Macroevolution of insect–plant associations: The relevance of host biogeography to host affiliation

Identifying the factors that have promoted host shifts by phytophagous insects at a macroevolutionary scale is critical to understanding the associations between plants and insects. We used molecular phylogenies of the beetle genus Blepharida and its host genus Bursera to test whether these insects have been using hosts with widely overlapping ranges over evolutionary time. We also quantified the importance of host range coincidence relative to host chemistry and host phylogenetic relatedness. Overall, the evolution of host use of these insects has not been among hosts that are geographically similar. Host chemistry is the factor that best explains their macroevolutionary patterns of host use. Interestingly, one exceptional polyphagous species has shifted among geographically close chemically dissimilar plants.

The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets

A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.

Miriamin, a defensive diterpene from the eggs of a land slug (Arion sp.)

The eggs of the land slug Arion sp. contain a diterpene, miriamin, characterized as a polyoxygenated geranylgeraniol derivative. In bioassays with a coccinellid beetle, Harmonia axyridis, miriamin was shown to be potently antifeedant, indicating that the compound plays a protective role in nature. It is suggested that mucilaginous soil-inhabiting organisms, given their intense exposure to pathogens and predators, may be a rich source of chemical defensive agents.

Polyazamacrolides from ladybird beetles: Ring-size selective oligomerization

The pupal defensive secretion of the 24-pointed ladybird beetle, Subcoccinella vigintiquatuorpunctata, consists of a mixture of macrocyclic polyamines, dominated by the three dimeric, 30-membered macrocycles 11-13, derived from the two building blocks 11-(2-hydoxyethylamino)-5-tetradecenoic acid (9) and 11-(2-hydoxyethylamino)-5,8-tetradecadienoic acid (10). Smaller amounts of the four possible cyclic trimers of 9 and 10 were also detected, corresponding to 45-membered macrocycles. Structural assignments were based on NMR-spectroscopic investigations and HPLC–MS analyses. In addition, the all-S absolute configuration of the S. vigintiquatuorpunctata macrocycles was determined by comparison of derivatives of the natural material with enantiomerically pure synthetic samples. Comparing this alkaloid mixture with that of the pupal defensive secretion in related ladybird beetle species indicates that the degree of oligomerization of the 2-hydroxyethylamino carboxylic acid building blocks can be carefully controlled by the insects.

Class 3 Hox genes in insects and the origin of zen.

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.

De novo biosynthesis of the aggregation pheromone components ipsenol and ipsdienol by the pine bark beetles Ips paraconfusus Lanier and Ips pini (Say) (Coleoptera: Scolytidae).

The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ips pini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1-14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified radiolabeled monoterpene alcohols from-males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.

Evidence of different thermoregulatory mechanisms between two sympatric Scarabaeus species using infrared thermography and micro-computer tomography

In endotherms insects, the thermoregulatory mechanisms modulate heat transfer from the thorax to the abdomen to avoid overheating or cooling in order to obtain a prolonged flight performance. Scarabaeus sacer and S. cicatricosus, two sympatric species with the same habitat and food preferences, showed daily temporal segregation with S. cicatricosus being more active during warmer hours of the day in opposition to S. sacer who avoid it. In the case of S. sacer, their endothermy pattern suggested an adaptive capacity for thorax heat retention. In S. cicatricosus, an active ‘heat exchanger’ mechanism was suggested. However, no empirical evidence had been documented until now. Thermographic sequences recorded during flight performance showed evidence of the existence of both thermoregulatory mechanisms. In S. sacer, infrared sequences showed a possible heat insulator (passive thermal window), which prevents heat transfer from meso- and metathorax to the abdomen during flight. In S. cicatricosus, infrared sequences revealed clear and effective heat flow between the thorax and abdomen (abdominal heat transfer) that should be considered the main mechanism of thermoregulation. This was related to a subsequent increase in abdominal pumping (as a cooling mechanism) during flight. Computer microtomography scanning, anatomical dissections and internal air volume measurements showed two possible heat retention mechanisms for S. sacer; the abdominal air sacs and the development of the internal abdominal sternites that could explain the thermoregulation between thorax and abdomen. Our results suggest that interspecific interactions between sympatric species are regulated by very different mechanisms. These mechanisms create unique thermal niches for the different species, thereby preventing competition and modulating spatio-temporal distribution and the composition of dung beetle assemblages.

