Association between molecular markers and behavioral phenotypes in the immatures of a butterfly.

Newly hatched caterpillars of the butterfly Heliconius erato phyllis routinely cannibalize eggs. In a manifestation of kin recognition they cannibalize sibling eggs less frequently than unrelated eggs. Previous work has estimated the heritability of kin recognition in H. erato phyllis to lie between 14 and 48%. It has furthermore been shown that the inheritance of kin recognition is compatible with a quantitative model with a threshold. Here we present the results of a preliminary study, in which we tested for associations between behavioral kin recognition phenotypes and AFLP and SSR markers. We implemented two experimental approaches: (1) a cannibalism test using sibling eggs only, which allowed for only two behavioral outcomes (cannibal and non-cannibal), and (2) a cannibalism test using two sibling eggs and one unrelated egg, which allowed four outcomes [cannibal who does not recognize siblings, cannibal who recognizes siblings, "super-cannibal" (cannibal of both eggs), and "super non-cannibal" (does not cannibalize eggs at all)]. Single-marker analyses were performed using χ2 tests and logistic regression with null markers as covariates. Results of the χ2 tests identified 72 associations for experimental design 1 and 73 associations for design 2. Logistic regression analysis of the markers found to be significant in the χ2 test resulted in 20 associations for design 1 and 11 associations for design 2. Experiment 2 identified markers that were more frequently present or absent in cannibals who recognize siblings and super non-cannibals; i.e. in both phenotypes capable of kin recognition.

Association between molecular markers and behavioral phenotypes in the immatures of a butterfly

Abstract Newly hatched caterpillars of the butterfly Heliconius erato phyllis routinely cannibalize eggs. In a manifestation of kin recognition they cannibalize sibling eggs less frequently than unrelated eggs. Previous work has estimated the heritability of kin recognition in H. erato phyllis to lie between 14 and 48%. It has furthermore been shown that the inheritance of kin recognition is compatible with a quantitative model with a threshold. Here we present the results of a preliminary study, in which we tested for associations between behavioral kin recognition phenotypes and AFLP and SSR markers. We implemented two experimental approaches: (1) a cannibalism test using sibling eggs only, which allowed for only two behavioral outcomes (cannibal and non-cannibal), and (2) a cannibalism test using two sibling eggs and one unrelated egg, which allowed four outcomes [cannibal who does not recognize siblings, cannibal who recognizes siblings, "super-cannibal" (cannibal of both eggs), and "super non-cannibal" (does not cannibalize eggs at all)]. Single-marker analyses were performed using χ2 tests and logistic regression with null markers as covariates. Results of the χ2 tests identified 72 associations for experimental design 1 and 73 associations for design 2. Logistic regression analysis of the markers found to be significant in the χ2 test resulted in 20 associations for design 1 and 11 associations for design 2. Experiment 2 identified markers that were more frequently present or absent in cannibals who recognize siblings and super non-cannibals; i.e. in both phenotypes capable of kin recognition.

Embryogenesis of Heliconius erato (Lepidoptera, Nymphalidae): a contribution to the anatomical development of an evo-devo model organism.

This study reports on the embryogenesis of Heliconius erato phyllis between blastoderm formation and the prehatching larval stage. Syncytial blastoderm formation occurred approximately 2 h after egg laying (AEL) and at about 4 h, the cellular blastoderm was formed. The germ band arose from the entire length of the blastoderm, and rapidly became compacted occupying approximately two-thirds of the egg length. At about 7 h AEL, protocephalon and protocorm differentiation occurred. Continued proliferation of the germ band was followed by penetration into the yolk mass, forming a C-shaped embryo at about 10 h. Approximately 12 h AEL, the gnathal, thoracic and abdominal segments became visible. The primordium of the mouthparts and thoracic legs formed as paired evaginations, while the prolegs formed as paired lobes. At about 30 h, the embryo reversed dorsoventrally. Approximately 32 h AEL, the protocephalon and gnathal segments fused, shifting the relative position of the rudimentary appendages in this region. At about 52 h, the embryo was U-shaped in lateral view and at approximately 56 h, the bristles began evagination from the larval cuticle. Larvae hatched at about 72 h. We found that H. erato phyllis followed an embryonic pattern consistent with long-germ embryogenesis. Thus, we believe that H. erato phyllis should be classified as a long-germ lepidopteran. The study of H. erato phyllis embryogenesis provided a structural glimpse into the morphogenetic events that occur in the Heliconius egg period. This study could help future molecular approaches to understanding the evolution of Heliconius development.

