. oleae and T. cacoeciae. Thus, the increased length of the adult emergence time is likely related to the susceptibility of Trichogramma species in their response to insecticide residues at the different exposure periods.</p><p>Residue analysis provides a measure of the nature and level of any chemical contamination within the environment and of its persistence. However, it is often difficult to correlate pesticide residues in the environment with effects on fauna and/or ecological processes. Furthermore, when using insecticides, the relationship between the toxicant and the unit of live is greater for smaller organisms [<xref ref-type="bibr" rid="scirp.58104-ref30">30</xref>] . Thus, we were interested to show whether the insect parasitoid has been exposed to direct toxic effect of chemicals and to identify its potential use for future biological control programmes in the environment.</p><p>For the pesticide risk assessment, the exposure characterization describes the actual contact of pesticides and its residue with the parasitoid and the objective was to describe exposure in terms of live, susceptibility and parasitism viability of Trichogramma species. This study show that exposure of Trichogramma species to spinosad is probably only of concern for 3 - 15 days after spraying, However, exposure to deltamethrin residues is likely to be persistent effect on the parasitoid.</p><p>Even experiments were carried out under laboratory conditions where the parasitoid was probably submitted to the highest insecticide pressure, Trichogramma release period might be expected, avoiding harmful insecticide residue, to make a better contribution to P. oleae second generation control. More generally this can suggests that an insecticide-free environment needs to be considered to ensure maximum parasitism viability of Trichogramma in the field. Thus, under field conditions, adults’ emergency from parasitized pupa the time of release, would not be affected by the residual activity of the insecticides if Trichogramma emergence occurred 24 days after treatment (case of T. oleae and T. cacoeciae) and 31 days after treatment (case of T. bourarachae).</p><p>Nevertheless, this prediction requires validation by field studies for example under particular dry conditions that may result in greater persistence of residues [<xref ref-type="bibr" rid="scirp.58104-ref14">14</xref>] .</p><p>Releasing native species is important since these species have the advantage that they are already adapted to the local environmental conditions of olive tree. However, the susceptibility of a Trichogramma parasitoid to the insecticide may be influenced by the ecological attributes of the introduced species [<xref ref-type="bibr" rid="scirp.58104-ref31">31</xref>] . This is, in fact, one of the reasons that make the introduction of Trichogramma species to the field conditions an interesting topic of study in the broader area of insecticide effects. This laboratory study must be validated with a field one studying the interactions between biotic performance of Trichogramma specie with the weather, foliage, selective forms of application and dosages of insecticides, to obtain an adequate relationship between effectiveness of treatment with the parasitoid fecundity. Accordingly, further studies are currently under way to gain better insights on Trichogramma species effectiveness for biological control programs.</p></sec><sec id="s5"><title>Acknowledgements</title><p>The authors wish to express their gratitude to Dr. Annette Herz (BBA, Germany) for her Technical Cooperation within the International project INCODEV (TRIPHELIO): Sustainable Control of Lepidopterous Pests in Olive Groves-Integration of Egg Parasitoids and Pheromones. They would also like to thank all technicians in the Unit of Mass production and maintain of Trichogramma in Olive Tree Institute of Sfax, Tunisia.</p></sec><sec id="s6"><title>Cite this paper</title><p>ImenBlibech,MohieddineKsantini,TaiebJardak,MohamedBouaziz, (2015) Effect of Insecticides on Trichogramma Parasitoids Used in Biological Control against Prays oleae Insect Pest. Advances in Chemical Engineering and Science,05,362-372. doi: 10.4236/aces.2015.53038</p></sec><sec id="s7"><title>NOTES</title></sec></body><back><ref-list><title>References</title><ref id="scirp.58104-ref1"><label>1</label><mixed-citation publication-type="other" xlink:type="simple">Hoffmann, M.P., Pitcher, S.A., Cheever, S.A., Gardner, J., Losey, J.E., Kuhar, T.P., Curtis, A.L. and Youngman, R.R. (2006) Efficacy of Inoculative Releases of Trichogramma ostriniae (Hymenoptera: Trichogrammatidae) against European Corn Borer Ostrinia nubilalis (Lepidoptera: Crambidae) in Field Corn. Biological Control, 36, 345-349.  
