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In response to herbivory, plants emit volatile compounds that play important roles in plant defense. Herbivore-induced plant volatiles (HIPVs) can deter herbivores, recruit natural enemies, and warn other plants of possible herbivore attack. Following HIPV detection, neighboring plants often respond by enhancing their anti-herbivore defenses, but a recent study found that herbivores can manipulate HIPV-interplant communication for their own benefit and suppress defenses in neighboring plants. Herbivores induce species-specific blends of HIPVs and how these different blends affect the specificity of plant defense responses remains unclear. Here we assessed how HIPVs from zucchini plants (Cucurbita pepo) challenged with different herbivore species affect resistance in neighboring plants. Volatile “emitter” plants were damaged by one of three herbivore species: saltmarsh caterpillars (Estigmene acrea), squash bugs (Anasa tristis), or striped cucumber beetles (Acalymma vittatum), or were left as undamaged controls. Neighboring “receiver” plants were exposed to HIPVs or control volatiles and then challenged by the associated herbivore species. As measures of plant resistance, we quantified herbivore feeding damage and defense-related phytohormones in receivers. We found that the three herbivore species induced different HIPV blends from squash plants. HIPVs induced by saltmarsh caterpillars suppressed defenses in receivers, leading to greater herbivory and lower defense induction compared to controls. In contrast, HIPVs induced by cucumber beetles and squash bugs did not affect plant resistance to subsequent herbivory in receivers. Our study shows that herbivore species identity affects volatile-mediated interplant communication in zucchini, revealing a new example of herbivore defense suppression through volatile cues.

Mating with the wrong species is surprisingly common in nature. Interspecific mating can lead to reproductive interference, where wasted time, energy, nutrients, or gametes reduces the fitness of one or both of the interacting species. However, the ecological and evolutionary forces that maintain this seemingly maladaptive behavior remain poorly understood, in part because the natural complexity of heterospecific encounters is often not considered experimentally. The goal of this study was to directly test if the negative effects of heterospecific mating can be mitigated by conspecific mating. We used two closely related species of squash bug, Anasa tristis and Anasa andresii, which are known to readily mate with each other despite clear negative fitness consequences. We gave all females opportunities to mate with conspecific males before and after encountering heterospecific males. We found that A. tristis females can alleviate temporary bouts of heterospecific interference when given opportunities to mate with conspecifics. However, we found the opposite for A. andresii females. Mating with conspecifics did not shelter female A. andresii from the consequences of heterospecific mating. Our study reveals the complex dynamics of reproductive interference and highlights scenarios where mating with the wrong species can have either minimal or long-lasting effects on fitness. We emphasize the benefit of assessing reproductive interference using experiments that not only vary heterospecific encounter rates, but that also quantify lifetime measures of fitness. Our study adds to the growing body of research highlighting the importance of reproductive interference and sheds light on why this seemingly paradoxical behavior continues to persist.

This study examined the ovipositional behavior of Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae) by evaluating how parasitoid density and access to nutrition influenced percent parasitism on egg masses of different sizes in laboratory tests. When three parasitoids were exposed to A. tristis egg masses with only three to five eggs, 72.7% of parasitoids became trapped in the eggs and failed to emerge successfully. These results suggest that competition between larvae within the egg may have reduced the fitness of the surviving parasitoid. Continual access to honey water did not significantly influence parasitism rates on A. armigera egg masses and only increased parasitism on A. tristis egg masses with 20–25 eggs. Overall, parasitism rates were higher on A. armigera egg masses than on A. tristis egg masses, and parasitoids were more likely to emerge successfully from A. armigera eggs than from A. tristis eggs. Parasitoids spent the same amount of time probing eggs of the two species, but they spent significantly more time drilling into A. tristis eggs than A. armigera eggs. Measurements taken using transmission electron microscopy determined that the average combined width of the epicuticle and exocuticle of the egg chorion was significantly greater for A. tristis eggs than for A. armigera eggs. This difference may account for the lower rates of parasitism and parasitoid emergence and for the increased time spent drilling into A. tristis eggs compared with A. armigera eggs.

Many insects harbor microbial symbiotic partners that offer protection against pathogens, parasitoids, and other natural enemies. Mounting evidence suggests that these symbiotic microbes can play key roles in determining infection outcomes in insect vectors, making them important players in the quest to develop novel vector control strategies. Using the squash bug Anasa tristis, we investigated how the presence of Caballeronia symbionts affected the persistence and intensity of phytopathogenic Serratia marcescens within the insect vector. We reared insects aposymbiotically and with different Caballeronia isolates, infected them with S. marcescens, and then sampled the insects periodically to assess the intensity and persistence of pathogen infection. Squash bugs harboring Caballeronia consistently had much lower-intensity infections and cleared S. marcescens significantly faster than their aposymbiotic counterparts. These patterns held even when we reversed the timing of exposure to symbiont and pathogen. Taken together, these results indicate that Caballeronia symbionts play an essential role in S. marcescens infection outcomes in squash bugs and could be used to alter vector competence to enhance agricultural productivity in the future. IMPORTANCE Insect-microbe symbioses have repeatedly been shown to profoundly impact an insect’s ability to vector pathogens to other hosts. The use of symbiotic microbes to control insect vector populations is of growing interest in agricultural settings. Our study examines how symbiotic microbes affect the dynamics of a plant pathogen infection within the squash bug vector Anasa tristis, a well-documented pest of squash and other cucurbit plants and a vector of Serratia marcescens, the causative agent of cucurbit yellow vine disease. We provide evidence that the symbiont Caballeronia prevents successful, long-term establishment of S. marcescens in the squash bug. These findings give us insight into symbiont-pathogen dynamics within the squash bug that could ultimately determine its ability to transmit pathogens and be leveraged to interrupt disease transmission in this system.