The comparative effectiveness of rodents and dung beetles as local seed dispersers in Mediterranean oak forests

The process of seed dispersal of many animal-dispersed plants is frequently mediated by a small set of biotic agents. However, the contribution that each of these dispersers makes to the overall recruitment may differ largely, with important ecological and management implications for the population viability and dynamics of the species implied in these interactions. In this paper, we compared the relative contribution of two local guilds of scatter-hoarding animals with contrasting metabolic requirements and foraging behaviours (rodents and dung beetles) to the overall recruitment of two Quercus species co-occurring in the forests of southern Spain. For this purpose, we considered not only the quantity of dispersed seeds but also the quality of the seed dispersal process. The suitability for recruitment of the microhabitats where the seeds were deposited was evaluated in a multi-stage demographic approach. The highest rates of seed handling and predation occurred in those microhabitats located under shrubs, mostly due to the foraging activity of rodents. However, the probability of a seed being successfully cached was higher in microhabitats located beneath a tree canopy as a result of the feeding behaviour of beetles. Rodents and beetles showed remarkable differences in their effectiveness as local acorn dispersers. Quantitatively, rodents were much more important than beetles because they dispersed the vast majority of acorns. However, they were qualitatively less effective because they consumed a high proportion of them (over 95%), and seeds were mostly dispersed under shrubs, a less suitable microhabitat for short-term recruitment of the two oak species. Our findings demonstrate that certain species of dung beetles (such as Thorectes lusitanicus), despite being quantitatively less important than rodents, can act as effective local seed dispersers of Mediterranean oak species. Changes in the abundance of beetle populations could thus have profound implications for oak recruitment and community dynamics.

Sampling scarab beetles in tropical forests: the effect of light source and night sampling periods

Light traps have been used widely to sample insect abundance and diversity, but their performance for sampling scarab beetles in tropical forests based on light source type and sampling hours throughout the night has not been evaluated. The efficiency of mercury-vapour lamps, cool white light and ultraviolet light sources in attracting Dynastinae, Melolonthinae and Rutelinae scarab beetles, and the most adequate period of the night to carry out the sampling was tested in different forest areas of Costa Rica. Our results showed that light source wavelengths and hours of sampling influenced scarab beetle catches. No significant differences were observed in trap performance between the ultraviolet light and mercury-vapour traps, whereas these two methods caught significantly more species richness and abundance than cool white light traps. Species composition also varied between methods. Large differences appear between catches in the sampling period, with the first five hours of the night being more effective than the last five hours. Because of their high efficiency and logistic advantages, we recommend ultraviolet light traps deployed during the first hours of the night as the best sampling method for biodiversity studies of those scarab beetles in tropical forests.

Composición, riqueza y abundancia de coleópteros (Coleoptera) asociados a bosques semideciduos y vegetaciones ruderales en la Sierra del Rosario, Cuba

En tres bosques semideciduos y en tres tipos de vegetación ruderal del Área Protegida de Recursos Manejados Mil Cumbres (Sierra del Rosario, Pinar del Río, Cuba), se analizan las comunidades de coleópteros y se las compara respecto a la composición, riqueza, abundancia, diversidad, equitatividad, ordenación y complementariedad. La composición de coleópteros conocida hasta el momento es de 166 especies, incluidas en 75 géneros y 34 familias. La vegetación ruderal presentaó mayor riqueza y abundancia que los bosques semideciduos. El bosque y la vegetación ruderal de Pan de Guajaibón exhibieron los valores más altos de riqueza, abundancia y número de especies únicas. Ambas formaciones vegetales de Sierra Chiquita presentaron la mayor diversidad y equitatividad. Las comunidades de coleópteros más afines estaban entre los bosques y entre la vegetaciones ruderales en Forneguera y Pan de Guajaibón. Cada bosque y tipo de vegetación ruderal presentaba especies exclusivas.

The “dehesa”, a key ecosystem in maintaining the diversity of Mediterranean saproxylic insects (Coleoptera and Diptera: Syrphidae)

The “dehesa” is a traditional Iberian agrosilvopastoral ecosystem characterized by the presence of old scattered trees that are considered as “keystone-structures”, which favor the presence of a wide range of biodiversity. We show the high diversity of saproxylic beetles and syrphids (Diptera) in this ecosystem, including red-listed species. We analyzed whether saproxylic species distribution in the “dehesa” was affected by tree density per hectare, dominant tree species or vegetation coverage. Species diversity did not correlate with tree density; however, it was affected by tree species and shrub coverage but in a different way for each taxon. The highest beetle diversity was linked to Quercus pyrenaica, the most managed tree species, with eight indicator species. In contrast, Q. rotundifolia hosted more species of saproxylic syrphids. Regarding vegetation coverage, shrub coverage was the only variable that affected insect richness, again in a different way for both taxa. In contrast, beetle species composition was only affected by dominant tree species whereas syrphid species composition was not affected by tree species or shrub coverage. We concluded that the high diversity of saproxylic insects in the “dehesa” is related to its long history of agrosilvopastoral management, which has generated landscape heterogeneity and preserved old mature trees. However, the richness and composition of different taxa of insects respond in different ways to tree species and vegetation coverage. Consequently, conservation strategies should try to maintain traditional management, and different saproxylic taxa should be used to monitor the effect of management on saproxylic diversity.