First record of Wolbachia in South American terrestrial isopods: Prevalence and diversity in two species of Balloniscus (Crustacea, Oniscidea).

Wolbachia are endosymbiotic bacteria that commonly infect arthropods, inducing certain phenotypes in their hosts. So far, no endemic South American species of terrestrial isopods have been investigated for Wolbachia infection. In this work, populations from two species of Balloniscus (B. sellowii and B. glaber) were studied through a diagnostic PCR assay. Fifteen new Wolbachia 16S rDNA sequences were detected. Wolbachia found in both species were generally specific to one population, and five populations hosted two different Wolbachia 16S rDNA sequences. Prevalence was higher in B. glaber than in B. sellowii, but uninfected populations could be found in both species. Wolbachia strains from B. sellowii had a higher genetic variation than those isolated from B. glaber. AMOVA analyses showed that most of the genetic variance was distributed among populations of each species rather than between species, and the phylogenetic analysis suggested that Wolbachia strains from Balloniscus cluster within Supergroup B, but do not form a single monophyletic clade, suggesting multiple infections for this group. Our results highlight the importance of studying Wolbachia prevalence and genetic diversity in Neotropical species and suggest that South American arthropods may harbor a great number of diverse strains, providing an interesting model to investigate the evolution of Wolbachia and its hosts.

On the reliability of a simple method for scoring phenotypes to estimate heritability: A case study with pupal color in Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae).

In this paper, two methods for assessing the degree of melanization of pupal exuviae from the butterfly Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae, Heliconiini) are compared. In the first method, which was qualitative, the exuviae were classified by scoring the degree of melanization, whereas in the second method, which was quantitative, the exuviae were classified by optical density followed by analysis with appropriate software. The heritability (h(2)) of the degree of melanization was estimated by regression and analysis of variance. The estimates of h (2) were similar with both methods, indicating that the qualitative method could be particularly suitable for field work. The low estimates obtained for heritability may have resulted from the small sample size (n = 7-18 broods, including the parents) or from the allocation-priority hypothesis in which pupal color would be a lower priority trait compared to morphological traits and adequate larval development.

On the reliability of a simple method for scoring phenotypes to estimate heritability: a case study with pupal color in Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae)

In this paper, two methods for assessing the degree of melanization of pupal exuviae from the butterfly Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae, Heliconiini) are compared. In the first method, which was qualitative, the exuviae were classified by scoring the degree of melanization, whereas in the second method, which was quantitative, the exuviae were classified by optical density followed by analysis with appropriate software. The heritability (h2 ) of the degree of melanization was estimated by regression and analysis of variance. The estimates of h 2 were similar with both methods, indicating that the qualitative method could be particularly suitable for field work. The low estimates obtained for heritability may have resulted from the small sample size (n = 7-18 broods, including the parents) or from the allocation-priority hypothesis in which pupal color would be a lower priority trait compared to morphological traits and adequate larval development.

Nuptial gifts and sexual behavior in two species of spider (Araneae, Trechaleidae, Paratrechalea).