Most insects maintain associations with microbes that shape their ecology and evolution. Such symbioses have important applied implications when the associated insects are pests or vectors of disease. The squash bug, Anasa tristis (Coreoidea: Coreidae), is a significant pest of human agriculture in its own right and also causes damage to crops due to its capacity to transmit a bacterial plant pathogen. Here, we demonstrate that complete understanding of these insects requires consideration of their association with bacterial symbionts in the family Burkholderiaceae. Isolation and sequencing of bacteria housed in the insects’ midgut crypts indicates that these bacteria are consistent and dominant members of the crypt-associated bacterial communities. These symbionts are closely related to Caballeronia spp. associated with other true bugs in the superfamilies Lygaeoidea and Coreoidea. Fitness assays with representative Burkholderiaceae strains indicate that the association can significantly increase survival and decrease development time, though strains do vary in the benefits that they confer to their hosts, with Caballeronia spp. providing the greatest benefit. Experiments designed to assess transmission mode indicate that, unlike many other beneficial insect symbionts, the bacteria are not acquired from parents before or after hatching but are instead acquired from the environment after molting to a later developmental stage. The bacteria do, however, have the capacity to escape adults to be transmitted to later generations, leaving the possibility for a combination of indirect vertical and horizontal transmission.

Cucurbit yellow vine disease (CYVD), caused by the squash bug (Anasa tristis)-transmitted bacterium Serratia marcescens, was first identified in Oklahoma and Texas in 1988 and in Georgia in 2012. S. marcescens is a highly diverse species found in many ecological niches. In previous studies, CYVD strains of S. marcescens formed a closely related group separate from non-CYVD strains based on biological and molecular characterization techniques. Multilocus sequence analysis (MLSA) of six housekeeping genes and repetitive elements-based polymerase chain reaction (rep-PCR) using the BOX and ERIC primers were used to assess the genetic diversity of CYVD strains of S. marcescens collected in Georgia together with a strain from Texas and seven non-CYVD strains of S. marcescens. rep-PCR results revealed genetic diversity among CYVD strains while MLSA results showed a 100% similarity across the six loci for all but one of the CYVD strains, which differed at the icd locus by five polymorphisms. For both methods, CYVD strains clustered separately from nonplant-pathogenic S. marcescens strains and were most similar to a rice endophyte strain. One CYVD strain isolated from a squash bug shared genetic similarities with non-CYVD strains, and may be the result of a recombination event between CYVD and non-CYVD strains.

A two-year study evaluated the effect of a flowering border of buckwheat Fagopyrum esculentum Moench on rates of egg parasitism, egg predation and adult parasitism on two squash bug species, Anasa tristis (DeGeer) and Anasa armigera Say, by comparing rates in squash fields with and without a flowering border. Furthermore, we evaluated whether there was an edge effect by comparing parasitism and predation rates in plots located in the corner of a squash field with plots located in the center of a squash field for fields with and without a flowering border. The egg parasitism rates were not affected by either treatment (flowering border or control) or plot location (edge or center). Anasa armigera egg masses only accounted for 4.3% of the total egg masses collected. The egg parasitism rates increased gradually throughout the season, peaking in the last week of August in 2017 at 45% for A. tristis egg masses. The most common egg parasitoid recovered was Gryon pennsylvanicum (Ashmead) followed by Ooencyrtus anasae (Ashmead). Adult parasitism was not affected by treatment, but A. tristis adult parasitism rates were higher in plots located on the edge of squash fields compared with plots located in the center of squash fields in 2016. Since adult parasitoid, Trichopoda pennipes (Fabricius) flies were observed visiting buckwheat flowers, future studies could explore the possibility that the flowering buckwheat may have a more impact on adult parasitism if there was a greater distance between fields with and without a flowering border.

Polycultures, or multicrops, are groupings of plants that grow more prolifically when planted together as compared to when planted alone as monocultures. One of the best known and widely utilized polycultures is the milpa cropping system - the cultivation of maize, beans, and squash together as “the three sisters.” Milpa has been utilized by the indigenous population of Central America for millennia due to its consistent abundant harvests; today it remains a cornerstone of the region’s tradition, diet, and economic growth. Likely contributing to this legacy is the known association of polycultures and heightened resistance to disease, yet the mechanism underlying this relationship in milpa has largely been unexplored. To assess the health of farm soil exposed to milpa monocrops, bicrops, and multicrops, we measured the growth of two bacterial strains: a Burkholderia strain symbiotic of Anasa tristis (the squash bug, an agricultural pest) and a plant pathogenic Serratia strain that is the primary causal agent of cucurbit yellow vine disease (CYVD). We found that after one week in both the polyculture (corn, bean, and squash) soil and the corn monoculture soil, the growth of Burkholderia was significantly inhibited. However, in both corn & bean and bean & squash biculture soils, the growth of the strain was significantly enhanced. The growth of the Serratia strain did not yield any significant increase or decrease after one week in any milpa soil. We conclude that the cultivation of milpa in its polyculture configuration demonstrates antibiotic activity towards the Burkholderia strain SQ4A. Our investigation supports findings that certain multicrop systems are less susceptible to disease than monocultures possibly due to their greater microbial biomass; thus we can infer a higher amount of root exudates present in the soil, of which a substantial amount may be anti-microbials.

