Host-Induced Plant Volatiles Mediate Ability of the Parasitoid Microplitis croceipes to Discriminate Between Unparasitized and Parasitized Heliothis virescens Larvae and Avoid Superparasitism.

In solitary endoparasitoids, oviposition in a host previously parasitized by a conspecific (superparasitism) leads to intraspecific competition, resulting in the elimination of all but one parasitoid offspring. Therefore, avoidance of parasitized hosts presents a strong selective advantage for such parasitoid species. Parasitoids use herbivore-induced plant volatiles (HIPVs) to find their hosts. In this study, we evaluated the ability of Microplitis croceipes (Hymenoptera: Braconidae) to discriminate between unparasitized and parasitized Heliothis virescens (Lepidoptera: Noctuidae) larvae using cotton plant odors as cues. A combination of behavioral and analytical techniques were used to test two hypotheses: (i) parasitoids will show preference for plant odors induced by unparasitized hosts over odors induced by parasitized hosts, and (ii) the parasitism status of herbivores affects HIPV emission in plants. Heliothis virescens larvae were parasitized for varying durations (0, 2 and 6-days after parasitism (DAP)). In four-choice olfactometer bioassays, female M. croceipes showed greater attraction to plant odors induced by unparasitized hosts compared to plant odors induced by parasitized hosts (2 and 6-DAP). Comparative gas chromatography-mass spectrometry analyses of cotton volatiles indicated reduced emission of 10 out of 21 identified compounds from plants infested by parasitized hosts compared with plants infested by unparasitized hosts. The results suggest that changes in plant volatile emission due to the parasitism status of infesting herbivores affect recruitment of parasitoids. Avoidance of superparasitism using plant odors optimizes host foraging in M. croceipes, and this strategy may be widespread in solitary parasitoid species.

Comparative Evaluation of Disease Induced by Three Strains of Tobacco etch virus in Capsicum annuum L.

Tobacco etch virus (TEV; genus Potyvirus) strains HAT, Mex21, and N were evaluated comparatively for their pathogenicity and effects on growth of Capsicum annuum L. 'Calwonder'. Each TEV strain induced an initial systemic symptom of vein-clearing but subsequent disease symptoms ranged from mild (HAT) to moderate (Mex21) to severe (N). Effects on plant growth parameters closely reflected disease symptoms induced by each TEV strain. HAT-infected Calwonder plants did not differ from the healthy control for plant height, internode lengths, and aboveground fresh weight of shoots. Root dry weight, however, was less for HAT-infected plants than for the healthy control. Mex21 affected plants more severely, with significantly shorter plant height (at 20, 30, and 40 days postinoculation), reduced root dry weight, and shortened internodes compared with HAT and healthy control treatments. Aboveground fresh weight of Mex21-infected plants was significantly less than for the healthy control. N induced significant negative effects relative to each of the other treatments for plant height, aboveground shoot fresh weight, root dry weight, and internode lengths. The effects on Calwonder fruit production mimicked disease severity and effects on plant growth for the respective TEV strains.

Identification of Key Plant-Associated Volatiles Emitted by Heliothis virescens Larvae that Attract the Parasitoid, Microplitis croceipes: Implications for Parasitoid Perception of Odor Blends.

Herbivores emit plant-associated volatile organic compounds (VOCs) after feeding on plants. These plant-associated VOCs can be used by parasitoids to locate their hosts. It is hypothesized that certain compounds play key roles in the attractiveness of host-associated odor blends. The larval parasitoid, Microplitis croceipes (Hymenoptera: Braconidae) and its herbivore host, Heliothis virescens (Lepidoptera: Noctuidae), a major pest of cotton plant were used as model species to identify key compounds mediating attraction of parasitoids to hosts. Comparative GC/MS analyses of cotton-fed vs. artificial diet-fed hosts indicated that 12 of 17 compounds in the headspace of H. virescens larvae were exclusive to plant-fed hosts, and thus considered to be plant-associated. In order to identify key attractive compounds, a full blend of 15 commercially available synthetic compounds was modified by removing each of the 10 plant-associated compounds emitted by host larvae. In Y-tube olfactometer bioassays testing parasitoid responses to modified blends, 1-octen-3-ol, decanal, (E)-ß-caryophyllene, α-humulene, α-farnesene, and ß-pinene were identified as key compounds contributing to attractiveness of the natural blend of VOCs emitted by cotton-fed hosts. The results showed that while various host-associated compounds act in concert to serve as useful host location cues, only a fraction of the natural blend mediates attraction in parasitoids. Furthermore, the role of a compound is better assessed in the context of other compounds, and odor blends are better perceived as a whole rather than as individual components.