http://dx.doi.org/10.1016/j.biocontrol.2005.10.008</mixed-citation></ref><ref id="scirp.58104-ref2"><label>2</label><mixed-citation publication-type="other" xlink:type="simple">Hegazi, E., Herz, A., Hassan, S.A., EKhafagi, W., Agamy, E., Zaitun, A., Gehan, A.E., Showeil, S., ElSaid, S. and Khamis, N. (2007) Field Efficiency of Indigenous Egg Parasitoids Hymenoptera, Trichogrammatidae) to Control the Olive Moth Prays oleae (Lepidoptera, Yponomeutidae) and the Jasmine Moth Palpita unionalis (Lepidoptera, Pyralidae) in an Olive Plantation in Egypt. Biological Control, 43, 171-187.  
http://dx.doi.org/10.1016/j.biocontrol.2007.07.009</mixed-citation></ref><ref id="scirp.58104-ref3"><label>3</label><mixed-citation publication-type="other" xlink:type="simple">Tzanakakis, M.E. (2003) Seasonal Development and Dormancy of Insects and Mites Feeding on Olive: A Review. Netherlands Journal of Zoology, 52, 87-224. http://dx.doi.org/10.1163/156854203764817670</mixed-citation></ref><ref id="scirp.58104-ref4"><label>4</label><mixed-citation publication-type="other" xlink:type="simple">Jardak, T. (1980) Etudes bioécologiques de Prays oleae BERN (Lepidoptera, Hyponomeutidae) et de ses parasites oophages du genre Trichogramma (Hymenoptera, Trichogrammatidae). Essais d’utilisation en lutte biologique. Thèse de 3ème cycle, Aix Marseille Université, 5-42.</mixed-citation></ref><ref id="scirp.58104-ref5"><label>5</label><mixed-citation publication-type="other" xlink:type="simple">Smith, S.M. (1996) Biological Control with Trichogramma: Advances, Successes and Potential of Their Use. Annual Review of Entomology, 41, 375-406. http://dx.doi.org/10.1146/annurev.en.41.010196.002111</mixed-citation></ref><ref id="scirp.58104-ref6"><label>6</label><mixed-citation publication-type="other" xlink:type="simple">Franz, J.M., Bogenschutz, H., Hassan, S.A., Huang, P., Naton, E., Suter, H. and Viggiani, G. (1980) Results of a Joint Pesticide Test Programme by the Working Group: Pesticides and Beneficial Arthropods. Entomophaga, 25, 231-236.  
http://dx.doi.org/10.1007/BF02371922</mixed-citation></ref><ref id="scirp.58104-ref7"><label>7</label><mixed-citation publication-type="other" xlink:type="simple">Hassan, S.A., Hafes, B., Degrande, P.E. and Herai, K. (1998) The Side-Effects Of Pesticides on the Egg Parasitoid Trichogramma cacoeciae Marchal (Hym., Trichogrammatidae), Acute Dose-Response and Persistence Tests. Journal of Applied Entomology, 122, 569-573. http://dx.doi.org/10.1111/j.1439-0418.1998.tb01547.x</mixed-citation></ref><ref id="scirp.58104-ref8"><label>8</label><mixed-citation publication-type="other" xlink:type="simple">Thompson, G.D., Dutton, R. and Sparks, T.C. (2000) Spinosad—A Case Study: An Example from a Natural Products Discovery Programme. Pest Management Science, 56, 696-702.  