Seasonal changes in egg parasitism and predation rates on sentinel (laboratory-reared) and wild (naturally occurring) egg masses of the squash bug, Anasa tristis (De Geer), were evaluated in squash fields in Maryland from June through September in 2013 and 2014. Rates of egg parasitism were significantly higher on wild egg masses than on sentinel egg masses. Squash bug nymphal emergence was significantly higher on sentinel egg masses than on wild egg masses. Between the first week of July and the first week of September of both survey years, squash bug nymphs emerged from 24.2% of wild eggs compared with 46.2% of sentinel eggs and parasitoids emerged from 55.7% of wild eggs compared with only 21.8% of sentinel eggs. Sentinel egg masses significantly underestimated the rate of natural egg parasitism. The egg parasitoid, Gryon pennsylvanicum (Ashmead), was responsible for over 99% of parasitism of squash bug eggs. There was a significant negative correlation between parasitoid emergence and nymphal emergence, suggesting that parasitoids were able to suppress squash bug populations. The average rate of parasitoid emergence peaked on wild egg masses on the fifth week of July at 72.8%, whereas the average rate of nymphal emergence from wild egg masses was

Individuals of different species sometimes mate in nature, and such behavior often carries costs, such as wasted gametes and inviable offspring. One context in which interspecific mating commonly occurs is when closely related species come into secondary contact. Here, we tested whether reproductive isolation is greater in an area of recent secondary contact than in allopatry for two closely related insect species, and we examined whether mating between individuals of these two species constitutes reproductive interference. In Florida, two species of squash bugs (Hemiptera: Coreidae: Anasa tristis DeGeer and Anasa andresii Guérin-Méneville) have been secondarily sympatric for ≥80 generations, and male A. andresii copulate with female A. tristis. Because hybridization is often costly for females, we predicted that secondarily sympatric females would be less likely to mate with heterospecifics than would allopatric females. We found no evidence of recent selection on reproductive isolation: females from both populations were equally likely to mate with heterospecifics, and heterospecific males did not make more mating attempts than conspecifics to achieve copulations. However, female A. tristis paired with heterospecifics produced many fewer eggs and offspring than females paired with conspecifics, and this did not differ according to whether females were from allopatric or sympatric populations. Our findings show that reproductive barriers between these species existed before secondary contact. We suggest that habitat use may limit encounter frequency, and that female choice, multiple mating, and postcopulatory processes may reduce costs for females. Consequently, we suggest that mating systems and ecological factors mediate the effects of reproductive interference.

This study evaluated how the size of the egg mass and the parasitoids prior exposure to eggs influenced parasitism rates by Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species, Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae). G. pennsylvanicum is the primary egg parasitoid of A. tristis. There were no published reports available on egg parasitism of A. armigera. In choice tests, there was no difference in host acceptance by G. pennsylvanicum of egg masses of the two squash bug species. In no-choice tests, overall parasitism rates were significantly higher on A. armigera egg masses than on A. tristis egg masses. Naive parasitoids had significantly higher parasitism rates than experienced parasitoids on egg masses of both squash bug species. In a comparison of parasitism rates of field-collected and laboratory-tested A. tristis egg masses of different sizes, parasitism rates were similar in the field and in the laboratory, with the exception of egg masses with > 25 eggs. Only 17.9% of eggs were parasitized in the laboratory, compared with 36.4% in the field. Results of this study indicate that transient egg limitation prevents G. pennsylvanicum from ovipositing in every available host egg in large squash bug egg masses. The low parasitism rate of G. pennsylvanicum on large egg masses may limit its effectiveness as a biological control agent of squash bugs.

The ovipositional preferences of two squash bug species, Anasa tristis (DeGeer) (Heteroptera: Coreidae) and Anasa armigera Say (Heteroptera: Coreidae), were evaluated in paired choice tests of different species and cultivars of plants in the family Cucurbitaceae. Females of A. tristis preferred to oviposit on the cultivar from which they were reared over three other cultivars of the same species. However, females did not show any ovipositional preference for different cultivars when they had no previous exposure to either cultivar. Females of A. tristis were equally likely to oviposit on Cucurbita pepo, Cucurbita maxima, and Cucurbita moschata but were significantly more likely to oviposit on C. pepo than on cucumber and watermelon. Females of A. armigera were equally likely to oviposit on C. pepo or cucumber, regardless of the species they were reared on. When A. armigera was reared on C. pepo, females were more likely to oviposit on C. maxima than on cucumber and less likely to oviposit on C. moschata than on cucumber.

Mating between species is common, but seldom adaptive. Mate preferences and receptivity to mating attempts may both contribute to such matings, also called reproductive interference. Here, we tested the hypothesis that both male mate preference for female body size and female receptivity contribute to reproductive interference between two secondarily sympatric insect species. In north-central Florida, the squash bug Anasa tristis co-occurs with a recently introduced, smaller congener, Anasa andresii. Male A. andresii are frequently found copulating with larger female A. tristis in the field. We found that male A. andresii prefer larger heterospecific females over conspecific females, that female A. tristis accept some mating attempts by heterospecific males, and that female A. tristis are more promiscuous with conspecifics than are female A. andresii. Our findings suggest that both male mate choice and female receptivity contribute to mating between these species.