Male delivering of a prey packed in silk as a nuptial gift is rare in spiders and restricted until now to Pisauridae. Here, we describe this behavioral pattern found in two Trechaleidae species, Paratrechalea azul Carico 2005, and Paratrechalea ornata (Mello-Leitão 1943), mainly based on field observations. We observed the following steps of sexual behavior: sperm induction, nuptial gift construction, mate searching, pre-copulatory courtship, copulation, and copulatory ending. In this group, a nuptial gift consists of a prey wrapped in silk, which appears as a white rounded shape. The male carries his nuptial gift in his chelicerae while searching for a female. When he finds a female, he shows a stereotyped courting behavior consisting of a hyperflexed posture that is also assumed by the receptive female while they face each other. The copulatory position and pattern is similar to that found in other Lycosoidea: the male mounts the female and makes a total of four palpal insertions while alternating sides. However, after each palpal insertion the male dismounts and returns to a frontal position while biting the gift. Copulatory courtship is evidenced by palpal and leg movements. The copulation ends by female initiative and she almost always retains the nuptial gift. No case of pre-copulatory or post-copulatory cannibalism has been recorded. Descriptions of nuptial gift construction by males and copulation in these species, as well as hypotheses about the origin of nuptial gift construction among spiders, are presented. These descriptions are the first records of such nuptial gift offering for Neotropical spiders and for non-Pisauridae species as well.

Pupal melanization in Heliconius erato phyllis (Lepidoptera; Nymphalidae): genetic and environmental effects.

This paper deals with estimates of heritability and the effect of two different kinds of environment on the melanization of pupae in the butterfly Heliconius erato phyllis. The results for heritability showed greater differences when the offspring were regressed on male or female values for pupal colour. Pupal colour followed a system of discrete scores, from 2 for the light pupae to 5 for the darkest one. Estimate of the heritability (h2) was 0.44 when the average score of the siblings were regressed on the mid-parental value. When regressed on the male parent, h2 was larger (0.54), being very low when regressed on the female parent (0.09). Estimate by the analysis of variance was also 0.44 when both male and female sibs were included in the analysis (for male sibs, h2=0.43 and for female sibs, 0.47). To test for the effect of the environment, each brood was divided in three, one being the control, the second subjected to a black environment when reaching the fifth instar and the third to a white environment. Those in the black environment originated dark pupae that scored 5 or 4; for those in the white environment, there was no difference with the controls. Caterpillars when entering the prepupal stage were also subjected to the black treatment (early and late prepupal stage, respectively, for treatments called black A and black B). A significant effect was observed only when early prepupal stage was subjected to the black environment.

Spreading the evolutionary synthesis: Theodosius Dobzhansky and genetics in Brazil

The so-called Evolutionary Synthesis, the present paradigm for evolutionary explanations, was established during the 1920s, 1930s and 1940s. One of the leading scientists contributing to this was Theodosius Dobzhansky, a Russian born geneticist who emigrated to the United States of America in 1927 to study with Thomas Hunt Morgan. He was also responsible for the development of Drosophila genetics in Brazil, which was the main organism employed in experimental studies of evolution. Dobzhansky had several opportunities to visit Brazil starting in 1943, to do field and laboratory work as well as teaching. All these activities were fundamental in the spreading of new concepts, methodology, and objectives of the Synthesis to a new audience. This paper discusses the results of the interaction between Dobzhansky and a group of young Brazilian researchers, particularly from the University of São Paulo.

Elytra colour polymorphism and randomness of matings in Chauliognathus fallax Germar 1824 from southern Brazil (Coleoptera, Cantharidae)

Beetles of the species Chauliognathus fallax Germar 1824 are polymorphic for elytron colouration with six morphs distinguished on the basis of black pigmentation on a yellow background. We investigated samples of C. fallax taken in eight consecutive weeks aiming to determine the frequency of the morphs which were grouped, for statistical analysis, into three classes according to elytra pigmentation as well as the possible occurrence of assortative mating. Our results showed a variation in the frequency of the classes throughout the season, both in males and females, with the maximum frequency of each class at the fourth and fifth week. The three classes (A, B, C) showed the same pattern of variation, and class B was always the more frequent. To test randomness of matings two approaches were taken: in the first, we compared the frequency of each class in copulating and non-copulating insects. In the second, the frequency of each class in the whole sample was taken as the probability of occurrence of the respective class; then, using the criterion of the probability of independent events we calculated the expected proportion of copulating insects for each pair of events. Both methods gave non-significant differences, suggesting that the matings were random.