Low doses of ivermectin cause sensory and locomotor disorders in dung beetles

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin’s effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung.

What can physical, biotic and chemical features of a tree hollow tell us about their associated diversity?

Tree hollows are keystone structures for saproxylic fauna and host numerous endangered species. However, not all tree hollows are equal. Many variables including physical, biotic and chemical ones, can characterise a tree hollow, however, the information that these could provide about the saproxylic diversity they harbour has been poorly explored. We studied the beetle assemblages of 111 Quercus species tree hollows in four protected areas of the Iberian Peninsula. Three physical variables related to tree hollow structure, and two biotic ones (presence of Cetoniidae and Cerambyx species recognised as ecosystem engineers) were measured in each hollow to explore their relative effect on beetle assemblages. Moreover, we analysed the chemical composition of the wood mould in 34 of the hollows, in order to relate beetle diversity with hollow quality. All the environmental variables analysed (physical and biological) showed a significant influence on saproxylic beetle assemblages that varied depending on the species. Furthermore, the presence of ecosystem engineers affected both physical and chemical features. Although wood mould volume, and both biotic variables could act as beetle diversity surrogate, we enhance the presence of Cetoniidae and Cerambyx activity (both easily observable in the field) as indicator variables, even more if both co-occur as each affect to different assemblages. Finally, assimilable carbon and phosphorous contents could act as indicator for past and present beetle activity inside the cavity that could become a useful tool in functional diversity studies. However, an extension of this work to other taxonomic groups would be desirable.

Intra-population variation and geographic correlation in Canthon humectus hidalgoensis using FTIR-ATR spectroscopy

In some cases external morphology is not sufficient to discern between populations of a species, as occurs in the dung beetle Canthon humectus hidalgoensis Bates; and much less to determine phenotypic distances between them. FTIR-ATR spectroscopy show several advantages over other identification techniques (e.g. morphological, genetic, and cuticular hydrocarbons analysis) due to the non-invasive manner of the sample preparation, the relative speed of sample analysis and the low-cost of this technology. The infrared spectrum obtained is recognized to give a unique ‘fingerprint’ because vibrational spectra are specific and unique to the molecular nature of the sample. In our study, results showed that proteins, amino acids and aromatic ethers of insect exocuticle have promising discriminative power to discern between different populations of C. h. hidalgoensis. Furthermore, the correlation between geographic distances between populations and the chemical distances obtained by proteins + amino acids + aromatic ethers was statistically significant, showing that the spectral and spatial information available of the taxa together with appropriated chemometric methods may help to a better understanding of the identity, structure, dynamics and diversity of insect populations.

Exploring several nonconventional interactions of ice-binding proteins

Traditionally, ice-binding proteins (IBPs), also known as antifreeze proteins (AFPs), have been defined by two universal activities: ice recrystallization inhibition and thermal hysteresis. However, there remains the possibility IBPs have other complementary functions given the diversity found within this protein group. This thesis explores some of these in both natural and applied settings, in the hopes of furthering our understanding of this remarkable group of proteins. Plant IBPs could function as part of a defensive strategy against ice nucleators produced by certain pathogens. To assess this hypothesis, recombinant IBPs from perennial ryegrass and purple false brome were combined with the ice nucleation protein (INP) from the plant pathogen, Pseudomonas syringae. Strikingly, the plant proteins depressed the freezing point of the bacterial INP, while a fish AFP could not, nor did the INPs have any effect on IBP activity. Thus, the interaction between these two different proteins suggests a role in plant defensive strategies against pathogenic bacteria as another IBP function. In addition, the potential use of hyperactive insect IBPs in organ preservation was investigated. Current kidney preservation techniques involve storing the organ at 4 °C for a maximum of 24 h prior to transplantation. Extending this “safe” time would have profound effects on renal transplants, however, ischemic injury is prevalent when storage periods are prolonged. Experiments described here allowed subzero preservation for 72 h with the addition of a beetle IBP to CryoStasis® solution. Kidneys stored using the traditional technique for 24 h and the method developed here for 72 h showed similar levels of biomarker enzymes, underscoring the potential utility of insect IBPs for future transplant purposes. Finally, IBP function in the freeze-tolerant gall fly, Eurosta solidaginis, was examined. Larvae representing the mid-autumn stage displayed ice-binding activity, suggesting an IBP is being expressed, possibly as a protective measure against freezing damage when fall temperatures can unpredictably drop. IBP activity was also observed in the larvae’s host plant, Solidago spp. Mass spectrometry analysis of ice-affinity purified plant extracts provided three candidate pathogenesis-related proteins that could be responsible for the detected activity, further demonstrating additional functions of IBPs.