Summary Viruses are widespread in both natural and agricultural plant communities and can significantly alter diverse traits of their host plants that mediate key interactions with other organisms. Yet, the impacts of plant viruses on broader community dynamics remain little studied. Here, we explore the effects of Cucumber mosaic virus, a common non-persistently transmitted plant virus, on short- and long-term interactions of herbivorous and predatory insects with squash (Cucurbita pepo) plants in a weedy field setting, as well as virus-induced changes in plant phenotypes that mediate these interactions. Cucumber mosaic virus has previously been shown to have numerous effects on host plants that likely influence interactions with arthropods, including reduced plant size, increased volatile emissions, and diminished plant quality and palatability for aphid vectors. Infection reduced the likelihood of many herbivorous insects arresting and feeding on plants, as well as the apparency of plants to herbivores that base in-flight foraging on visual cues. In particular, infection drastically reduced numbers of a specialist squash herbivore (Anasa tristis) on plants in the field – a pattern likely driven by a reduction of phagostimulatory sugar levels in leaf tissue and concurrent increase in amino acid levels, as nymphal development was not obviously impacted by infection status. Relative to effects on herbivores, virus infection had little impact on the ability of predatory insects to locate aphid prey, although an experiment examining plant visitation in the absence of aphids revealed reduced numbers of foraging Syrphidae (Diptera) and Coccinellidae (Coleoptera) on infected plants but increased visitation and oviposition by Chrysopidae (Neuroptera). CMV infection may reduce overall herbivore pressure on infected plants through effects on palatability and apparency, yet predators appear to locate herbivorous prey that do occur on infected plants as efficiently as those on healthy plants. Virus infection can significantly influence plant interactions with the insect community (including non-vector as well as vector insects) with potential implications both for disease spread and for broader community dynamics.

Plant chemicals mediating interactions with insect herbivores seem a likely target for manipulation by insectvectored plant pathogens. Yet, little is currently known about the chemical ecology of insect-vectored diseases or their effects on the ecology of vector and nonvector insects. We recently reported that a widespread plant pathogen, Cucumber mosaic virus (CMV), greatly reduces the quality of host-plants (squash) for aphid vectors, but that aphids are nevertheless attracted to the odors of infected plants—which exhibit elevated emissions of a volatile blend otherwise similar to the odor of healthy plants. This finding suggests that exaggerating existing host-location cues can be a viable vector attraction strategy for pathogens that otherwise reduce host quality for vectors. Here we report additional data regarding the effects of CMV infection on plant interactions with a common nonvector herbivore, the squash bug, Anasa tristis, which is a pest in this system. We found that adult A. tristis females preferred to oviposit on healthy plants in the field, and that healthy plants supported higher populations of nymphs. Collectively, our recent findings suggest that CMV-induced changes in host plant chemistry influence the behavior of both vector and non-vector herbivores, with significant implications both for disease spread and for broader community-level interactions.

The squash bug, Anasa tristis (De Geer), is a major indigenous pest of Cucurbita species across the United States and a vector of cucurbit yellow vine disease. The seasonal phenology of the squash bug in central Kentucky and its natural enemies were studied using summer squash planted sequentially throughout the 2005 and 2006 growing seasons. The squash bug was first detected on 5 June 2005 and 3 June 2006. In both years, peak numbers of all squash bug stages occurred in July and August. Our field data, substantiated by published degree-day models for squash bug development, suggest one complete and a partial second generation of squash bugs in 2005 and one complete generation of squash bugs in 2006. The most abundant ground-active predators in squash fields included Araneae, Carabidae, Staphylinidae, and Geocoridae. Coleomegilla maculata (De Geer) and Geocoris punctipes (Say) were the most abundant foliage-inhabiting predators. Direct field observations of predators feeding on squash bugs or their eggs included G. punctipes, Pagasa fusca (Stein), and Nabis sp. The parasitoids Trichopoda pennipes (Fabricius) and Gyron pennsylvanicum (Ashmead) were found also. Squash bug egg masses were monitored to determine predation and parasitism rates in the field. In four studies during 2005 and 2006, predation rates were low (7% or less), and parasitism ranged from 0 to 31%. Overall, squash bug egg mortality increased as the season progressed.

Cucurbit yellow vine disease (CYVD), which can inflict heavy losses to watermelon, pumpkin, cantaloupe, and squash in U.S. production areas from the midwest to northeastern states, causes phloem discoloration, foliar yellowing, wilting, and plant decline. Bacteria were cultured from the phloem of crown sections of symptomatic plants of Citrullus lanatas and Cucurbita pepo. Those bacteria testing positive in CYVD-specific polymerase chain reaction (PCR) were all gram negative and appeared morphologically identical, producing creamy white, smooth, entire, convex colonies on Luria-Bertani or nutrient agar. Characterized cucurbit-derived strains of Serratia marcescens were introduced into greenhouse-grown squash plants by puncture inoculation and into field-grown squash plants by enclosure with S. marcescens-fed squash bugs, Anasa tristis. Up to 60% of the bacteria-inoculated plants in the greenhouse and up to 17% of field plants caged with inoculative squash bugs developed phloem discoloration and tested positive for S. marcescens by CYVD-specific PCR. None of the controls developed phloem discoloration or tested positive by PCR. Of the diseased field plants, 12% (2 of 35) also yellowed, wilted, and collapsed, exhibiting full symptom development of CYVD. However, neither plant collapse nor decline was observed in the greenhouse-grown, puncture-inoculated plants. The morphology, growth habit, and PCR reaction of bacteria cultured from crown tissue of a subset of plants in each experimental group were indistinguishable from those of the inoculum bacteria. Evidence presented from our studies confirms that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-A. tristis relationship described here is the first instance in which the squash bug has been identified as a vector of a plant pathogen. Our experiments represent a completion of the steps of Koch's postulates, demonstrating that S. marcescens is the causal agent of CYVD and that the squash bug, A. tristis, is a vector of the pathogen.