http://dx.doi.org/10.1002/1526-4998(200008)56:8&lt;696::AID-PS182&gt;3.0.CO;2-5</mixed-citation></ref><ref id="scirp.58104-ref9"><label>9</label><mixed-citation publication-type="other" xlink:type="simple">C&amp;#244;nsoli, F.L., Parra, J.R.P. and Hassan, S.A. (2009) Side-Effects of Insecticides Used in Tomato Fields on the Egg Parasitoid Trichogramma pretiosum Riley (Hym., Trichogrammatidae), a Natural Enemy of Tuta absoluta (Meyrick) (Lep., Gelechiidae). Journal of Applied Entomology, 122, 43-47. http://dx.doi.org/10.1111/j.1439-0418.1998.tb01459.x</mixed-citation></ref><ref id="scirp.58104-ref10"><label>10</label><mixed-citation publication-type="other" xlink:type="simple">Bret, B.L., Larson, L.L., Schoonover, J.R., Sparks, T.C. and Thompson, G.D. (1997) Biological Properties of Spinosad. Down to Earth, 52, 6-13.</mixed-citation></ref><ref id="scirp.58104-ref11"><label>11</label><mixed-citation publication-type="other" xlink:type="simple">Saunders, D.G. and Bret, B.L. (1997) Fate of Spinosad in the Environment. Down to Earth, 52, 14-20.</mixed-citation></ref><ref id="scirp.58104-ref12"><label>12</label><mixed-citation publication-type="other" xlink:type="simple">Thomson, L.J., Glenn, D.C. and Hoffmann, A.A. (2000) Effects of Sulfur on Trichogramma Egg Parasitoids in Vineyards: Measuring Toxic Effects and Establishing Release Windows. Australian Journal of Experimental Agriculture, 40, 1165-1171. http://dx.doi.org/10.1071/EA00074</mixed-citation></ref><ref id="scirp.58104-ref13"><label>13</label><mixed-citation publication-type="other" xlink:type="simple">Thompson, G. and Hutchins, S. (1999) Spinosad. Pesticide Outlook, 10, 78-81.</mixed-citation></ref><ref id="scirp.58104-ref14"><label>14</label><mixed-citation publication-type="other" xlink:type="simple">Williams, T., Valle, J. and Vinuela, E. (2003) Is the Naturally-Derived Insecticide Spinosad&amp;reg; Compatible with Insect Natural Enemies? Biocontrol Science and Technology, 13, 459-475. http://dx.doi.org/10.1080/0958315031000140956</mixed-citation></ref><ref id="scirp.58104-ref15"><label>15</label><mixed-citation publication-type="other" xlink:type="simple">Pietrantonio, P.V. and Benedict, J.H. (1999) Effect of New Cotton Insecticide Chemistries, Tebufenozide, Spinosad and Chlorfenapyr, on Orius insidiosus and Two Cotesia Species. Southwestern Entomologist, 24, 21-29.</mixed-citation></ref><ref id="scirp.58104-ref16"><label>16</label><mixed-citation publication-type="other" xlink:type="simple">Tillman, P.G. and Mulrooney, J.E. (2000) Effect of Selected Insecticides on the Natural Enemies Coleomegilla maculata and Hippodamia convergens (Coleoptera: Coccinelidae), Geocoris punctipes (Hemiptera: Lygaeidae), and Bracon mellitor, Cardiochiles nigriceps and Cotesia marginiventris (Hymenoptera: Braconidae) in Cotton. Journal of Economic Entomology, 93, 1638-1643. http://dx.doi.org/10.1603/0022-0493-93.6.1638</mixed-citation></ref><ref id="scirp.58104-ref17"><label>17</label><mixed-citation publication-type="other" xlink:type="simple">Mason, P.G., Erlandson, M.A., Elliott, R.H. and Harris, B.J. (2002) Potential Impact of Spinosad on Parasitoids of Mamestra configurata (Lepidoptera: Noctuidae). Canadian Entomologist, 134, 59-68. 
