Abdominal-B regulates structure and development of the Harmonia axyridis cremaster.

The ladybird Harmonia axyridis is an insect that exhibits pupal attachment to plants, which facilitates development and environmental adaptation. The cremaster is highly specialized for this behavior. However, the underlying molecular regulation of the cremaster remains unclear; therefore, we performed experiments to investigate the transcriptional regulation of cremaster development. First, we examined the morphological structure of the cremaster to reveal its function in pupal attachment of H. axyridis. Next, we analyzed the Hox gene Ha-Abd-B using RNA interference (RNAi) to determine its function in regulating cremaster formation; Ha-Abd-B up-regulation promoted effective pupal attachment, whereas successful RNAi caused severe down-regulation of this gene, and pupae were unable to attach. Furthermore, successful RNAi and subsequent Ha-Abd-B down-regulation caused phenotypic changes in cremaster structure, including its complete disappearance from some individuals. Finally, we observed unique development of the cremaster and dynamic expression of Ha-Abd-B during pre-pupal development; consequently, we hypothesized that there was specific pre-pupal development of the cremaster. Overall, based on these results, the specialized cremasteric structure located on the posterior side of H. axyridis was determined to be a key organ for pupal attachment. Cremaster identification in H. axyridis is regulated by Ha-Abd-B and exhibits preferential development. Pupal attachment of H. axyridis reveals an environmental adaptation of this species; thus, this study and future molecular studies will help determine the role of Hox genes in regulation of insect attachment and further our understanding of the multiple functions of Hox genes.

Aspartate-ß-alanine-NBAD pathway regulates pupal melanin pigmentation plasticity of ladybird Harmonia axyridis.

Phenotypic plasticity is observed in many animal species and it is effective for them to cope with many types of environmental threats. The multicolored Asian ladybird Harmonia axyridis shows a cuticular pigmentation plasticity that can be rapidly induced by temperature changes, and in the form of changeable melanin spot patterns to adjust heat-absorbing. Here, H. axyridis with thermal stimulation were selected for determining the molecular regulations behind it. First, we confirmed the melanin level changes of H. axyridis pupa could be induced by temperature, and then screened the efficient time window for thermal sensing of H. axyridis pre-pupa; it is suggested that the late stage of pre-pupa (late stage of 4th instar larva) is the critical period to sense thermal signals and adjust its pupal melanin spot area size to adapt to upcoming thermal conditions. The Ha-ADC (aspartate decarboxylase) and Ha-ebony (NBAD synthase) of aspartate-ß-alanine-NBAD pathway were then proved in regulation of cuticular melanization for pupa through RNA interference experiments; knockdown of these two genes enlarged the melanin spot size. Finally, we designed a random injection of Ha-ADC at different pre-pupal stages, to further study the regulation window during this process. Combined with all evidence observed, we suggested the spot size determination can be regulated very close to the time point of pupation, and genes of the aspartate-ß-alanine-NBAD pathway play an important role at the molecular level. In brief, H. axyridis exhibits a flexible active physiological regulation through transcriptional modification to thermal changes.

The L-DOPA/Dopamine Pathway Transgenerationally Regulates Cuticular Melanization in the Pea Aphid Acyrthosiphon pisum.

Maternal phenotypic regulations between different generations of aphid species help aphids to adapt to environmental challenges. The pea aphid Acyrthosiphon pisum has been used as a biological model for studies on phenotypic regulation for adaptation, and its alternative phenotypes are typically and physiologically based on maternal effects. We have observed an artificially induced and host-related maternal effect that may be a new aspect to consider in maternal regulation studies using A. pisum. Marked phenotypic changes in the cuticular melanization of daughter A. pisum were detected via tyrosine hydroxylase knockdown in the mothers during their period of host plants alternations. This phenotypic change was found to be both remarkable and repeatable. We performed several studies to understand its regulation and concluded that it may be controlled via the dopamine pathway. The downregulation and phenotypes observed were verified and described in detail. Additionally, based on histological and immunofluorescence analyses, the phenotypic changes caused by cuticular dysplasia were physiologically detected. Furthermore, we found that this abnormal development could not be reversed after birth. Transcriptome sequencing confirmed that this abnormal development represents a systemic developmental failure with numerous transcriptional changes, and chemical interventions suggested that transgenerational signals were not transferred through the nervous system. Our data show that transgenerational regulation (maternal effect) was responsible for the melanization failure. The developmental signals were received by the embryos from the mother aphids and were retained after birth. APTH RNAi disrupted the phenotypic determination process. We demonstrate that non-neuronal dopamine regulation plays a crucial role in the transgenerational phenotypic regulation of A. pisum. These results enhance our understanding of phenotyping via maternal regulation in aphids.

Superficially Similar Adaptation Within One Species Exhibits Similar Morphological Specialization but Different Physiological Regulations and Origins.