Summary • Compared with leaf-feeding herbivores, little is known about how sap-feeding herbivores affect plant growth, morphology and reproduction. This study examines effects of the sap-feeding squash bug ( Anasa tristis ) on watermelon ( Citrullus lanatus ) root, leaf and reproductive structures. • Plants at the four-leaf stage were exposed to different densities of caged squash bugs for 67 d (to plant maturity). • Initial effects were on roots. Herbivory was associated with a significant reduction in mean total root length, root surface area and number of root tips, as well as an increase in root diameters. Herbivore-exposed plants had significantly more leaves, although leaf lifespan was decreased. Both total plant dry mass and fruit dry mass per unit leaf area were significantly greater in controls. Significant effects of herbivores on plant reproductive traits included delayed flower formation (by 7–12 d), change in floral sex ratio (in favor of femaleness), increased fruit abortion, and smaller fruit size. • Developmental consequences were related to changes in plant water relations, including decreased water-use efficiency. Water use in treated plants was three to nine times greater per unit of plant dry mass than in controls.

Chagas’ disease is a parasitic disease caused by Trypanosoma cruzi (T. cruzi). It is a major health concern for the USA, although, mainly endemic to rural, poverty-stricken areas, of Latin America. It is estimated 300,000 people in the USA and 8 to 14 million in Latin America are infected. The parasites are transmitted by the reduviid “kissing” bug. The kissing bug takes a blood meal and defecates on the host, which allows feces infected with T. cruzi to enter the wound. Currently, the source of T. cruzi transmission in the USA is unknown. Therefore, the objective of this study was to determine if squash bugs, common relatives of the kissing bug, can act as vectors for T. cruzi and potentially transmit the parasites to mammalian hosts. Hence, 400 squash bugs were collected from a variety of curcubit plants, identified and separated by genus/species. PCR analysis was performed on DNA isolated from bug midgut contents to determine if T. cruzi was present. Morphological examination confirmed that all samples...

This publication discusses squash bugs, damaged caused by them, and their control.

The early development and feeding behavior of larvae of Anasa tristis (DeGeer) on cucumber, Cucumis sativus L., and pumpkin, Cucurbita pepo L., were compared. First-instar A. tristis development is similar on pumpkin, cucumber, and water alone. Second-instar A. tristis develop to 3rd instar on pumpkin but do not gain substantial weight on cucumber or water alone. Reduction in feeding is at least partly responsible and could be entirely responsible for lack of growth of 2nd-instar A. tristis on cucumber. Both initiation of feeding and feeding duration are reduced. However, 2nd-instar A. tristis can extract nutrients from plants that are not suitable for development. Feeding behavior of 2nd-instar A. tristis may reflect a balance between subsisting on available plants and moving to a suitable plant.

The Squash bug, Anasa tristis (De Geer), is a major piercing/sucking pest of cucurbits, causing extensive damage to plants and fruits, and transmitting phytopathogens. No genomic resources to facilitate field and laboratory studies of this pest were available; therefore the first de novo exome for this destructive pest was assembled. RNA was extracted from insects challenged with bacterial and fungal immunoelicitors, insects fed on different cucurbit species, and insects from all life stages from egg to adult. All treatments and replicates were separately barcoded for subsequent analyses, then pooled for sequencing in a single lane using the Illumina HiSeq2000 platform. Over 211 million 100-base tags generated in this manner were trimmed, filtered, and cleaned, then assembled into a de novo reference transcriptome using the Broad Institute Trinity assembly algorithm. The assembly was annotated using NCBIx NR, BLAST2GO, KEGG and other databases. Of the >130,000 total assemblies 37,327 were annotated identifying the sequences of candidate gene silencing targets from immune, endocrine, reproductive, cuticle, and other physiological systems. Expression profiling of the adult immune response was accomplished by aligning the 100-base tags from each biological replicate from each treatment and controls to the annotated reference assembly of the A. tristis transcriptome.

Field releases of the indigenous egg parasitoid Crayon pennsylvanicum (Ashmead) resulted in a 4-fold reduction in squash bug, Anasa tristis (De Geer), densities on ‘Jack-O-Lantern’ pumpkins in 1991. However, pest populations subjected to parasitoids were consistently higher, and fruit yields and quality lower, than on plants that were treated with the insecticide, esfenvalerate, When the squash bug-resistant ‘Green-Striped Cushaw’ was used, the difference in pest densities on parasitoid-released and insecticide-treated plants was only half of that observed on the susceptible cultivar. In 1992, under abnormally wet, cool conditions, squash bug densities were higher on insecticide-treated plants and lower on plants where parasitoids were released than in 1991. Fruit quality for both cultivars was substantially reduced in all treatments in 1992, The wet, cool conditions apparently had less of an effect on pest densities and fruit yields on resistant pumpkins than on susceptible pumpkins. The expected net income for insecticide-treated pumpkins was substantially lower in 1992 than in 1991. Where biological control and host plant resistance were combined, loss in expected net income in 1992 was considerably less than in 1991. Although costs associated with biological control were very high relative to the revenues, integrating biological control with host plant resistance may have some potential as an alternative management strategy.

A laboratory experiment was conducted to determine whether leaf exudates could be used as a source of adult nutrition for the squash bug, Auasa tristis (De Geer), egg parasitoid, Gryon pennsylvanicum (Ashmead), and to compare adult and progeny fitness traits when female Wasps were provided with squash leaves, a standard honey diet, a combination of leaves and honey, or water only Results showed that fecundity, progeny developmental rates, and progeny survival did not differ significantly when females were exposed to different dietary sources. Adult longevity was shorter on squash leaves without honey than on leaves with honey or the honey–only diet. However, reproductive fitness was not reduced because oviposition had been completed before death of the adult females. When only water was provided, parasitoid females lived only a few days. Observations showed that squash leaves have two kinds of trichomes (hair–like and peg–like), both of which produce droplets that are fed on by both sexes of G. pennsylvanicum. Nutrient analyses revealed that exudates from the hair–like trichomes are composed predominantly of monosaccharides (mainly glucose and galactose), and that the peg–like trichomes produce glucose and some protein. Thus, these trichomes appear to serve as extrafloral nectaries and may provide an important energy source for augmentatively released G. pennsylvanicum .

