Evaluating the role of coevolution in a horizontally transmitted mutualism

Mutualism depends on the alignment of host and symbiont fitness. Horizontal transmission can readily decouple fitness interests, yet horizontally transmitted mutualisms are common in nature. We hypothesized that pairwise coevolution and specialization in host-symbiont interactions underlies the maintenance of cooperation in a horizontally transmitted mutualism. Alternatively, we predicted selection by multiple host species may select for cooperative traits in a generalist symbiont through diffuse coevolution. We tested for signatures of pairwise coevolutionary change between the squash bug Anasa tristis and its horizontally acquired bacterial symbiont Caballeronia spp. by measuring local adaptation. We found no evidence for local adaptation between sympatric combinations of A. tristis squash bugs and Caballeronia spp. across their native geographic range. To test for diffuse coevolution, we performed reciprocal inoculations to test for specialization between three Anasa host species and Caballeronia spp. symbionts isolated from conspecific hosts. We observed no evidence of specialization across host species. Our results demonstrate generalist dynamics underlie the interaction between Anasa insect hosts and their Caballeronia spp. symbionts. Specifically, diffuse coevolution between multiple host species with a shared generalist symbiont may maintain cooperative traits despite horizontal transmission.

The Importance of Environmentally Acquired Bacterial Symbionts for the Squash Bug (Anasa tristis), a Significant Agricultural Pest

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.

The importance of environmentally-acquired bacterial symbionts for the squash bug (Anasa tristis), a significant agricultural pest

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 midgut crypts in these insects indicates that these bacteria are consistent and dominant members of the crypt-associated bacterial communities. These symbionts are closely related to Caballeronia spp. associated other true bugs in the superfamiles 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 development 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.

Ovipositional Behavior of the Egg Parasitoid Gryon pennsylvanicum (Hymenoptera: Scelionidae) on Two Squash Bug Species Anasa tristis (Hemiptera: Coreidae) and Anasa armigera: Effects of Parasitoid Density, Nutrition, and Host Egg Chorion on Parasitism Rates

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.

Use of Flowering Plants to Enhance Parasitism and Predation Rates on Two Squash Bug Species Anasa tristis and Anasa armigera (Hemiptera: Coreidae)

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.

Bacterial Growth in Milpa Polyculture and Monoculture Soils [Emory University]

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.

The impact of tillage regime and row cover use on insect pests and yield in organic cucurbit production

Control of cucurbit pests, such as striped cucumber beetle (Acalymma vittatum), spotted cucumber beetle (Diabrotica undecimpunctata howardi) and squash bug (Anasa tristis), in organic systems is difficult due to a lack of effective insecticide options. This has led to the development of many integrated pest management techniques, such as use of row covers, crop rotation and cover crops. This study explored the novel use of strip tillage and row covers to reduce pest pressure in summer squash (Cucurbita pepe) and muskmelon (Cucumis melo) production systems. Results showed that although strip tillage reduced striped cucumber beetle and squash bug numbers, there was a yield reduction in both crops compared with the plasticulture system. Row cover increased marketable yield in both systems, with the highest yield being in the plasticulture system. Unmarketable fruit directly attributed to insect damage was higher in the plasticulture systems, but was not significantly different when compared with the strip tillage system. Although there are many documented positive attributes of strip tillage, results from this study indicate that a combination of plasticulture and row cover may be a superior system for organic cucurbit production.