http://dx.doi.org/10.4039/Ent13459-1</mixed-citation></ref><ref id="scirp.58104-ref18"><label>18</label><mixed-citation publication-type="other" xlink:type="simple">Pawlisz, A.V., Busnarda, J., McLauchlin, A., Caux, P.Y. and Kent, R.A. (1998) Canadian Water Quality Guidelines for Deltamethrin. Environmental Toxicology and Water Quality, 13, 175-210. 
http://dx.doi.org/10.1002/(SICI)1098-2256(1998)13:3&lt;175::AID-TOX1&gt;3.0.CO;2-4</mixed-citation></ref><ref id="scirp.58104-ref19"><label>19</label><mixed-citation publication-type="journal" xlink:type="simple"><name name-style="western"><surname>Arambourg</surname><given-names> Y. </given-names></name>,<etal>et al</etal>. (<year>1966</year>)<article-title>La teigne de l’olivier. A. S</article-title><source> Balachowsky Entomologie appliquée à l’agriculture</source><volume> 2</volume>,<fpage> 181</fpage>-<lpage>193</lpage>.<pub-id pub-id-type="doi"></pub-id></mixed-citation></ref><ref id="scirp.58104-ref20"><label>20</label><mixed-citation publication-type="book" xlink:type="simple">Hassan, S.A., Halsall, N., Gray, A.P., Kuehner, C., Moll, M.F., Bakker, M., Roembke, J., Yousef, A., Nasr, F. and Abdelgader, H. (2000) A Laboratory Method to Evaluate the Side Effects of Plant Protection Products on Trichogramma cacoeciae Marchal (Hym., Trichogrammatidae). In: Candolfi, M.P., et al., Eds., Guidelines to Evaluate Side-Effects of Plant Protection Products to Non-Target Arthropods, IOBC/WPRS, 107-119.</mixed-citation></ref><ref id="scirp.58104-ref21"><label>21</label><mixed-citation publication-type="other" xlink:type="simple">Shu, C.P.C., Harles, P., Orr, D.B. and Van Duyn, J.W. (2000) Effect of Insecticides on Trichogramma Exiguum (Trichogrammatidae: Hymenoptera) Preimaginal Development and Adult Survival. Journal of Economic Entomology, 93, 577-583. http://dx.doi.org/10.1603/0022-0493-93.3.577</mixed-citation></ref><ref id="scirp.58104-ref22"><label>22</label><mixed-citation publication-type="other" xlink:type="simple">Hommay, G., Gertz, C., Kienlen, J.C., Pizzol, J. and Chavigny, P. (2002) Comparison between the Control Efficacy of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) and Two Trichogramma cacoeciae Marshal Strains against Grape Moth (Lobesia botrana Den and Schiff.), Depending on Release Density. Biocontrol Science and Technology, 12, 569-581. http://dx.doi.org/10.1080/0958315021000016234</mixed-citation></ref><ref id="scirp.58104-ref23"><label>23</label><mixed-citation publication-type="other" xlink:type="simple">Melo, R.L., Pratissoli, D., Polanczyk, R.A., Melo, D.F., Barros, R. and Milanez, A.M. (2007) Biology and Thermal Requirements of Trichogramma atopovirilia Oatman &amp; Platner (Hymenoptera: Trichogrammatidae) Parasitizing Eggs of Diaphania hyalinata L. (Lepidoptera: Pyralidae). Neotropical Entomology, 36, 431-435. 