The squash bug, Anasa tristis (DeGeer), is an endemic species of the Americas that feeds on plants in the family Cucurbitaceae. The pest is particularly abundant and damaging on plants in the genus Cucurbita (i.e., zucchini, summer squash, and pumpkins). Squash bug has become problematic in recent years due to changes in insecticide use strategie s by conventional growers, dramatic increases in organic vegetable production, and increasing incidences of cucurbit yellow vine disease, a phloem-clogging bacterial disease transmitted by the bug. A review of insect biology, description of life stages, host plants, damage, and management options for squash bug is presented.

Laboratory experiments were conducted to quantify effects of resistant and susceptible host plants on the preimaginal and adult biologies of the squash bug, Anasa tristis (De Geer), and its egg parasitoid, Gryon pennsylvanicum (Ashmead). ‘Early Prolific Straightneck’ squash, ‘Green Striped Cushaw’ pumpkin, and ‘Waltham Butternut’ squash served as test plants. Preimaginal development of the squash bug (egg to adult) was similar on all three cultivars (range: 31–35 d). However, survivorship to the adult was significantly lower on ‘Green Striped Cushaw’ and ‘Waltham Butternut’ than on ‘Early Prolific Straightneck’. Statistical differences in survivorship were not observed in second-generation squash bugs, suggesting that some selection may have occurred. Squash bug reproduction and longevity were similar on all three cultivars. However, the reduced juvenile survivorship on ‘Green Striped Cushaw’ and ‘Waltham Butternut’ resulted in significantly lower rmax values on these cultivars than on the more susceptible ‘Early Prolific Straightneck’. The dietary history of adult squash bugs did not significantly affect development, preimaginal survival, or reproduction of G. pennsylvanicum . However, adult longevity of the parasitoid was significantly reduced when its larvae developed in eggs of squash bugs that were reared on the most resistant cultivar (‘Waltham Butternut’).

Mated, 1-d-old female Gryon pennsylvanicum (Ashmead) exhibit a diel pattern of activity and rest. Daily activity (e.g., oviposition, walking, grooming, flying, feeding) is greatest during morning hours (0815-1215 hours CST). Two forms of resting behavior occur. “Motionless” is a temporary state occurring mostly during afternoon hours. The more pronounced resting phase, “tucking,” occurs in aggregated individuals of both sexes and is a form of rest previously undescribed in the parasitic Hymenoptera. The period of tucking persists from before lights go off to about 1 h after the lights go on. In individual wasps, activity resumes abruptly at about 0830 hours; by 0930 hours, all adults become active. Observations of a field population show a similar pattern of diel activity. The significance of these data for biological control programs is discussed.

Six squash cultivars, Cucurbita spp., were evaluated in the field during 1983 and 1984, and five cucurbit hosts were evaluated during 1985 for ovipositional preference by squash bugs, Anasa Tristis (De Geer). Yellow straightneck ‘Hyrific’ and crookneck squashes were found to be the most preferred in the squash study. In the cucurbit study, two cultivars of C. pepo L., yellow straightneck ‘Hyrific’ squash and ‘Jack o’ Lantern’ pumpkin, were most preferred. Relatively few egg masses were found on watermelon, Citrullus lanatus (Thunberg) Matsumura & Nakai ‘Crimson Sweet’; muskmelon, Cucumis mela L. ‘Hales Best #36’; and cucumber, Cucumis sativus L. ‘Poinsett.’ The abaxial leaf surfaces of all hosts in both studies were the favored ovipositional site of squash bugs. This phenomenon is important in developing squash bug sampling strategies and in insecticide applications.

Commercial plantings of summer squash in Charlemont, Franklin County, MA, were decimated in 1999 by 100% incidence of a yellowing disease resembling cucurbit yellow vine disease (CYVD) (1). Both plantings were established in the same field during the third week of May, one with transplants and the second by direct-seeding. Each planting consisted of four 30-m rows each of yellow zucchini (Cucurbita pepo cv. Gold Rush), summer squash (C. pepo cv. Seneca Prolific), and zucchini (C. pepo cv. Condor). Crops were produced organically and pyrethrum was used to control a high infestation of squash bugs, Anasa tristis (De Geer) (Heteroptera:Coreidae), a putative vector of CYVD (3). Just prior to fruit set, during the first two weeks of June, plants began showing symptoms of foliar chlorosis, plant stunting, or both. All of the plants in the field eventually wilted and collapsed. Cross-sections of the below-ground stem and primary root revealed a honey-brown phloem discoloration and healthy appearing xylem, symptoms characteristic of CYVD. Plants yielded marketable fruit for only about 1 week. When plant samples were tested by polymerase chain reaction (PCR) with CYVD bacterium specific primers (2), a band of the expected size for the CYVD bacterium, identified as Serratia marcescens based on 16s rDNA and groE sequence analyses (4), was amplified in every case. Since all plant samples collected were symptomatic and PCR positive for S. marcescens, asymptomatic greenhouse plants were run simultaneously as a control. All control plants tested negative. A third planting, similar to the two disease-affected plantings and containing the same three squash cultivars from the same seed lot, was established at about the same time approximately 3 km away. No symptoms of CYVD occurred at this site, further evidence that the pathogen is not seed-borne (1). Furthermore, squash bugs were not observed in this field. In 2000, the disease was observed in a planting of ‘Atlantic Giant’ pumpkin in Erving, Franklin County, MA, and confirmed by PCR. Until now, CYVD has been reported only in the states of Oklahoma, Texas, and Tennessee. Confirmation of the disease in Massachusetts significantly increases the known geographical range of CYVD to include the New England area. References: (1) B. D. Bruton et al. Plant Dis. 82:512–520, 1998. (2) U. Melcher et al. Phytopathology 89:S95, 1999. (3) S. D. Pair et al. Pages 145–148 in: Proc. 19th Ann. Hort. Conf., Okla. State Univ. (4) J. Rascoe et al. Phytopathology 90:S63, 2000.