Animals have developed numerous strategies to contend with environmental pressures. We observed that the same adaptation strategy may be used repeatedly by one species in response to a certain environmental challenge. The ladybird Harmonia axyridis displays thermal phenotypic plasticity at different developmental stages. It is unknown whether these superficially similar temperature-induced specializations share similar physiological mechanisms. We performed various experiments to clarify the differences and similarities between these processes. We examined changes in the numbers and sizes of melanic spots in pupae and adults, and confirmed similar patterns for both. The dopamine pathway controls pigmentation levels at both developmental stages of H. axyridis. However, the aspartate-ß-alanine pathway controls spot size and number only in the pupae. An upstream regulation analysis revealed the roles of Hox genes and elytral veins in pupal and adult spot formation. Both the pupae and the adults exhibited similar morphological responses to temperatures. However, they occurred in different body parts and were regulated by different pathways. These phenotypic adaptations are indicative of an effective thermoregulatory system in H. axyridis and explains how insects contend with certain environmental pressure based on various control mechanisms.

Effects of mummy consumption on fitness and oviposition site selection on Harmonia axyridis.

Intraguild predation (IGP) has been commonly reported between predators and parasitoids used as biological control agents as predators consuming parasitoids within their hosts. However, the effect of parasitoid-mummy consumption on the fitness of the predator and subsequent oviposition site selection have not been well studied. In our study, we conducted two laboratory experiments to examine the influence of Aphidius gifuensis Ashmead (Hymenoptera: Braconidae) mummies as prey on fitness and subsequently oviposition site selection of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Results indicate that when H. axyridis was reared on A. gifuensis mummies only, its larval development was prolonged, and body weight of the 4th instar larvae and newly emerged adults, and fecundity decreased. Moreover, H. axyridis did not exhibit oviposition preference on plants infested with unparasitized aphids or aphids parasitized for shorter than 9 days. However, compared with plants with mummies (parasitized ≥9 days), H. axyridis laid more eggs on plants with unparasitized aphids. In contrast, H. axyridis previously fed with A. gifuensis mummies did not show a significant oviposition preference between plants with unparasitized aphids and those with mummies (parasitized ≥9 days). Overall, our results suggest that mummy consumption reduced the fitness of H. axyridis. Although H. axyridis avoided laying eggs on plants with A. gifuensis mummies, prior feeding experience on A. gifuensis mummies could alter the oviposition site preference. Thus, in biological control practice, prior feeding experience of H. axyridis should be carefully considered for reduction of IGP and increase of fitness of H. axyridis on A. gifuensis.

Starvation Stress Causes Body Color Change and Pigment Degradation in Acyrthosiphon pisum.

The pea aphid, Acyrthosiphon pisum (Harris), shows body color shifting from red to pale under starvation in laboratory conditions. These body color changes reflect aphid's adaptation to environmental stress. To understand the color-shifting patterns, the underlying mechanism and its biological or ecological functions, we measured the process of A. pisum's body color shifting patterns using a digital imagery and analysis system; we conducted a series of biochemical experiments to determine the mechanism that causes color change and performed biochemical and molecular analyses of the energy reserves during the color shifting process. We found that the red morph of A. pisum could shift their body color to pale red, when starved; this change occurred rapidly at a certain stress threshold. Once A. pisum initiated the process, the shifting could not be stopped or reversed even after food was re-introduced. We also discovered that the orange-red pigments may be responsible for the color shift and that the shift might be caused by the degradation of these pigments. The carbohydrate and lipid content correlated to the fading of color in red A. pisum. A comparative analysis revealed that these reddish pigments might be used as backup energy. The fading of color reflects a reorganization of the energy reserves under nutritional stress in A. pisum; surprisingly, aphids with different body colors exhibit diverse strategies for storage and consumption of energy reserves.

Use of tyrosine hydroxylase RNAi to study Megoura viciae (Hemiptera: Aphididae) sequestration of its host's l-DOPA for body melanism.

Melanism in insects is important for their physical protection, immunoreactions, and sclerotization. The vetch aphid, Megoura viciae (Buckton), has relatively strong tanning in its prothorax, head, antennae, cornicles, and legs. It was hypothesized that M. viciae may sequester the high level of l-DOPA in its host Vicia faba to help in its melanization process for ecological adaptation. To confirm this hypothesis, the amount of l-DOPA in M. viciae was modified and quantified. We first generated a Trifolium repens (clover, low l-DOPA containing) host to cut off the extra l-DOPA intake by M. viciae. The rate-limiting tyrosine hydroxylase gene of M. viciae (MV-TH) was then cloned and analyzed. To further reduce the l-DOPA level in the insect, RNAi was used to downregulate the transcriptional level of MV-TH. Our results confirmed that M. viciae could indeed sequester l-DOPA in its body, and its ample storage of this amino acid could be the reason for the strong tanning of its body. M. viciae reared on T. repens could upregulate its MV-TH to enhance l-DOPA biosynthesis and thus maintain a high level of l-DOPA. The MV-TH repression by RNAi lasted for about 3 days, successfully decreasing the l-DOPA level. Aside from a slight decrease in exuvia tanning, no other obvious change in body appearance was detected in the RNAi-treated insect. Although M. viciae can obtain most of its l-DOPA directly from its original host, its internal l-DOPA synthetase is still functional, especially when extra l-DOPA is removed from the diet. This capability to enhance its shield ensures the ecological adaptation of this insect.