http://dx.doi.org/10.1590/S1519-566X2007000300011</mixed-citation></ref><ref id="scirp.58104-ref24"><label>24</label><mixed-citation publication-type="other" xlink:type="simple">Herz, A., Hassan, S.A., Hegazi, E., Khafagi, W.E., Nasr, F.N., Youssef, A.A., Agamy, E., Blibech, I., Ksentini, I., Ksantini, M., Jardak, T., Bento, A., Pereira, J.A., Torres, L., Souliotis, C., Moschos, T. and Milonas, P. (2007) Egg Parasitoids of the Genus Trichogramma (Hymenoptera, Trichogrammatidae) in Olive Groves of the Mediterranean Region. Biological Control, 40, 48-56. http://dx.doi.org/10.1016/j.biocontrol.2006.08.002</mixed-citation></ref><ref id="scirp.58104-ref25"><label>25</label><mixed-citation publication-type="journal" xlink:type="simple"><name name-style="western"><surname>Hassan</surname><given-names> S.A. </given-names></name>,<etal>et al</etal>. (<year>1994</year>)<article-title>Guideline for the Evaluation of Side Effect of Plant Protection Products on Trichogramma cacoeciae Marchal (Hym., Trichogrammatidae)</article-title><source> IOBC Bulletin</source><volume> 2</volume>,<fpage> 119</fpage>-<lpage>128</lpage>.<pub-id pub-id-type="doi"></pub-id></mixed-citation></ref><ref id="scirp.58104-ref26"><label>26</label><mixed-citation publication-type="other" xlink:type="simple">Li, K.H., Xu, X., Li, Y.F., Meng, Q.Z. and Zhou, L.C (1986) Determination of Toxicity of 29 Chemicals to Trichogramma japonicum at Various Developmental Stages. Natural Enemies of Insects, 8, 187-194.</mixed-citation></ref><ref id="scirp.58104-ref27"><label>27</label><mixed-citation publication-type="other" xlink:type="simple">Canête, C.L. (2005) Seletividade de inseticidas a espécies de Trichogramma (Hymenoptera: Trichogrammatidae). Tesis (Doutorado em Ciências-Zoologia), Universidade Federal do Paraná, Curitiba, 117 p.</mixed-citation></ref><ref id="scirp.58104-ref28"><label>28</label><mixed-citation publication-type="journal" xlink:type="simple"><name name-style="western"><surname>Hassan</surname><given-names> S.A. </given-names></name>,<etal>et al</etal>. (<year>1998</year>)<article-title>The Suitability of Trichogramma cacoeciae as an Indicator Species for Testing the Side Effects of Pesticides on Beneficial Arthropods, Compared to Other Hymenopterous Parasitoids</article-title><source> IOBC/WPRS Bulletin</source><volume> 21</volume>,<fpage> 89</fpage>-<lpage>92</lpage>.<pub-id pub-id-type="doi"></pub-id></mixed-citation></ref><ref id="scirp.58104-ref29"><label>29</label><mixed-citation publication-type="other" xlink:type="simple">Cisneros, J., Goulson, D., Derwent, L.C., Penagos, D.I., Hernández, O. and Williams, T. (2002) Toxic Effects of Spinosad on Predatory Insects. Biological Control, 23, 156-163. http://dx.doi.org/10.1006/bcon.2001.1000</mixed-citation></ref><ref id="scirp.58104-ref30"><label>30</label><mixed-citation publication-type="other" xlink:type="simple">Araya, J.E., Araya, M. and Guerrero, M.A. (2010) Effects of Some Insecticides Applied in Sublethal Concentrations on the Survival and Longevity of Aphidius ervi (Haliday) (Hymenoptera: Aphidiidae) Adults. Chilean Journal of Agricultural Research, 70, 221-227. http://dx.doi.org/10.4067/S0718-58392010000200005</mixed-citation></ref><ref id="scirp.58104-ref31"><label>31</label><mixed-citation publication-type="other" xlink:type="simple">Carrillo, D., Jorge, E., Pe&amp;#241;a, M. and Rogers, E. (2009) Relative Susceptibility of Haeckeliania sperata (Hymenoptera: Trichogrammatidae) to Pesticides Used in Citrus and Ornamental Systems in Florida. Journal of Economic Entomology, 102, 905-912. http://dx.doi.org/10.1603/029.102.0307</mixed-citation></ref></ref-list></back></article>