The squash bug, Anasa tristis DeGeer, undergoes a reproductive diapause from late summer to spring in northeastern Kansas. In the laboratory, diapause was induced in 100% of adult females reared under photoperiods shorter than 14:10 (L:D) and in a variably lower percentage of the population under all longer photoperiods. The critical photoperiod for diapause induction falls between 14:10 and 14.5:9.5; this range compares closely with prevailing natural daylengths when 50% of the adult population enters diapause in the field. Between October and March, short daylengths maintained, and long daylengths terminated, diapause in field-sampled adults. Under natural daylength at 26°C, the duration of diapause became progressively shorter with advancing sample date. In nature, the photoperiodic maintenance of diapause is completed in most of the population by late May. A prolonged diapause probably serves to prevent premature postdiapause development during the thermally variable spring conditions encountered in Kansas. Some implications of these findings for biological control and pest management programs are discussed.

Laboratory investigations of the gregarious squash bug egg parasitoids Ooencyrtus anasae (Ashmead) and O . sp. were conducted at 20.8, 23.0, and 26.6°C. Females of both species emerged with undeveloped ovaries. Oocyte development proceeded during the preovipositional period (ca. 35 h at 26.6°C) but did not exceed half of the total fecundity at initial oviposition. Preovipositional periods, time to peak oviposition, and time to 75% oviposition were generally similar for both parasitoids at 20.8°C. However, reproductive responses differed at temperatures above 20.8°C. In O. anasae , oviposition occurred significantly earlier, and at a similarly higher rate, at 23.0 and 26.6°C than at 20.8°C, whereas significant changes in preovipositional and ovipositional biology of O . sp. occurred only after an increase from 23.0 to 26.6°C. O . sp. had a significantly higher fecundity and longer ovipositional period at each temperature than did O. anasae. Mean fecundities did not change significantly with increasing temperature. Both parasitoids had an unusually long postreproductive period (ca. 1 mo) and a longevity of 40 to 50 d. At 26.6°C, O . sp. had higher gross and net reproductive rates than O. anasae. However, O. anasae had a shorter generation time (21.4 versus 23.8 d) and higher innate capacity for increase (0.146 versus 0.134).

The squash bug (SB), Anasa tristis DeGeer, completes 1.5 generations per yr in northeastern Kansas. Overwintered adults mate and oviposit on cucurbitaceous host plants from early June to mid-August. Most eggs are deposited from June to early July when female survivorship is highest, but some oviposition occurs through mid-August. First-generation adults begin to emerge between the 1st and 3rd wk of July. Oviposition commences 7–10 d after emergence. Females that emerge before late July avoid diapause and produce a second generation. Thereafter the incidence of diapause increases progressively, reaching 100% in first- and second-generation females that emerge in early September. Diapausing females do not mate or reproduce until the following spring. SB males also appear to undergo diapause but, unlike females, they overwinter with mature reproductive organs. The primary expression of male diapause is suppressed reproductive behavior. Reproductive periods of overwintered and first summer-generation adults overlap. The extent of this overlap depends in part on spring temperature conditions that influence field entry by overwintered adults; the oviposition schedule; and development of first-generation progeny. During cool springs, a large proportion of first-generation SB emerge late in the season and enter diapause. In these years, smaller, less-damaging, populations occur.

Laboratory investigations of the gregarious squash bug egg parasitoids Ooencyrtus anasae and O . n. sp. near anasae ( O . sp.) were conducted at 20.8, 23.0, and 26.6°C. In both species, total developmental periods (egg to eclosed adult) were inversely related to temperature. Temperature had no significant influence on survivorship, progeny production, or sex ratio. At each temperature, O. anasae developed and emerged about a day earlier and produced a significantly higher percentage of female progeny (77%) than did O . sp. (60%). Both parasitoids deposited an average of three (2–7) progeny per host. However, O. anasae consistently deposited more female eggs per host than did O . sp. Proportion of females produced per host by O. anasae tended to increase directly with number of hosts parasitized, but no such relationship was observed in O . sp. Total preimaginal survivorship in both parasitoids was about 89%. In O . sp., male progeny that developed without females emerged about a day later at all temperatures and had a lower pharate adult survivorship than did males that developed in hosts with female siblings.

A recent paper by Wigglesworth (1943) has dealt extensively with the metabolism of hemoglobin by certain blood-sucking Hemiptera and other insects. The data presented were of great interest from the standpoint of comparative biochemistry and aroused the writer's curiosity regarding the possible metabolic breakdown of the related pyrrole pigment, chlorophyll, by plant-feeding Hemiptera.

The squash bug, Anasa tristis DeG., is commonly parasitized by a tachinid fly, Trichopoda pennipes Fabr. Within the body cavity of the host the parasite spends its three larval instars, the first of which may be found free in either the thoracic or abdominal cavity of the bug. Prior to the first molt, the maggot makes its way to the thorax, if not already there, and attaches itself to a tracheal trunk of the host with the result that its own tracheal system, through its posterior spiracles, has direct access to an adequate oxygen supply. Of 309 recorded cases, 304 were attached to metathoracic tracheae, on either the right or left side. The other five were connected to tracheae leading from the mesothoracic spiracles.