Duration of plant damage by host larvae affects attraction of two parasitoid species (Microplitis croceipes and Cotesia marginiventris) to cotton: implications for interspecific competition.

Volatile organic compounds (VOCs) released by herbivore-damaged plants can guide parasitoids to their hosts. The quantity and quality of VOC blends emitted by plants may be affected by the duration of plant damage by herbivores, which could have potential ramifications on the recruitment of competing parasitoids. We used two parasitoid species, Microplitis croceipes and Cotesia marginiventris (Hymenoptera: Braconidae), to address the question of whether duration of plant damage affects parasitoid use of plant VOCs for host location. Both wasp species are larval endoparasitoids of Heliothis virescens (Lepidoptera: Noctuidae), an important pest of cotton. Attraction of the two parasitoid species to odors emitted by undamaged (UD), fresh (6 h infestation) damage (FD), and old (24 h infestation) damage (OD) cotton plants infested by H. virescens larvae was investigated using a headspace volatile collection system coupled with four-choice olfactometer bioassay. Both sexes of M. croceipes showed a preference for FD- and OD-plant odors over UD-plants. On the other hand, more C. marginiventris females were attracted to UD- and FD-plants than to OD-plants. GC/MS analyses showed qualitative and quantitative differences in the VOC profiles of UD, FD, and OD-plants, which may explain the observed preferences of the parasitoids. These results suggest a temporal partitioning in the recruitment of M. croceipes and C. marginiventris to H. virescens-damaged cotton, and may have potential implications for interspecific competition between the two parasitoid species.

Kudzu Bug (Megacopta cribraria) and Associated Egg Parasitoids Emergence Rates in Alabama are Predicted by Weather Indices.

The kudzu bug, Megacopta cribraria (Fabricius) (Hemiptera: Plataspidae), is a major economic pest of soybean in the southeastern United States. With climate warming, this pest is expected to move northward and cause additional crop damage. Parasitoid biocontrol is a potential method of integrated pest management for kudzu bug. Two species of egg parasitoid wasps have been observed emerging from kudzu bug egg masses in the southeastern United States: Paratelenomus saccharalis (Dodd) (Hymenoptera: Platygastridae) and Ooencyrtus nezarae (Ishii) (Hymenoptera: Encyrtidae). This paper used egg mass emergence data collected between 2018 and 2020 in Alabama soybean fields and compared the data to weather indices. Indices included the number of days with minimum temperatures below zero, accumulated rainfall (mm m-2), as well as species specific metrics of accumulated growing degree days, accumulated daily minimum temperature (°C), and accumulated daily maximum temperature (°C). Emergence of the generalist parasitoid, O. nezarae, was highly correlated with kudzu bug nymph abundance, accumulated degree day, accumulated daily temperatures, and precipitation. Ooencyrtus nezarae emergence was predicted in a stepwise regression equation by aggregated degree day and date of collection, which indicates that seasonality may be a predictor of its presence. In contrast, collections of the specialist parasitoid, P. saccharalis, were near-zero throughout the collection period, suggesting that this species may no longer be a usable biocontrol agent in the southeastern United States as a result of external limiting factors.

The role of herbivore- and plant-related experiences in intraspecific host preference of a relatively specialized parasitoid.

