The genetic, morphological, and physiological characterization of a dark larval cuticle mutation in the butterfly, Bicyclus anynana

Abstract

Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.

Figure 1. Method of measuring the cuticle brightness value (B-value) of caterpillars.

a. 5th instar dl. b. 5^th instar wt. Both original images have been separated from the background using the “lasso” tool, averaged using the “average” tool, and then converted to gray scale in Adobe Photoshop.

Figure 2. Graphical representation of B-values of offspring from three types of parental cross.

a. Average B-values (± SD) of offspring from three types of parental cross. Each cross is represented by five families arranged vertically by instar. b. Scatter plot to show overlap and spread of B-values for individuals from the families of the three types of parental cross.

Figure 3. Representative photographs of larval families from the three types of cross.

Columns represent siblings from the same family and rows represent the same individual photographed as a 2^nd, 3^rd, 4^th and 5^th instar, as a prepupa (pp) and as a pupa (p).

Figure 4. Topical application of JHA to dl leads to a supernumerary instar.

a. normal 5^th instar larva that was treated with acetone. b. 6^th supernumerary instar larva that was treated with JHA on the 4^th day of the 4^th instar.

Figure 5. Light microscope and TEM imaging of dl and wt cuticles.

a,b. Light micrograph of the dorsal cuticle of a fifth instar wt (a) and dl (b) larvae showing darkly pigmented bristles and star-like structures with concentrated pigmentation. c,d. TEM micrograph of dorsal cuticle of fifth instar wt (c) and dl (d) showing a star-like structure containing a homogeneous layer of diffuse pigment that is darker in dl (d).