1. For species of insects that deposit their eggs on foliage, as Leptinotarsa decemlineata, Crioceris asparagi, and Anasa tristis, the rates of metabolism as measured by the CO2 output or the oxygen intake indicate, during embryonic development, a short formative period, followed by a very active extended growth. Those species that deposit their eggs in the soil, as Cotinis nitida and Popillia japonica, show that the formative period is greatly extended and the growth period similarly lengthened. 2. It has been shown from metabolism experiments that intensive histolysis occurs during the prepupal period and becomes less intensive during pupal development. 3. Metabolism experiments show a greater amount of energy change during embryonic development as compared to the energy developed during metamorphosis. This is shown by the greater CO2 output and by the oxygen intake. 4. Low respiratory quotients, varying from 0.42 to 0.71, have been obtained during the embryonic and pupal development of insects, resembling similar low quotients obtained with hibernating forms. 5. Changes from a neutral to an acid reaction (pH 6.8 to 5.9) have been observed to take place in some species during prepupal and early pupal development. As metamorphosis is completed a converse change occurs (pH 5.9 to 6.8). Changes in pH from an acid to neutral reaction were also observed to take place in some species, as Cotinis nitida and Hylemyia cilicrura, during embryonic development.

1. The work presented in this paper has proved the presence of size dimorphism in the adult spermatozoa of Anasa tristis. Measurements of the length of the chromatic rod of several hundred spermatozoa from each testis, plotted as a frequency curve, have demonstrated two modes, one between 27.54µ and 28.08µ, and the other between 30.24µ and 30.78µ, and the data offer adequate support for the conclusion that length dimorphism exists.2. This dimorphism in length of adult spermatozoa probably bears a fundamental relation to the dimorphism in chromosome number revealed by spermatogenesis studies.

The squash bug, Anasa tristis DeG., has been a desirable subject for one whose interests are with the Hemipterous forms. Specimens used in the following studies were collected in early Octoher and were kept in a moderately cool basement room in a Riley insect cage. They were fed squash of the Acorn variety from a supply in storage and remained active throughout the fall and well into the winter.

In writing this article the chief aim is to endeavor to supply a reference work on the external morphological characters of a typical Heteropterous insect. For this reason the common squash bug has been selected as it is widely distributed, well known as a pest, and is readily obtainable for study.

does not appear. First page follows. Introduction Since the early years of the nineteenth century it has been known (e.g., see (Dufour, 1833)4 that the posterior end of the midgut in certain groups of Heteroptera is characterized by the presence of saclike appendages opening into it. These evaginations, called “gastric caeca” or simply “caeca,” apparently serve a purpose different from that of the so-called gastric caeca of the Acrididae, and they appear almost always to contain specific bacteria. The bacteria from different Heteroptera may vary in their morphology, but they are relatively constant for a given insect species. The caeca themselves also vary a great deal in their morphology according to species, showing simple to complex arrangements, and apparently reflect basic phylogenetic relationships. Although there have been a number of studies (e.g., those by (Forbes, 1892); (Glasgow, 1914); (Kuskop, 1924); (Rosenkranz, 1939); (Schneider, 1940) very little has been accomplished toward revealing the nature, kind, or function of the caecal bacteria. Of their function we are most ignorant; certainly it can be said that the role of the bacteria, and their relation to the host insect, are not clearly understood. (Glasgow (1914)) assumed that the caecal bacteria not only inhibited the development of “foreign” or adventitious bacteria but excluded them altogether. Others (e.g., (Kuskop, 1924) have thought they might play a nutritional role. In size and shape, the bacteria, as they occur in different species of Heteroptera, range from small coccuslike bacilli to large vermiform or spirochete-like forms. Attempts to cultivate the bacteria on ordinary bacteriological media have not met with much success. (Glasgow (1914)) reported the cultivation of the symbiote from the caeca of the squash bug, Anasa tristis (De Geer), and of certain other coreids, in nutrient broth and agar; and Steinhaus

The spermatogenesis of Leptocoris hcematoloma (family Coreidoe) very closely resembles that of Anasa tristis, as described by Paulmier. During the past few years it has been used extensively here in the course on cytology to demonstrate the behavior of the X-chromosome. As a matter of record, it seems worth while to give a very brief account of the spermatocytic divisions. Furthermore, the material is very fine for the study of tetrad formation, and it is hoped later to publish an account of this process. The material on which these observations were made was collected in November and December, I914, on the plant Cardiospermum halicacabum, the balloon vine which is very common in this vicinity. It may be of interest, however, to know that this is the only plant on which we have found this bug. The testes, which are large yellow pear-shaped bodies located at the base of the third pair of walking legs were dissected out in Ringer's solution and fixed immediately in Bouin's fluid. They were stained with iron haematoxylin and counterstained with eosin and light-green. Other fixing agents were used, but this proved to be the best. The material is excellent for the study of spermatogenesis, as the chromosomes are not numerous, and vary greatly in size. It is possible to follow accurately the courses of the smallest, largest and accessory chromosomes throughout their entire history. The spermatogonial complex of Leptocoris hcematoloma consists of thirteen chromosomes (Figs. I and 2). These vary in size, but can be classified in three groups. The members of two pairs 1Contributions from the Zoological Laboratory of the University of Texas, No. I38. 316

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During July, 1895, while occupied as an Assistant inthe Illinois State Laboratory of Natural History, instudying some fungous diseases of the chinch-bug I used many squash-bugs (Anasa tristis DeG.) for experimental purposes, their larger size making them convenient for use in certain investigations for which the chinch-bug is poorly adapted. These squash-bugs were kept in a laboratory breeding-cage, and daily supplied with fresh food and a suitable amount of moisture. They were soonobserved to be dying in considerable numbers, although I could detect nothing unhealthful in their surroundings.