Parasitoids use odor cues from infested plants and herbivore hosts to locate their hosts. Specialist parasitoids of generalist herbivores are predicted to rely more on herbivore-derived cues than plant-derived cues. Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) is a relatively specialized larval endoparasitoid of Heliothis virescens (F.) (Lepidoptera: Noctuidae), which is a generalist herbivore on several crops including cotton and soybean. Using M. croceipes/H. virescens as a model system, we tested the following predictions about specialist parasitoids of generalist herbivores: (i) naive parasitoids will show innate responses to herbivore-emitted kairomones, regardless of host plant identity and (ii) herbivore-related experience will have a greater influence on intraspecific oviposition preference than plant-related experience. Inexperienced (naive) female M. croceipes did not discriminate between cotton-fed and soybean-fed H. virescens in oviposition choice tests, supporting our first prediction. Oviposition experience alone with either host group influenced subsequent oviposition preference while experience with infested plants alone did not elicit preference in M. croceipes, supporting our second prediction. Furthermore, associative learning of oviposition with host-damaged plants facilitated host location. Interestingly, naive parasitoids attacked more soybean-fed than cotton-fed host larvae in two-choice tests when a background of host-infested cotton odor was supplied, and vice versa. This suggests that plant volatiles may have created an olfactory contrast effect. We discussed ecological significance of the results and concluded that both plant- and herbivore-related experiences play important role in parasitoid host foraging.

Do age and mating status affect olfactory response of the parasitoid, Microplitis croceipes (Hymenoptera: Braconidae) to host-related plant odors?

Background: Parasitic wasps (parasitoids) use volatile organic compounds released by herbivore-infested plants to locate their hosts. Response of parasitoids to plant odors may be plastic and dependent on their physiological state. Using Microplitis croceipes (Hymenoptera: Braconidae), a relatively specialized larval endoparasitoid of Heliothis virescens (Lepidoptera: Noctuidae), we asked whether age and mating status of parasitoids affect their olfactory response to host-related odors. Methods: Four odor stimuli of varying complexity were selected based on previous reports of parasitoid response to cotton volatiles: cis-3-hexenol (a green leaf volatile), α-pinene (a constitutive monoterpene), a 50/50 v/v binary mixture ( cis-3-hexenol + α-pinene), and H. virescens-infested cotton odors. Female M. croceipes used in Y-tube olfactometer bioassays were either mated or unmated, and grouped 1-3, 4-6, and 7-9 d-old. Female parasitoids used in electroantennogram (EAG) recording were mated and grouped 1-3, 4-6, 7-9 and 10-12 d-old. Results: In Y-tube olfactometer bioassays, neither age nor mating status played a major role in the attraction of parasitoids to test odor stimuli, with two exceptions: 4-6 d-old mated parasitoids showed attraction to the binary mixture, and 1-3 d-old mated parasitoids showed attraction to H. virescens-infested cotton. Age did not affect EAG response of parasitoids to test stimuli. Conclusions: The present results suggest that age and mating status do not play a major role in modulating olfactory responses of M. croceipes to host-related plant odors. Instead, plasticity of olfactory response may be limited in M. croceipes due to strong innate sensitivity to host-related odor cues.

Plant-associated odor perception and processing in two parasitoid species with different degrees of host specificity: Implications for host location strategies.

Microplitis croceipes and Cotesia marginiventris (Hymenoptera: Braconidae) are parasitoids of lepidopteran larvae with different degrees of host specificity. Both parasitoid species rely on host-related plant volatiles as odor cues to locate their herbivore hosts. To better understand mechanisms of odor processing in parasitoids, we tested responses of olfactory sensory neurons (OSNs) in the antennal sensilla placodea of female parasitoids to select plant volatiles and mixtures. The compounds tested include two green leaf volatiles (i.e., cis-3-hexenol and hexanal) and three herbivore-induced plant volatiles (i.e., cis-3-hexenyl butyrate, cis-3-hexenyl acetate and linalool). Single-sensillum recording showed that the test compounds elicited activity in large and small amplitude neurons housed in the short sensilla placodea of both parasitoid species. In general, C. marginiventris showed greater OSN responses to a low dose while M. croceipes showed greater responses to a high dose of test compounds. Binary mixtures of cis-3-hexenol and linalool inhibited OSN activity in M. croceipes, but not in C. marginiventris. These differences may have implications for odor discrimination in the two parasitoid species. In addition, anterograde neurobiotin stainings were performed to map glomerular projections of OSNs in the antennal lobe of the parasitoids. In M. croceipes, a mixture of cis-3-hexenol and linalool inhibited activity of the glomerulus activated by cis-3-hexenol alone. In C. marginiventris, a mixture of cis-3-hexenol and cis-3-hexenyl acetate showed intense labeling in their respective glomeruli, possibly suggesting a synergistic interaction. These differences in detection and coding of single compounds and mixtures may impact host location strategies in the two parasitoid species.

Sugar Diet Affects Odor Reception but Variation in Sugar Concentration Plays Minimal Role in the Response of the Parasitoid, Microplitis croceipes (Hymenoptera: Braconidae), to Host-Related Plant Volatiles.

Parasitoids utilize various sugar resources in nature, and rely on odor cues from plants to locate their food and hosts. However, lack of sugar in the diet may negatively impact odor reception in parasitoids, thus affecting foraging efficiency. We used Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), a larval endoparasitoid of Heliothis virescens (F.) (Lepidoptera: Noctuidae), as a model species to test the hypothesis that variation in sugar diet of parasitoids affects their olfactory response to host-related odors. Heliothis virescens is a major pest of cotton and other important crops. Response of female M. croceipes fed different diet treatments (i.e., 40%, 20%, 10%, or 0% sucrose/water solution [w/v]) to select cotton volatiles were tested in electroantennogram (EAG) and Y-tube olfactometer bioassays. The following cotton plant odors were tested: cis-3-hexenol, α-pinene, 50/50 v/v binary mixture of cis-3-hexenol and α-pinene, and H. virescens-infested cotton. Sucrose-fed parasitoids showed higher EAG response to the binary mixture and host-infested plant volatile extract, compared with sucrose-starved (0% sucrose) parasitoids. However, there was no significant difference in EAG response of parasitoids to odor treatments among individuals fed 40%, 20%, or 10% sucrose. In a Y-tube olfactometer, female M. croceipes fed 40% sucrose were significantly more attracted to host-infested cotton than to a control (no plant). However, parasitoids were not significantly attracted to other odor stimuli. These results suggest that the availability of sugar diet affects odor reception in M. croceipes but variation in sugar concentration probably plays a minimal role in olfactory response of M. croceipes to host-related odors.

Electroantennogram response of the parasitoid, Microplitis croceipes to host-related odors: The discrepancy between relative abundance and level of antennal responses to volatile compound.

Herbivores emit volatile organic compounds (VOCs) after feeding on plants. Parasitoids exploit these VOCs as odor cues to locate their hosts. In nature, host-related odors are emitted as blends of various compounds occurring in different proportions, and minor blend components can sometimes have profound effects on parasitoid responses. In a previous related study, we identified and quantified VOCs emitted by cotton plant-fed Heliothis virescens (Lepidoptera: Noctuidae) larvae, an herbivore host of the parasitoid Microplitis croceipes (Hymenoptera: Braconidae). In the present study, the olfactory response of female M. croceipes to synthetic versions of 15 previously identified compounds was tested in electroantennogram (EAG) bioassays. Using M. croceipes as a model species, we further asked the question: does the relative abundance of a volatile compound match the level of antennal response in parasitoids? Female M. croceipes showed varying EAG responses to test compounds, indicating different levels of bioactivity in the insect antenna. Eight compounds, including decanal, 1-octen-3-ol, 3-octanone, 2-ethylhexanol, tridecane, tetradecane, α-farnesene and bisabolene, elicited EAG responses above or equal to the 50 th percentile rank of all responses. Interestingly, decanal, which represented only 1% of the total amount of odors emitted by cotton-fed hosts, elicited the highest (0.82 mV) EAG response in parasitoids. On the other hand, ( E)-ß-caryophyllene, the most abundant (29%) blend component, elicited a relatively low (0.17 mV) EAG response. The results suggest that EAG response to host-related volatiles in parasitoids is probably more influenced by the ecological relevance or functional role of the compound in the blend, rather than its relative abundance.